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Lehtonen J, Hakonen AH, Hassinen A, Lurås SI, Kaustio M, Glumoff V, Hinrichsen F, Li W, Sulonen AM, Wickman S, Almusa H, Polso M, Palomäki M, Kivirikko S, Avela K, Heiskanen K, Pietiäinen V, Aittomäki K, Saarela J. Genome sequencing reveals CCDC88A variants in malformations of cortical development and immune dysfunction. Hum Mol Genet 2025:ddaf081. [PMID: 40401444 DOI: 10.1093/hmg/ddaf081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2024] [Revised: 03/24/2025] [Accepted: 04/22/2025] [Indexed: 05/23/2025] Open
Abstract
Malformations of cortical development (MCDs) encompass a diverse group of genetic and clinical disorders. Here, we aimed to determine a genetic etiology for two siblings manifesting MCD, microcephaly, epilepsy, intellectual disability, and susceptibility to infections. A missense variant (NM_018084:c.929A > C, p.Asp310Ala) and an intragenic deletion (exons 14-16) in CCDC88A were identified as compound heterozygous in patients by genome sequencing. Truncating homozygous CCDC88A variants are known to cause an ultra-rare syndrome manifesting with MCD, microcephaly, seizures, and severe neurological impairment. CCDC88A encodes girdin, which is essential for various cell functions, such as actin remodeling and cell proliferation. Western blot analysis showed that the missense variant allele was expressed in fibroblasts at a level compatible with a heterozygous allele, whereas a truncated protein from the deletion allele was barely detectable. Proliferation and wound-healing assays revealed that girdin-deficient fibroblasts proliferated faster and migrated slower than controls. High-content imaging highlighted girdin-deficient fibroblasts as smaller and their actin remodeling disrupted, leading to perinuclear accumulation of endolysosomal organelles. To confirm these cellular phenotypes resulted from girdin loss, CRISPR-Cas9 edited knockout models of healthy fibroblasts were created, replicating the observations in patient cells. Additionally, the siblings exhibited reduced monocytoid and plasmacytoid dendritic cells, suggesting compromised immunity due to girdin deficiency. In summary, the study describes the first case of a CCDC88A missense variant and intragenic deletion associated with MCD. It demonstrates altered immunity and girdin-related cellular changes, such as cell morphology and proliferation-migration dichotomy, in patient and knockout fibroblasts, reinforcing the pathogenic relevance of these variants.
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Affiliation(s)
- Johanna Lehtonen
- Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo Science Park, Gaustadalléen 2, Oslo 0349, Norway
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
- Folkhälsan Research Center, Biomedicum 1, Haartmaninkatu 8, Helsinki 00290, Finland
- Department of Medical Genetics, Oslo University Hospital, Building 25, Kirkeveien 166 (Ullevål), Oslo 0450, Norway
| | - Anna H Hakonen
- Department of Clinical Genetics, HUSLAB, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Topeliuksenkatu 32, Helsinki 00290, Finland
| | - Antti Hassinen
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Sanne Iversen Lurås
- Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo Science Park, Gaustadalléen 2, Oslo 0349, Norway
| | - Meri Kaustio
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Virpi Glumoff
- Medical Research Laboratory Unit, Faculty of Medicine, University of Oulu, Pentti Kaiteran katu 1, Oulu 90570, Finland
| | - Francisca Hinrichsen
- Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo Science Park, Gaustadalléen 2, Oslo 0349, Norway
| | - Weiwei Li
- Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo Science Park, Gaustadalléen 2, Oslo 0349, Norway
| | - Anna-Maija Sulonen
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Sanna Wickman
- Department of Pediatric Neurology, Hyvinkää Hospital, Helsinki and Uusimaa Hospital District, Sairaalankatu 1, Hyvinkää 05850, Finland
| | - Henrikki Almusa
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Minttu Polso
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Maarit Palomäki
- Department of Radiology, Helsinki University Hospital, Stenbäckinkatu 9, Helsinki 00290, Finland
| | - Sirpa Kivirikko
- Department of Clinical Genetics, HUSLAB, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Topeliuksenkatu 32, Helsinki 00290, Finland
| | - Kristiina Avela
- Department of Clinical Genetics, HUSLAB, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Topeliuksenkatu 32, Helsinki 00290, Finland
- Turku University Hospital, University of Turku, Kiinamyllynkatu 4-8, Turku 20520, Finland
| | - Kaarina Heiskanen
- New Children's Hospital, HUS, Helsinki University Hospital and University of Helsinki, Stenbäckinkatu 9, Helsinki 00290, Finland
| | - Vilja Pietiäinen
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
| | - Kristiina Aittomäki
- Department of Medical and Clinical Genetics, University of Helsinki, Fabianinkatu 33, Helsinki 00100, Finland
| | - Janna Saarela
- Centre for Molecular Medicine Norway (NCMM), University of Oslo, Oslo Science Park, Gaustadalléen 2, Oslo 0349, Norway
- Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Biomedicum 2, Tukholmankatu 8, Helsinki 00290, Finland
- Department of Medical Genetics, Oslo University Hospital, Building 25, Kirkeveien 166 (Ullevål), Oslo 0450, Norway
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Park Y, Matsumoto S, Ogata K, Ma B, Kanada R, Isaka Y, Arichi N, Liang X, Maki R, Kozasa T, Okuno Y, Ohno H, Ishihama Y, Toyoshima F. Receptor-independent regulation of Gα13 by alpha-1-antitrypsin C-terminal peptides. J Biol Chem 2025; 301:108136. [PMID: 39730062 PMCID: PMC11815680 DOI: 10.1016/j.jbc.2024.108136] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2024] [Revised: 12/17/2024] [Accepted: 12/19/2024] [Indexed: 12/29/2024] Open
Abstract
Alpha-1-antitrypsin (AAT), a circulating serine protease inhibitor, is an acute inflammatory response protein with anti-inflammatory functions. The C-terminal peptides of AAT are found in various tissues and have been proposed as putative bioactive peptides with multiple functions, but its mechanism of action remains unclear. We previously reported that a mouse AAT C-terminal peptide of 35 amino acids (mAAT-C1-35) penetrates plasma membrane and associates guanine nucleotide-binding protein subunit alpha 13 (Gα13). Here, we show that mAAT-C1-35 binds directly to the guanosine diphosphate (GDP)-bound form of Gα13 through the N-terminal region (mAAT-C1-17), thereby facilitating the interaction of Gα13・GDP with its effector proteins. The minimal sequence (mAAT-C3-16) and essential amino acid residue (Phe11) of mAAT-C1-17 were identified as being necessary for this effect. A molecular dynamics simulation for the Gα13・GDP-mAAT-C1-17 complex model showed that binding of mAAT-C1-17 to the region surrounded by switch regions of Gα13 stabilizes the flexible switch II and III regions, thereby maintaining their active conformation. In addition, mAAT-C1-35 activates the Gα13 signaling pathway in cells where Phe11 is required. Our study reveals the structure-based mechanism of action of AAT-C peptides in the regulation of Gα13 and demonstrates that AAT-C peptides represent a biological peptide capable of activating G protein signals in a manner that is independent of G-protein-coupled receptors.
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Affiliation(s)
- Yonghak Park
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Mammalian and Regulatory Networks, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Shigeyuki Matsumoto
- Department of Biomedical Data Intelligence, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
| | - Kosuke Ogata
- Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Biao Ma
- HPC- and AI-driven Drug Development Platform Division, RIKEN Center for Computational Science, Kobe, Hyogo, Japan
| | - Ryo Kanada
- HPC- and AI-driven Drug Development Platform Division, RIKEN Center for Computational Science, Kobe, Hyogo, Japan
| | - Yuta Isaka
- HPC- and AI-driven Drug Development Platform Division, RIKEN Center for Computational Science, Kobe, Hyogo, Japan
| | - Norihito Arichi
- Department of Bioorganic Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Xiaowen Liang
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Mammalian and Regulatory Networks, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Ritsuko Maki
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Tohru Kozasa
- Department of Biochemistry, Yokohama University of Pharmacy, Yokohama, Japan
| | - Yasushi Okuno
- Department of Biomedical Data Intelligence, Graduate School of Medicine, Kyoto University, Kyoto, Japan; HPC- and AI-driven Drug Development Platform Division, RIKEN Center for Computational Science, Kobe, Hyogo, Japan
| | - Hiroaki Ohno
- Department of Bioorganic Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Yasushi Ishihama
- Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Fumiko Toyoshima
- Department of Biosystems Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan; Department of Mammalian and Regulatory Networks, Graduate School of Biostudies, Kyoto University, Kyoto, Japan; Department of Homeostatic Medicine, Medical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan.
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Rajala RVS, Rajala A. Girdin: A Class I Phosphatidylinositol 3-Kinase-Binding Protein in the Retina. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1468:299-303. [PMID: 39930212 DOI: 10.1007/978-3-031-76550-6_49] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/08/2025]
Abstract
Girdin is a nonreceptor guanine nucleotide exchange factor but functions as a guanine nucleotide exchange factor for the G protein Gαi. It plays a crucial role in regulating cell migration and adhesion, as well as protecting cells from apoptosis. Previous research has demonstrated that Girdin activates Akt through a pathway involving phosphatidylinositol 3-kinase (PI3K) in HeLa cells. Our earlier studies revealed that light-induced activation of the PI3K/Akt pathway in the retina is controlled by G-protein-coupled receptor activation, specifically the rhodopsin-dependent insulin receptor. In our current study, we investigated the localization and interaction of Girdin with the p85α-subunit of PI3K. Our findings indicate that Girdin is present in photoreceptor cells and interacts with the p85α-subunit of PI3K in response to light stimulation. Activation of the PI3K/Akt pathway promotes the survival of photoreceptor cells. These results suggest that Girdin might be a novel factor contributing to the protection of photoreceptors in response to light, potentially offering new insights into neuroprotection for these cells.
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Affiliation(s)
- Raju V S Rajala
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
- Department of Biochemistry and Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
- Dean McGee Eye Institute, Oklahoma City, OK, USA.
| | - Ammaji Rajala
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA
- Dean McGee Eye Institute, Oklahoma City, OK, USA
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Dwyer MB, Aumiller JL, Wedegaertner PB. Going Rogue: Mechanisms, Regulation, and Roles of Mutationally Activated G α in Human Cancer. Mol Pharmacol 2024; 106:198-215. [PMID: 39187387 PMCID: PMC11493338 DOI: 10.1124/molpharm.124.000743] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Revised: 08/19/2024] [Accepted: 08/21/2024] [Indexed: 08/28/2024] Open
Abstract
G protein-coupled receptors (GPCRs) couple to heterotrimeric G proteins, comprised of α and βγ subunits, to convert extracellular signals into activation of intracellular signaling pathways. Canonically, GPCR-mediated activation results in the exchange of GDP for GTP on G protein α subunits (Gα) and the dissociation of Gα-GTP and G protein βγ subunits (Gβγ), both of which can regulate a variety of signaling pathways. Hydrolysis of bound GTP by Gα returns the protein to Gα-GDP and allows reassociation with Gβγ to reform the inactive heterotrimer. Naturally occurring mutations in Gα have been found at conserved glutamine and arginine amino acids that disrupt the canonical G protein cycle by inhibiting GTP hydrolysis, rendering these mutants constitutively active. Interestingly, these dysregulated Gα mutants are found in many different cancers due to their ability to sustain aberrant signaling without a need for activation by GPCRs. This review will highlight an increased recognition of the prevalence of such constitutively activating Gα mutations in cancers and the signaling pathways activated. In addition, we will discuss new knowledge regarding how these constitutively active Gα are regulated, how different mutations are biochemically distinct, and how mutationally activated Gα are unique compared with GPCR-activated Gα Lastly, we will discuss recent progress in developing inhibitors directly targeting constitutively active Gα mutants. SIGNIFICANCE STATEMENT: Constitutively activating mutations in G protein α subunits (Gα) widely occur in and contribute to the development of many human cancers. To develop ways to inhibit dysregulated, oncogenic signaling by these mutant Gα, it is crucial to better understand mechanisms that lead to constitutive Gα activation and unique mechanisms that regulate mutationally activated Gα in cells. The prevalence of activating mutations in Gα in various cancers makes Gα proteins compelling targets for the development of therapeutics.
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Affiliation(s)
- Morgan B Dwyer
- Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Jenna L Aumiller
- Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
| | - Philip B Wedegaertner
- Department of Biochemistry and Molecular Biology, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania
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Janicot R, Garcia-Marcos M. Get Ready to Sharpen Your Tools: A Short Guide to Heterotrimeric G Protein Activity Biosensors. Mol Pharmacol 2024; 106:129-144. [PMID: 38991745 PMCID: PMC11331509 DOI: 10.1124/molpharm.124.000949] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 06/27/2024] [Accepted: 07/01/2024] [Indexed: 07/13/2024] Open
Abstract
G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors encoded in the human genome, and they initiate cellular responses triggered by a plethora of extracellular stimuli ranging from neurotransmitters and hormones to photons. Upon stimulation, GPCRs activate heterotrimeric G proteins (Gαβγ) in the cytoplasm, which then convey signals to their effectors to elicit cellular responses. Given the broad biological and biomedical relevance of GPCRs and G proteins in physiology and disease, there is great interest in developing and optimizing approaches to measure their signaling activity with high accuracy and across experimental systems pertinent to their functions in cellular communication. This review provides a historical perspective on approaches to measure GPCR-G protein signaling, from quantification of second messengers and other indirect readouts of activity to biosensors that directly detect the activity of G proteins. The latter is the focus of a more detailed overview of the evolution of design principles for various optical biosensors of G protein activity with different experimental capabilities. We will highlight advantages and limitations of biosensors that detect different G protein activation hallmarks, like dissociation of Gα and Gβγ or nucleotide exchange on Gα, as well as their suitability to detect signaling mediated by endogenous versus exogenous signaling components or in physiologically relevant systems like primary cells. Overall, this review intends to provide an assessment of the state-of-the-art for biosensors that directly measure G protein activity to allow readers to make informed decisions on the selection and implementation of currently available tools. SIGNIFICANCE STATEMENT: G protein activity biosensors have become essential and widespread tools to assess GPCR signaling and pharmacology. Yet, investigators face the challenge of choosing from a growing list of G protein activity biosensors. This review provides an overview of the features and capabilities of different optical biosensor designs for the direct detection of G protein activity in cells, with the aim of facilitating the rational selection of systems that align with the specific scientific questions and needs of investigators.
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Affiliation(s)
- Remi Janicot
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine (R.J., M.G.-M.) and Department of Biology, College of Arts & Sciences (M.G.-M.), Boston University, Boston, Massachusetts
| | - Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine (R.J., M.G.-M.) and Department of Biology, College of Arts & Sciences (M.G.-M.), Boston University, Boston, Massachusetts
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Ruan ML, Ni WX, Chu JCH, Lam TL, Law KC, Zhang Y, Yang G, He Y, Zhang C, Fung YME, Liu T, Huang T, Lok CN, Chan SLF, Che CM. Iridium(III) carbene complexes as potent girdin inhibitors against metastatic cancers. Proc Natl Acad Sci U S A 2024; 121:e2316615121. [PMID: 38861602 PMCID: PMC11194514 DOI: 10.1073/pnas.2316615121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2023] [Accepted: 04/27/2024] [Indexed: 06/13/2024] Open
Abstract
Many cancer-driving protein targets remain undruggable due to a lack of binding molecular scaffolds. In this regard, octahedral metal complexes with unique and versatile three-dimensional structures have rarely been explored as inhibitors of undruggable protein targets. Here, we describe antitumor iridium(III) pyridinium-N-heterocyclic carbene complex 1a, which profoundly reduces the viability of lung and breast cancer cells as well as cancer patient-derived organoids at low micromolar concentrations. Compound 1a effectively inhibits the growth of non-small-cell lung cancer and triple-negative breast cancer xenograft tumors, impedes the metastatic spread of breast cancer cells, and can be modified into an antibody-drug conjugate payload to achieve precise tumor delivery in mice. Identified by thermal proteome profiling, an important molecular target of 1a in cellulo is Girdin, a multifunctional adaptor protein that is overexpressed in cancer cells and unequivocally serves as a signaling hub for multiple pivotal oncogenic pathways. However, specific small-molecule inhibitors of Girdin have not yet been developed. Notably, 1a exhibits high binding affinity to Girdin with a Kd of 1.3 μM and targets the Girdin-linked EGFR/AKT/mTOR/STAT3 cancer-driving pathway, inhibiting cancer cell proliferation and metastatic activity. Our study reveals a potent Girdin-targeting anticancer compound and demonstrates that octahedral metal complexes constitute an untapped library of small-molecule inhibitors that can fit into the ligand-binding pockets of key oncoproteins.
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Affiliation(s)
- Mei-Ling Ruan
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
- National Key Laboratory of Green Pesticide, College of Chemistry, Central China Normal University, Wuhan430079, China
| | - Wen-Xiu Ni
- Department of Medicinal Chemistry, Shantou University Medical College, Shantou515041, Guangdong, China
- Chemistry and Chemical Engineering of Guangdong Laboratory, Shantou515041, Guangdong, China
| | - Jacky C. H. Chu
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Tsz-Lung Lam
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Kwok-Chung Law
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Yiwei Zhang
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Guanya Yang
- AI And Life Sciences Institute (Hong Kong) Limited, Hong Kong Science Park, Shatin, Hong Kong, China
| | - Ying He
- AI And Life Sciences Institute (Hong Kong) Limited, Hong Kong Science Park, Shatin, Hong Kong, China
| | - Chunlei Zhang
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Yi Man Eva Fung
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Tao Liu
- Department of Medicinal Chemistry, Shantou University Medical College, Shantou515041, Guangdong, China
- Chemistry and Chemical Engineering of Guangdong Laboratory, Shantou515041, Guangdong, China
| | - Tao Huang
- Department of Medicinal Chemistry, Shantou University Medical College, Shantou515041, Guangdong, China
- Chemistry and Chemical Engineering of Guangdong Laboratory, Shantou515041, Guangdong, China
| | - Chun-Nam Lok
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
| | - Sharon Lai-Fung Chan
- Department of Applied Biology and Chemical Biology, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China
| | - Chi-Ming Che
- Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Shatin, Hong Kong, China
- State Key Laboratory of Synthetic Chemistry and Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
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Roy S, Sinha S, Silas AJ, Ghassemian M, Kufareva I, Ghosh P. Growth factor-dependent phosphorylation of Gα i shapes canonical signaling by G protein-coupled receptors. Sci Signal 2024; 17:eade8041. [PMID: 38833528 PMCID: PMC11328959 DOI: 10.1126/scisignal.ade8041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2022] [Accepted: 05/17/2024] [Indexed: 06/06/2024]
Abstract
A long-standing question in the field of signal transduction is how distinct signaling pathways interact with each other to control cell behavior. Growth factor receptors and G protein-coupled receptors (GPCRs) are the two major signaling hubs in eukaryotes. Given that the mechanisms by which they signal independently have been extensively characterized, we investigated how they may cross-talk with each other. Using linear ion trap mass spectrometry and cell-based biophysical, biochemical, and phenotypic assays, we found at least three distinct ways in which epidermal growth factor affected canonical G protein signaling by the Gi-coupled GPCR CXCR4 through the phosphorylation of Gαi. Phosphomimicking mutations in two residues in the αE helix of Gαi (tyrosine-154/tyrosine-155) suppressed agonist-induced Gαi activation while promoting constitutive Gβγ signaling. Phosphomimicking mutations in the P loop (serine-44, serine-47, and threonine-48) suppressed Gi activation entirely, thus completely segregating growth factor and GPCR pathways. As expected, most of the phosphorylation events appeared to affect intrinsic properties of Gαi proteins, including conformational stability, nucleotide binding, and the ability to associate with and to release Gβγ. However, one phosphomimicking mutation, targeting the carboxyl-terminal residue tyrosine-320, promoted mislocalization of Gαi from the plasma membrane, a previously uncharacterized mechanism of suppressing GPCR signaling through G protein subcellular compartmentalization. Together, these findings elucidate not only how growth factor and chemokine signals cross-talk through the phosphorylation-dependent modulation of Gαi but also how such cross-talk may generate signal diversity.
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Affiliation(s)
- Suchismita Roy
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Saptarshi Sinha
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Ananta James Silas
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Majid Ghassemian
- Department of Chemistry and Biochemistry, Biomolecular and Proteomics Mass Spectrometry Facility, University of California San Diego, San Diego, CA 92093, USA
| | - Irina Kufareva
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, CA 92093, USA
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
- Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, CA 92093, USA
- Department of Medicine, University of California San Diego, CA 92093, USA
- Moore’s Comprehensive Cancer Center, University of California San Diego, CA 92093, USA
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8
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Shewani K, Madhu MK, Murarka RK. Mechanistic insights into G-protein activation via phosphorylation mediated non-canonical pathway. Biophys Chem 2024; 309:107234. [PMID: 38603989 DOI: 10.1016/j.bpc.2024.107234] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2024] [Revised: 03/21/2024] [Accepted: 04/02/2024] [Indexed: 04/13/2024]
Abstract
Activation of heterotrimeric G-proteins (Gαβγ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of Gα that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce Gα activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the Gαi3, a subtype of Gα-protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. In addition to weakened salt bridges at the domain interface, phosphorylations induced the unfolding of helix αF, which contributed to increased flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated Gαi3. Although the larger domain separation in the phosphorylated system provided an unobstructed path for the nucleotide, the accelerated release of GDP was attributed to increased fluctuations in several conserved regions like P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.
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Affiliation(s)
- Kunal Shewani
- Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhopal 462066, MP, India
| | - Midhun K Madhu
- Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhopal 462066, MP, India
| | - Rajesh K Murarka
- Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhopal 462066, MP, India.
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Garcia-Marcos M. Heterotrimeric G protein signaling without GPCRs: The Gα-binding-and-activating (GBA) motif. J Biol Chem 2024; 300:105756. [PMID: 38364891 PMCID: PMC10943482 DOI: 10.1016/j.jbc.2024.105756] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2023] [Revised: 01/30/2024] [Accepted: 02/02/2024] [Indexed: 02/18/2024] Open
Abstract
Heterotrimeric G proteins (Gαβγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.
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Affiliation(s)
- Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, Massachusetts, USA; Department of Biology, College of Arts & Sciences, Boston University, Boston, Massachusetts, USA.
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10
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Lao-Peregrin C, Xiang G, Kim J, Srivastava I, Fall AB, Gerhard DM, Kohtala P, Kim D, Song M, Garcia-Marcos M, Levitz J, Lee FS. Synaptic plasticity via receptor tyrosine kinase/G-protein-coupled receptor crosstalk. Cell Rep 2024; 43:113595. [PMID: 38117654 PMCID: PMC10844890 DOI: 10.1016/j.celrep.2023.113595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 11/15/2023] [Accepted: 12/01/2023] [Indexed: 12/22/2023] Open
Abstract
Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and the physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK) TrkB and the G-protein-coupled receptor (GPCR) metabotropic glutamate receptor 5 (mGluR5) together mediate hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode switch that drives BDNF-dependent sustained, oscillatory Ca2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gβγ, released by TrkB, and Gαq-GTP, released by mGluR5, to enable physiologically relevant RTK/GPCR crosstalk.
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Affiliation(s)
| | - Guoqing Xiang
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA; Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA
| | - Jihye Kim
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA
| | - Ipsit Srivastava
- Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA
| | - Alexandra B Fall
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA
| | - Danielle M Gerhard
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA
| | - Piia Kohtala
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA
| | - Daegeon Kim
- Department of Life Sciences, Yeongnam University, Gyeongsan, Gyeongbuk 38451, South Korea
| | - Minseok Song
- Department of Life Sciences, Yeongnam University, Gyeongsan, Gyeongbuk 38451, South Korea
| | - Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Joshua Levitz
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA; Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA.
| | - Francis S Lee
- Department of Psychiatry, Weill Cornell Medicine. New York, NY 10065, USA.
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11
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Luebbers A, Gonzalez-Hernandez AJ, Zhou M, Eyles SJ, Levitz J, Garcia-Marcos M. Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP. Structure 2024; 32:47-59.e7. [PMID: 37989308 PMCID: PMC10872408 DOI: 10.1016/j.str.2023.10.010] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Revised: 09/23/2023] [Accepted: 10/25/2023] [Indexed: 11/23/2023]
Abstract
It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαβγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gβγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.
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Affiliation(s)
- Alex Luebbers
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | | | - Myles Zhou
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Stephen J Eyles
- Mass Spectrometry Core Facility, Institute for Applied Life Sciences (IALS), University of Massachusetts Amherst, Amherst, MA 01003, USA
| | - Joshua Levitz
- Department of Biochemistry, Weill Cornell Medicine, New York, NY 10064, USA; Department of Psychiatry, Weill Cornell Medicine, New York, NY 10065, USA
| | - Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA; Department of Biology, College of Arts & Sciences, Boston University, Boston, MA 02115, USA.
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12
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Campagna CM, McMahon H, Nechipurenko I. The G protein alpha chaperone and guanine-nucleotide exchange factor RIC-8 regulates cilia morphogenesis in Caenorhabditis elegans sensory neurons. PLoS Genet 2023; 19:e1011015. [PMID: 37910589 PMCID: PMC10642896 DOI: 10.1371/journal.pgen.1011015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2023] [Revised: 11/13/2023] [Accepted: 10/12/2023] [Indexed: 11/03/2023] Open
Abstract
Heterotrimeric G (αβγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.
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Affiliation(s)
- Christina M. Campagna
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
| | - Hayley McMahon
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
| | - Inna Nechipurenko
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
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13
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Pepanian A, Binbay FA, Roy S, Nubbemeyer B, Koley A, Rhodes CA, Ammer H, Pei D, Ghosh P, Imhof D. Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators. J Med Chem 2023; 66:12396-12406. [PMID: 37587416 PMCID: PMC11000586 DOI: 10.1021/acs.jmedchem.3c00873] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/18/2023]
Abstract
Noncanonical G protein activation and inactivation, particularly for the Gαi/s protein subfamilies, have long been a focus of chemical research. Combinatorial libraries were already effectively applied to identify modulators of the guanine-nucleotide exchange, as can be exemplified with peptides such as KB-752 and GPM-1c/d, the so-called guanine-nucleotide exchange modulators. In this study, we identified novel bicyclic peptides from a combinatorial library screening that show prominent properties as molecular switch-on/off modulators of Gαi signaling. Among the series of hits, the exceptional paradigm of GPM-3, a protein and state-specific bicyclic peptide, is the first chemically identified GAP (GTPase-activating protein) modulator with a high binding affinity for Gαi protein. Computational analyses identified and assessed the structure of the bicyclic peptides, novel ligand-protein interaction sites, and their subsequent impact on the nucleotide binding site. This approach can therefore lead the way for the development of efficient chemical biological probes targeting Gαi protein modulation within a cellular context.
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Affiliation(s)
- Anna Pepanian
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenbeurg 4, Bonn 53121, Germany
| | - Furkan Ayberk Binbay
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenbeurg 4, Bonn 53121, Germany
| | - Suchismita Roy
- Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, United States
| | - Britta Nubbemeyer
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenbeurg 4, Bonn 53121, Germany
| | - Amritendu Koley
- Department of Chemistry and Biochemistry, The Ohio State University, 578 Biological Sciences Building, 484 W 12th Avenue, Columbus, Ohio 43210, United States
| | - Curran A Rhodes
- Department of Chemistry and Biochemistry, The Ohio State University, 578 Biological Sciences Building, 484 W 12th Avenue, Columbus, Ohio 43210, United States
| | - Hermann Ammer
- Institute of Pharmacology Toxicology and Pharmacy, Veterinary Faculty, Ludwig Maximilian University of Munich, Königinstr. 16, Munich 80539, Germany
| | - Dehua Pei
- Department of Chemistry and Biochemistry, The Ohio State University, 578 Biological Sciences Building, 484 W 12th Avenue, Columbus, Ohio 43210, United States
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, United States
- Department of Medicine, University of California San Diego, La Jolla, California 92093, United States
| | - Diana Imhof
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenbeurg 4, Bonn 53121, Germany
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14
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Lao-Peregrin C, Xiang G, Kim J, Srivastava I, Fall AB, Gerhard DM, Kohtala P, Kim D, Song M, Garcia-Marcos M, Levitz J, Lee FS. Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.28.555210. [PMID: 37693535 PMCID: PMC10491144 DOI: 10.1101/2023.08.28.555210] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/12/2023]
Abstract
Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK), TrkB, and the G protein-coupled receptor (GPCR), metabotropic glutamate receptor 5 (mGluR5), together mediate a novel form of hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode-switch that drives BDNF-dependent sustained, oscillatory Ca 2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gβγ, released by TrkB, and Gα q -GTP, released by mGluR5, to enable a previously unidentified form of physiologically relevant RTK/GPCR crosstalk.
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15
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Campagna CM, McMahon H, Nechipurenko I. The G protein alpha Chaperone and Guanine-Nucleotide Exchange Factor RIC-8 Regulates Cilia Morphogenesis in Caenorhabditis elegans Sensory Neurons. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.25.554856. [PMID: 37662329 PMCID: PMC10473713 DOI: 10.1101/2023.08.25.554856] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Heterotrimeric G (αβγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite severe defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction.
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Affiliation(s)
- Christina M. Campagna
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
| | - Hayley McMahon
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
| | - Inna Nechipurenko
- Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America
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16
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Park JC, Luebbers A, Dao M, Semeano A, Nguyen AM, Papakonstantinou MP, Broselid S, Yano H, Martemyanov KA, Garcia-Marcos M. Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits. Mol Cell 2023; 83:2540-2558.e12. [PMID: 37390816 PMCID: PMC10527995 DOI: 10.1016/j.molcel.2023.06.006] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Revised: 04/28/2023] [Accepted: 06/02/2023] [Indexed: 07/02/2023]
Abstract
G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαβγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gβγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gβγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gβγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gβγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.
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Affiliation(s)
- Jong-Chan Park
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Alex Luebbers
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Maria Dao
- U.F. Scripps Biomedical Research, University of Florida, Jupiter, FL 33458, USA
| | - Ana Semeano
- Department of Pharmaceutical Sciences, Center for Drug Discovery, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA 02115, USA
| | - Anh Minh Nguyen
- Department of Pharmaceutical Sciences, Center for Drug Discovery, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA 02115, USA
| | - Maria P Papakonstantinou
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Stefan Broselid
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Hideaki Yano
- Department of Pharmaceutical Sciences, Center for Drug Discovery, School of Pharmacy and Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA 02115, USA
| | | | - Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA; Department of Biology, College of Arts & Sciences, Boston University, Boston, MA 02115, USA.
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17
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Jia X, Xu F, Lu S, Jie H, Guan W, Zhou Y. An unusual signal transducer GIV/Girdin engages in the roles of adipocyte-derived hormone leptin in liver fibrosis. Biochim Biophys Acta Mol Basis Dis 2023; 1869:166797. [PMID: 37478565 DOI: 10.1016/j.bbadis.2023.166797] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 06/06/2023] [Accepted: 06/22/2023] [Indexed: 07/23/2023]
Abstract
Obese patients usually have hyperleptinemia and are prone to develop liver fibrosis. Leptin is intimately linked to liver fibrogenesis, a multi-receptor-driven disease. Gα-Interacting Vesicle-associated protein (GIV) functions as a multimodular signal transducer and a guanine nucleotide exchange factor for Gi controling key signalings downstream of diverse receptors. This study aimed to examine the roles of GIV in leptin-caused liver fibrosis and employed the culture-activated hepatic stellate cells (HSCs) and leptin-deficient mice, respectively. Results indicated that leptin upregulated GIV expression in HSCs. GIV was involved in leptin-induced HSC activation and liver fibrosis. GIV mediated leptin regulation of TIMP1, MMP9, PDGFβ receptor and TGFβ receptor and was required for leptin stimulating the pathways of Erk1/2, Akt1, and Smad3. GIV was also a mediator for leptin-regulation of Cyclin D1 and Caspase-3 activity but GIV reduced Caspase-3 level independently of leptin in vivo. Erk1/2 signaling, Egr1 and c-Jun were associated with the effect of leptin on GIV expression in HSCs. Leptin-induced Erk1/2 signaling increased Egr1 and p-c-Jun levels and promoted their binding to GIV promoter at the sites between -190 bp and -180 bp and between -382 bp and - 376 bp, respectively. Egr1 knockdown lessened leptin-upregulation of GIV in vitro and in vivo. In human cirrhotic livers, the increase in GIV protein level parallelled with the elevated p-Erk1/2 and Egr1 levels in HSCs. In summary, the unusual signal transducer GIV was identified as an important mediator in leptin-induced liver fibrosis. GIV may have significant implications in liver fibrosis progression of obese patients with hyperleptinaemia.
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Affiliation(s)
- Xin Jia
- Department of Biochemistry & Molecular Biology, Medical School, Nantong University, Qi xiou Road 19, Nantong 226001, Jiangsu, China
| | - Feifan Xu
- Department of Clinical Laboratory, Affiliated Nantong Hospital of Shanghai University (The Sixth People's Hospital of Nantong), 500 Yonghe Road, Nantong 226011, Jiangsu, China
| | - Sidan Lu
- Department of Biochemistry & Molecular Biology, Medical School, Nantong University, Qi xiou Road 19, Nantong 226001, Jiangsu, China
| | - Huang Jie
- Department of Pharmacology, School of Pharmacy, Nantong University, Qi xiou Road 19, Nantong 226001, Jiangsu, China
| | - Wei Guan
- Department of Pharmacology, School of Pharmacy, Nantong University, Qi xiou Road 19, Nantong 226001, Jiangsu, China.
| | - Yajun Zhou
- Department of Biochemistry & Molecular Biology, Medical School, Nantong University, Qi xiou Road 19, Nantong 226001, Jiangsu, China.
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18
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Rajanala K, Wedegaertner PB. Gβγ signaling regulates microtubule-dependent control of Golgi integrity. Cell Signal 2023; 106:110630. [PMID: 36805843 PMCID: PMC10079639 DOI: 10.1016/j.cellsig.2023.110630] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/09/2023] [Accepted: 02/13/2023] [Indexed: 02/18/2023]
Abstract
Gβγ subunits regulate several non-canonical functions at distinct intracellular organelles. Previous studies have shown that Gβγ signaling at the Golgi is necessary to mediate vesicular protein transport function and to regulate mitotic Golgi fragmentation. Disruption of Golgi structure also occurs in response to microtubule depolymerizing agents, such as nocodazole. In this study, we use siRNA against Gβ1/2 or specific Gγ subunits to deplete their expression, and show that their knockdown causes a significant reduction in nocodazole-induced Golgi fragmentation. We establish that knockdown of Gβγ or inhibition of Gβγ with gallein resulted in decreased activation of protein kinase D (PKD) in response to nocodazole treatment. We demonstrate that restricting the amount of free Gβγ available for signaling by either inhibiting Gαi activation using pertussis toxin or by knockdown of the non-GPCR GEF, Girdin/GIV protein, results in a substantial decrease in nocodazole-induced Golgi fragmentation and PKD phosphorylation. Our results also indicate that depletion of Gβγ or inhibition with gallein or pertussis toxin significantly reduces the microtubule disruption-dependent Golgi fragmentation phenotype observed in cells transfected with mutant SOD1, a major causative protein in familial amyotrophic lateral sclerosis (ALS). These results provide compelling evidence that Gβγ signaling is critical for the regulation of Golgi integrity.
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Affiliation(s)
- Kalpana Rajanala
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Sidney Kimmel Medical College, Philadelphia, PA 19107, United States of America
| | - Philip B Wedegaertner
- Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Sidney Kimmel Medical College, Philadelphia, PA 19107, United States of America.
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19
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Zhao J, DiGiacomo V, Ferreras-Gutierrez M, Dastjerdi S, Ibáñez de Opakua A, Park JC, Luebbers A, Chen Q, Beeler A, Blanco FJ, Garcia-Marcos M. Small-molecule targeting of GPCR-independent noncanonical G-protein signaling in cancer. Proc Natl Acad Sci U S A 2023; 120:e2213140120. [PMID: 37098067 PMCID: PMC10160980 DOI: 10.1073/pnas.2213140120] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2022] [Accepted: 03/06/2023] [Indexed: 04/26/2023] Open
Abstract
Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class small-molecule inhibitor of noncanonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking noncanonical G-protein signaling in tumor cells and inhibiting proinvasive traits of metastatic cancer cells. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable noncanonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.
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Affiliation(s)
- Jingyi Zhao
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
| | - Vincent DiGiacomo
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
| | | | - Shiva Dastjerdi
- Department of Chemistry, Boston University, College of Arts & Sciences, Boston, MA02115
| | | | - Jong-Chan Park
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
| | - Alex Luebbers
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
| | - Qingyan Chen
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
| | - Aaron Beeler
- Department of Chemistry, Boston University, College of Arts & Sciences, Boston, MA02115
| | - Francisco J. Blanco
- Centro de Investigaciones Biológicas-Centro Superior de Investigaciones Cientificas, Madrid, Spain
| | - Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Boston University, Chobanian & Avedisian School of Medicine, Boston, MA02118
- Department of Biology, College of Arts & Sciences, Boston University, Boston, MA02115
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20
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Luebbers A, Zhou M, Eyles SJ, Garcia-Marcos M. Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.20.537566. [PMID: 37131787 PMCID: PMC10153262 DOI: 10.1101/2023.04.20.537566] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
It is well-established that activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters is a key mechanism underlying neuromodulation. Much less is known about how G-protein regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls neurological processes like pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding Gαi subunits and regulating G-protein signaling are not known. Here, we combined hydrogen-deuterium exchange mass-spectrometry, protein folding predictions, bioluminescence resonance energy transfer assays, and biochemical experiments to identify the first loop of the PHD domain of GINIP as an obligatory requirement for Gαi binding. Surprisingly, our results support a model in which GINIP undergoes a long-range conformational change to accommodate Gαi binding to this loop. Using cell-based assays, we demonstrate that specific amino acids in the first loop of the PHD domain are essential for the regulation of Gαi-GTP and free Gβγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.
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Affiliation(s)
- Alex Luebbers
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Myles Zhou
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
| | - Stephen J Eyles
- Mass Spectrometry Core Facility, Institute for Applied Life Sciences (IALS), University of Massachusetts Amherst, Amherst, MA 01003, USA
| | - Mikel Garcia-Marcos
- Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA
- Department of Biology, College of Arts & Sciences, Boston University, Boston, MA 02115, USA
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21
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Wang H, Yuan YC, Chang C, Izumi T, Wang HH, Yang JK. The signaling protein GIV/Girdin mediates the Nephrin-dependent insulin secretion of pancreatic islet β cells in response to high glucose. J Biol Chem 2023; 299:103045. [PMID: 36822326 PMCID: PMC10040812 DOI: 10.1016/j.jbc.2023.103045] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 02/10/2023] [Accepted: 02/12/2023] [Indexed: 02/23/2023] Open
Abstract
Glucose-stimulated insulin secretion of pancreatic β cells is essential in maintaining glucose homeostasis. Recent evidence suggests that the Nephrin-mediated intercellular junction between β cells is implicated in the regulation of insulin secretion. However, the underlying mechanisms are only partially characterized. Herein we report that GIV is a signaling mediator coordinating glucose-stimulated Nephrin phosphorylation and endocytosis with insulin secretion. We demonstrate that GIV is expressed in mouse islets and cultured β cells. The loss of function study suggests that GIV is essential for the second phase of glucose-stimulated insulin secretion. Next, we demonstrate that GIV mediates the high glucose-stimulated tyrosine phosphorylation of GIV and Nephrin by recruiting Src kinase, which leads to the endocytosis of Nephrin. Subsequently, the glucose-induced GIV/Nephrin/Src signaling events trigger downstream Akt phosphorylation, which activates Rac1-mediated cytoskeleton reorganization, allowing insulin secretory granules to access the plasma membrane for the second-phase secretion. Finally, we found that GIV is downregulated in the islets isolated from diabetic mice, and rescue of GIV ameliorates the β-cell dysfunction to restore the glucose-stimulated insulin secretion. We conclude that the GIV/Nephrin/Akt signaling axis is vital to regulate glucose-stimulated insulin secretion. This mechanism might be further targeted for therapeutic intervention of diabetic mellitus.
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Affiliation(s)
- Hao Wang
- Beijing Key Laboratory of Diabetes Research and Care, Beijing Diabetes Institute, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
| | - Ying-Chao Yuan
- Beijing Key Laboratory of Diabetes Research and Care, Beijing Diabetes Institute, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Cong Chang
- College of Biology, Hunan University, Changsha, Hunan, China; Hunan Food and Drug Vocational College, Changsha, Hunan, China
| | - Tetsuro Izumi
- Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan
| | - Hong-Hui Wang
- College of Biology, Hunan University, Changsha, Hunan, China.
| | - Jin-Kui Yang
- Beijing Key Laboratory of Diabetes Research and Care, Beijing Diabetes Institute, Beijing Tongren Hospital, Capital Medical University, Beijing, China.
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22
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Zhao J, DiGiacomo V, Ferreras-Gutierrez M, Dastjerdi S, de Opakua AI, Park JC, Luebbers A, Chen Q, Beeler A, Blanco FJ, Garcia-Marcos M. Small-molecule targeting of GPCR-independent non-canonical G protein signaling inhibits cancer progression. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.18.529092. [PMID: 36824907 PMCID: PMC9949157 DOI: 10.1101/2023.02.18.529092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/21/2023]
Abstract
Activation of heterotrimeric G-proteins (Gαβγ) by G-protein-coupled receptors (GPCRs) is a quintessential mechanism of cell signaling widely targeted by clinically-approved drugs. However, it has become evident that heterotrimeric G-proteins can also be activated via GPCR-independent mechanisms that remain untapped as pharmacological targets. GIV/Girdin has emerged as a prototypical non-GPCR activator of G proteins that promotes cancer metastasis. Here, we introduce IGGi-11, a first-in-class smallmolecule inhibitor of non-canonical activation of heterotrimeric G-protein signaling. IGGi-11 binding to G-protein α-subunits (Gαi) specifically disrupted their engagement with GIV/Girdin, thereby blocking non-canonical G-protein signaling in tumor cells, and inhibiting pro-invasive traits of metastatic cancer cells in vitro and in mice. In contrast, IGGi-11 did not interfere with canonical G-protein signaling mechanisms triggered by GPCRs. By revealing that small molecules can selectively disable non-canonical mechanisms of G-protein activation dysregulated in disease, these findings warrant the exploration of therapeutic modalities in G-protein signaling that go beyond targeting GPCRs.
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23
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Abd El-Hafeez AA, Sun N, Chakraborty A, Ear J, Roy S, Chamarthi P, Rajapakse N, Das S, Luker KE, Hazra TK, Luker GD, Ghosh P. Regulation of DNA damage response by trimeric G-proteins. iScience 2023; 26:105973. [PMID: 36756378 PMCID: PMC9900518 DOI: 10.1016/j.isci.2023.105973] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Revised: 08/14/2022] [Accepted: 01/10/2023] [Indexed: 01/14/2023] Open
Abstract
Upon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of the two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DSBs hinges on GIV/Girdin, a guanine nucleotide-exchange modulator of heterotrimeric Giα•βγ protein. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1's BRCT-modules via both phospho-dependent and -independent mechanisms. Using another non-overlapping C-terminal motif GIV binds and activates Gi and enhances the "free" Gβγ→PI-3-kinase→Akt pathway, which promotes survival and is known to suppress HR, favor NHEJ. Absence of GIV, or loss of either of its C-terminal motifs enhanced cell death upon genotoxic stress. Because GIV selectively binds other BRCT-containing proteins suggests that G-proteins may fine-tune sensing, repair, and survival after diverse types of DNA damage.
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Affiliation(s)
- Amer Ali Abd El-Hafeez
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
- Pharmacology and Experimental Oncology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt
| | - Nina Sun
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
| | - Anirban Chakraborty
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Jason Ear
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
- Biological Sciences Department, California State Polytechnic University, Pomona, CA 91768, USA
| | - Suchismita Roy
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
| | - Pranavi Chamarthi
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
| | - Navin Rajapakse
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
| | - Soumita Das
- Department of Pathology, University of California San Diego, La Jolla, CA 92093, USA
| | - Kathryn E. Luker
- Center for Molecular Imaging, Department of Radiology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA
| | - Tapas K. Hazra
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555, USA
| | - Gary D. Luker
- Center for Molecular Imaging, Department of Radiology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA
- Department of Biomedical Engineering, University of Michigan, 2200 Bonisteel, Blvd., Ann Arbor, MI 48109-2099, USA
- Department of Microbiology and Immunology, University of Michigan, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200, USA
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA
- Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA
- Moores Comprehensive Cancer Center, University of California San Diego, La Jolla, CA 92093, USA
- Veterans Affairs Medical Center, La Jolla, CA, USA
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24
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Qiao L, Ghosh P, Rangamani P. Design principles of improving the dose-response alignment in coupled GTPase switches. NPJ Syst Biol Appl 2023; 9:3. [PMID: 36720885 PMCID: PMC9889403 DOI: 10.1038/s41540-023-00266-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Accepted: 01/17/2023] [Indexed: 02/02/2023] Open
Abstract
"Dose-response alignment" (DoRA), where the downstream response of cellular signaling pathways closely matches the fraction of activated receptor, can improve the fidelity of dose information transmission. The negative feedback has been experimentally identified as a key component for DoRA, but numerical simulations indicate that negative feedback is not sufficient to achieve perfect DoRA, i.e., perfect match of downstream response and receptor activation level. Thus a natural question is whether there exist design principles for signaling motifs within only negative feedback loops to improve DoRA to near-perfect DoRA. Here, we investigated several model formulations of an experimentally validated circuit that couples two molecular switches-mGTPase (monomeric GTPase) and tGTPase (heterotrimeric GTPases) - with negative feedback loops. In the absence of feedback, the low and intermediate mGTPase activation levels benefit DoRA in mass action and Hill-function models, respectively. Adding negative feedback has versatile roles on DoRA: it may impair DoRA in the mass action model with low mGTPase activation level and Hill-function model with intermediate mGTPase activation level; in other cases, i.e., the mass action model with a high mGTPase activation level or the Hill-function model with a non-intermediate mGTPase activation level, it improves DoRA. Furthermore, we found that DoRA in a longer cascade (i.e., tGTPase) can be obtained using Hill-function kinetics under certain conditions. In summary, we show how ranges of activity of mGTPase, reaction kinetics, the negative feedback, and the cascade length affect DoRA. This work provides a framework for improving the DoRA performance in signaling motifs with negative feedback.
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Affiliation(s)
- Lingxia Qiao
- Department of Mechanical and Aerospace Engineering, Jacob's School of Engineering, University of California San Diego, La Jolla, CA, USA
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA.
- Moores Comprehensive Cancer Center, University of California San Diego, La Jolla, CA, USA.
- Department of Medicine, School of Medicine, University of California San Diego, La Jolla, CA, USA.
| | - Padmini Rangamani
- Department of Mechanical and Aerospace Engineering, Jacob's School of Engineering, University of California San Diego, La Jolla, CA, USA.
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25
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Kaur G, Verma SK, Singh D, Singh NK. Role of G-Proteins and GPCRs in Cardiovascular Pathologies. Bioengineering (Basel) 2023; 10:bioengineering10010076. [PMID: 36671648 PMCID: PMC9854459 DOI: 10.3390/bioengineering10010076] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2022] [Revised: 12/19/2022] [Accepted: 01/03/2023] [Indexed: 01/09/2023] Open
Abstract
Cell signaling is a fundamental process that enables cells to survive under various ecological and environmental contexts and imparts tolerance towards stressful conditions. The basic machinery for cell signaling includes a receptor molecule that senses and receives the signal. The primary form of the signal might be a hormone, light, an antigen, an odorant, a neurotransmitter, etc. Similarly, heterotrimeric G-proteins principally provide communication from the plasma membrane G-protein-coupled receptors (GPCRs) to the inner compartments of the cells to control various biochemical activities. G-protein-coupled signaling regulates different physiological functions in the targeted cell types. This review article discusses G-proteins' signaling and regulation functions and their physiological relevance. In addition, we also elaborate on the role of G-proteins in several cardiovascular diseases, such as myocardial ischemia, hypertension, atherosclerosis, restenosis, stroke, and peripheral artery disease.
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Affiliation(s)
- Geetika Kaur
- Integrative Biosciences Center, Wayne State University, Detroit, MI 48202, USA
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI 48202, USA
| | - Shailendra Kumar Verma
- Integrative Biosciences Center, Wayne State University, Detroit, MI 48202, USA
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI 48202, USA
| | - Deepak Singh
- Lloyd Institute of Engineering and Technology, Greater Noida 201306, India
| | - Nikhlesh K. Singh
- Integrative Biosciences Center, Wayne State University, Detroit, MI 48202, USA
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI 48202, USA
- Correspondence:
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26
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Van Trigt WK, Kelly KM, Hughes CCW. GNAQ mutations drive port wine birthmark-associated Sturge-Weber syndrome: A review of pathobiology, therapies, and current models. Front Hum Neurosci 2022; 16:1006027. [PMID: 36405075 PMCID: PMC9670321 DOI: 10.3389/fnhum.2022.1006027] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Accepted: 10/12/2022] [Indexed: 11/06/2022] Open
Abstract
Port-wine birthmarks (PWBs) are caused by somatic, mosaic mutations in the G protein guanine nucleotide binding protein alpha subunit q (GNAQ) and are characterized by the formation of dilated, dysfunctional blood vessels in the dermis, eyes, and/or brain. Cutaneous PWBs can be treated by current dermatologic therapy, like laser intervention, to lighten the lesions and diminish nodules that occur in the lesion. Involvement of the eyes and/or brain can result in serious complications and this variation is termed Sturge-Weber syndrome (SWS). Some of the biggest hurdles preventing development of new therapeutics are unanswered questions regarding disease biology and lack of models for drug screening. In this review, we discuss the current understanding of GNAQ signaling, the standard of care for patients, overlap with other GNAQ-associated or phenotypically similar diseases, as well as deficiencies in current in vivo and in vitro vascular malformation models.
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Affiliation(s)
- William K. Van Trigt
- Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, Irvine, CA, United States
| | - Kristen M. Kelly
- Department of Dermatology, School of Medicine, University of California, Irvine, Irvine, CA, United States
| | - Christopher C. W. Hughes
- Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, Irvine, CA, United States
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27
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Akturk A, Day M, Tarchini B. RGS12 polarizes the GPSM2-GNAI complex to organize and elongate stereocilia in sensory hair cells. SCIENCE ADVANCES 2022; 8:eabq2826. [PMID: 36260679 PMCID: PMC9581478 DOI: 10.1126/sciadv.abq2826] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Accepted: 08/31/2022] [Indexed: 06/10/2023]
Abstract
Inhibitory G proteins (GNAI/Gαi) bind to the scaffold G protein signaling modulator 2 (GPSM2) to form a conserved polarity complex that regulates cytoskeleton organization. GPSM2 keeps GNAI in a guanosine diphosphate (GDP)-bound state, but how GPSM2-GNAI is generated or relates to heterotrimeric G protein signaling remains unclear. We find that RGS12, a GTPase-activating protein (GAP), is required to polarize GPSM2-GNAI at the hair cell apical membrane and to organize mechanosensory stereocilia in rows of graded heights. Accordingly, RGS12 and the guanine nucleotide exchange factor (GEF) DAPLE are asymmetrically co-enriched at the hair cell apical junction, and Rgs12 mouse mutants are deaf. GPSM2 and RGS12 share GoLoco motifs that stabilize GNAI(GDP), and GPSM2 outcompetes RGS12 to bind GNAI. Our results suggest that polarized GEF/GAP junctional activity might dissociate heterotrimeric G proteins, generating free GNAI(GDP) for GPSM2 at the adjacent apical membrane. GPSM2-GNAI(GDP), in turn, imparts asymmetry to the forming stereocilia to enable sensory function in hair cells.
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Affiliation(s)
- Anil Akturk
- The Jackson Laboratory, Bar Harbor, ME 04609, USA
| | - Matthew Day
- The Jackson Laboratory, Bar Harbor, ME 04609, USA
| | - Basile Tarchini
- The Jackson Laboratory, Bar Harbor, ME 04609, USA
- School of Medicine, Tufts University, Boston, MA 02111, USA
- Graduate School of Biomedical Science and Engineering (GSBSE), University of Maine, Orono, ME 04469, USA
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28
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MAS-related G protein-coupled receptors X (MRGPRX): Orphan GPCRs with potential as targets for future drugs. Pharmacol Ther 2022; 238:108259. [DOI: 10.1016/j.pharmthera.2022.108259] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Revised: 07/30/2022] [Accepted: 08/01/2022] [Indexed: 11/20/2022]
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29
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Villaseca S, Romero G, Ruiz MJ, Pérez C, Leal JI, Tovar LM, Torrejón M. Gαi protein subunit: A step toward understanding its non-canonical mechanisms. Front Cell Dev Biol 2022; 10:941870. [PMID: 36092739 PMCID: PMC9449497 DOI: 10.3389/fcell.2022.941870] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 08/01/2022] [Indexed: 11/13/2022] Open
Abstract
The heterotrimeric G protein family plays essential roles during a varied array of cellular events; thus, its deregulation can seriously alter signaling events and the overall state of the cell. Heterotrimeric G-proteins have three subunits (α, β, γ) and are subdivided into four families, Gαi, Gα12/13, Gαq, and Gαs. These proteins cycle between an inactive Gα-GDP state and active Gα-GTP state, triggered canonically by the G-protein coupled receptor (GPCR) and by other accessory proteins receptors independent also known as AGS (Activators of G-protein Signaling). In this review, we summarize research data specific for the Gαi family. This family has the largest number of individual members, including Gαi1, Gαi2, Gαi3, Gαo, Gαt, Gαg, and Gαz, and constitutes the majority of G proteins α subunits expressed in a tissue or cell. Gαi was initially described by its inhibitory function on adenylyl cyclase activity, decreasing cAMP levels. Interestingly, today Gi family G-protein have been reported to be importantly involved in the immune system function. Here, we discuss the impact of Gαi on non-canonical effector proteins, such as c-Src, ERK1/2, phospholipase-C (PLC), and proteins from the Rho GTPase family members, all of them essential signaling pathways regulating a wide range of physiological processes.
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30
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Marivin A, Ho RXY, Garcia-Marcos M. DAPLE orchestrates apical actomyosin assembly from junctional polarity complexes. J Biophys Biochem Cytol 2022; 221:213115. [PMID: 35389423 PMCID: PMC8996326 DOI: 10.1083/jcb.202111002] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 01/28/2022] [Accepted: 02/17/2022] [Indexed: 12/25/2022] Open
Abstract
Establishment of apicobasal polarity and the organization of the cytoskeleton must operate coordinately to ensure proper epithelial cell shape and function. However, the precise molecular mechanisms by which polarity complexes directly instruct the cytoskeletal machinery to determine cell shape are poorly understood. Here, we define a mechanism by which the PAR polarity complex (PAR3–PAR6–aPKC) at apical cell junctions leads to efficient assembly of the apical actomyosin network to maintain epithelial cell morphology. We found that the PAR polarity complex recruits the protein DAPLE to apical cell junctions, which in turn triggers a two-pronged mechanism that converges upon assembly of apical actomyosin. More specifically, DAPLE directly recruits the actin-stabilizing protein CD2AP to apical junctions and, concomitantly, activates heterotrimeric G protein signaling in a GPCR-independent manner to favor RhoA-myosin activation. These observations establish DAPLE as a direct molecular link between junctional polarity complexes and the formation of apical cytoskeletal assemblies that support epithelial cell shape.
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Affiliation(s)
- Arthur Marivin
- Department of Biochemistry, Boston University School of Medicine, Boston, MA
| | - Rachel Xi-Yeen Ho
- Department of Biochemistry, Boston University School of Medicine, Boston, MA
| | - Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, MA
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31
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Nubbemeyer B, George AAP, Kühl T, Pepanian A, Beck MS, Maghraby R, Boushehri MS, Muehlhaupt M, Pfeil EM, Annala SK, Ammer H, Imhof D, Pei D. Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators. ACS Chem Biol 2022; 17:463-473. [PMID: 35042325 PMCID: PMC11002716 DOI: 10.1021/acschembio.1c00929] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.
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Affiliation(s)
- Britta Nubbemeyer
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Ajay Abisheck Paul George
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
- BioSolveIT GmbH, An der Ziegelei 79, 53757, Sankt Augustin, Germany
| | - Toni Kühl
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Anna Pepanian
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Maximilian Steve Beck
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Rahma Maghraby
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Maryam Shetab Boushehri
- Pharmaceutical Technology and Biopharmacy, University of Bonn, Gerhard-Domagk-Str. 3, 53121, Bonn, Germany
| | - Maximilian Muehlhaupt
- Institute of Pharmacology, Toxicology and Pharmacy, Veterinary Faculty, Ludwig Maximilian University of Munich, Königinstr. 16, 80539, Munich, Germany
| | - Eva Marie Pfeil
- Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115, Bonn, Germany
| | - Suvi Katariina Annala
- Molecular, Cellular and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115, Bonn, Germany
| | - Hermann Ammer
- Institute of Pharmacology, Toxicology and Pharmacy, Veterinary Faculty, Ludwig Maximilian University of Munich, Königinstr. 16, 80539, Munich, Germany
| | - Diana Imhof
- Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany
| | - Dehua Pei
- Department of Chemistry and Biochemistry, The Ohio State University, 578 Biosciences Building, 484 W 12 Avenue, Columbus, OH 43210, USA
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32
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Sayed IM, Ibeawuchi SR, Lie D, Anandachar MS, Pranadinata R, Raffatellu M, Das S. The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses. Gut Microbes 2022; 13:1991776. [PMID: 34719317 PMCID: PMC8565811 DOI: 10.1080/19490976.2021.1991776] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Host engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effectors of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. In-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knockout mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ELMO1 generates differential pro-inflammatory cytokines between pathogenic and nonpathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1-SifA interaction affects bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1-SifA interaction increases TNF-α and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map and induces inflammation after challenge with microbes or microbial ligands. ELMO1 generates a differential response through interaction with the WxxxE motif, which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response.
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Affiliation(s)
- Ibrahim M Sayed
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
| | | | - Dominique Lie
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
| | | | - Rama Pranadinata
- Department of Pathology, University of California San Diego, La Jolla, CA, USA
| | - Manuela Raffatellu
- Department of Pediatrics, Division of Host-Microbe Systems and Therapeutics, University of California San Diego, LA Jolla, CA, USA,Center for Mucosal Immunology, Chiba University-UC San Diego, La Jolla, CAUSA
| | - Soumita Das
- Department of Pathology, University of California San Diego, La Jolla, CA, USA,CONTACT Soumita Das Department of Pathology, University of California, San Diego, 9500 Gilman Drive, Mc 0644, George Palade Laboratory, Office Rm 256, San Diego, Ca, 92093-0644, USA
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Li Z, Sun C, Wang F, Wang X, Zhu J, Luo L, Ding X, Zhang Y, Ding P, Wang H, Pu M, Li Y, Wang S, Qin Q, Wei Y, Sun J, Wang X, Luo Y, Chen D, Qiu W. Molecular mechanisms governing circulating immune cell heterogeneity across different species revealed by single-cell sequencing. Clin Transl Med 2022; 12:e689. [PMID: 35092700 PMCID: PMC8800483 DOI: 10.1002/ctm2.689] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2021] [Revised: 11/30/2021] [Accepted: 12/15/2021] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Immune cells play important roles in mediating immune response and host defense against invading pathogens. However, insights into the molecular mechanisms governing circulating immune cell diversity among multiple species are limited. METHODS In this study, we compared the single-cell transcriptomes of immune cells from 12 species. Distinct molecular profiles were characterized for different immune cell types, including T cells, B cells, natural killer cells, monocytes, and dendritic cells. RESULTS Our data revealed the heterogeneity and compositions of circulating immune cells among 12 different species. Additionally, we explored the conserved and divergent cellular crosstalks and genetic regulatory networks among vertebrate immune cells. Notably, the ligand and receptor pair VIM-CD44 was highly conserved among the immune cells. CONCLUSIONS This study is the first to provide a comprehensive analysis of the cross-species single-cell transcriptome atlas for peripheral blood mononuclear cells (PBMCs). This research should advance our understanding of the cellular taxonomy and fundamental functions of PBMCs, with important implications in evolutionary biology, developmental biology, and immune system disorders.
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Affiliation(s)
- Zhibin Li
- Department of NeurologyThe Third Affiliated Hospital of Sun Yat‐Sen UniversityGuangzhouChina
| | - Chengcheng Sun
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | - Fei Wang
- BGI‐ShenzhenShenzhenChina
- Department of BiomedicineAarhus UniversityAarhusDenmark
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life Sciences, BGI‐Qingdao, BGI‐ShenzhenQingdaoChina
| | - Xiran Wang
- National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original BacteriaSouth China Agricultural UniversityGuangzhouChina
- Guangdong Laboratory for Lingnan Modern AgricultureGuangzhouChina
| | - Jiacheng Zhu
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | - Lihua Luo
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | - Xiangning Ding
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | - Yanan Zhang
- Tsinghua‐Berkeley Shenzhen InstituteTsinghua UniversityShenzhenChina
| | - Peiwen Ding
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | - Haoyu Wang
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | | | | | - Shiyou Wang
- BGI‐ShenzhenShenzhenChina
- College of Life SciencesUniversity of Chinese Academy of SciencesBeijingChina
| | | | | | - Jian Sun
- National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original BacteriaSouth China Agricultural UniversityGuangzhouChina
- Guangdong Laboratory for Lingnan Modern AgricultureGuangzhouChina
| | - Xiangdong Wang
- Department of Pulmonary and Critical Care MedicineZhongshan HospitalShanghaiChina
- Fudan University Shanghai Medical CollegeShanghaiChina
| | - Yonglun Luo
- BGI‐ShenzhenShenzhenChina
- Department of BiomedicineAarhus UniversityAarhusDenmark
- Lars Bolund Institute of Regenerative MedicineQingdao‐Europe Advanced Institute for Life Sciences, BGI‐Qingdao, BGI‐ShenzhenQingdaoChina
- Steno Diabetes Center AarhusAarhus University HospitalAarhusDenmark
| | | | - Wei Qiu
- Department of NeurologyThe Third Affiliated Hospital of Sun Yat‐Sen UniversityGuangzhouChina
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Reynoso S, Castillo V, Katkar GD, Lopez-Sanchez I, Taheri S, Espinoza C, Rohena C, Sahoo D, Gagneux P, Ghosh P. GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility. eLife 2021; 10:69160. [PMID: 34409938 PMCID: PMC8376251 DOI: 10.7554/elife.69160] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Accepted: 08/05/2021] [Indexed: 12/25/2022] Open
Abstract
For a sperm to successfully fertilize an egg, it must first undergo capacitation in the female reproductive tract and later undergo acrosomal reaction (AR) upon encountering an egg surrounded by its vestment. How premature AR is avoided despite rapid surges in signaling cascades during capacitation remains unknown. Using a combination of conditional knockout (cKO) mice and cell-penetrating peptides, we show that GIV (CCDC88A), a guanine nucleotide-exchange modulator (GEM) for trimeric GTPases, is highly expressed in spermatocytes and is required for male fertility. GIV is rapidly phosphoregulated on key tyrosine and serine residues in human and murine spermatozoa. These phosphomodifications enable GIV-GEM to orchestrate two distinct compartmentalized signaling programs in the sperm tail and head; in the tail, GIV enhances PI3K→Akt signals, sperm motility and survival, whereas in the head it inhibits cAMP surge and premature AR. Furthermore, GIV transcripts are downregulated in the testis and semen of infertile men. These findings exemplify the spatiotemporally segregated signaling programs that support sperm capacitation and shed light on a hitherto unforeseen cause of infertility in men.
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Affiliation(s)
- Sequoyah Reynoso
- Department of Pathology, School of Medicine, University of California San Diego, San Diego, United States
| | - Vanessa Castillo
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, San Diego, United States
| | - Gajanan Dattatray Katkar
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, San Diego, United States
| | - Inmaculada Lopez-Sanchez
- Department of Medicine, School of Medicine, University of California San Diego, San Diego, United States
| | - Sahar Taheri
- Department of Computer Science and Engineering, Jacob's School of Engineering, University of California San Diego, San Diego, United States
| | - Celia Espinoza
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, San Diego, United States
| | - Cristina Rohena
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, San Diego, United States
| | - Debashis Sahoo
- Department of Computer Science and Engineering, Jacob's School of Engineering, University of California San Diego, San Diego, United States.,Moore's Comprehensive Cancer Center, University of California San Diego, San Diego, United States.,Department of Pediatrics, School of Medicine, University of California San Diego, San Diego, United States
| | - Pascal Gagneux
- Department of Pathology, School of Medicine, University of California San Diego, San Diego, United States
| | - Pradipta Ghosh
- Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, San Diego, United States.,Department of Medicine, School of Medicine, University of California San Diego, San Diego, United States.,Moore's Comprehensive Cancer Center, University of California San Diego, San Diego, United States.,Veterans Affairs Medical Center, Washington DC, United States
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Abd El-hafeez AA, Sun N, Chakraborty A, Ear J, Roy S, Chamarthi P, Rajapakse N, Das S, Luker KE, Hazra TK, Luker GD, Ghosh P. Regulation of DNA damage response by trimeric G-protein Signaling.. [DOI: 10.1101/2021.07.21.452842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/02/2023]
Abstract
AbstractUpon sensing DNA double-strand breaks (DSBs), eukaryotic cells either die or repair DSBs via one of two competing pathways, i.e., non-homologous end-joining (NHEJ) or homologous recombination (HR). We show that cell fate after DNA damage hinges on the guanine nucleotide-exchange modulator of heterotrimeric G-protein, Giα•βγ, GIV/Girdin. GIV suppresses HR by binding and sequestering BRCA1, a key coordinator of multiple steps within the HR pathway, away from DSBs; it does so using a C-terminal motif that binds BRCA1’s BRCT-modules via both phospho-dependent and -independent mechanisms. GIV promotes NHEJ, and binds and activates Gi and enhances the ‘free’ Gβγ→PI-3-kinase→Akt pathway, thus revealing the enigmatic origin of prosurvival Akt signals during dsDNA repair. Absence of GIV, or the loss of either of its two functions impaired DNA repair, and induced cell death when challenged with numerous cytotoxic agents. That GIV selectively binds few other BRCT-containing proteins suggests convergent signaling such that heterotrimeric G-proteins may finetune sensing, repair, and outcome after DNA damage.GRAPHIC ABSTRACTHIGHLIGHTSNon-receptor G protein modulator, GIV/Girdin binds BRCA1Binding occurs in both canonical and non-canonical modesGIV sequesters BRCA1 away from dsDNA breaks, suppresses HRActivation of Gi by GIV enhances Akt signals, favors NHEJIN BRIEFIn this work, the authors show that heterotrimeric G protein signaling that is triggered by non-receptor GEF, GIV/Girdin, in response to double-stranded DNA breaks is critical for decisive signaling events which favor non-homologous end-joining (NHEJ) and inhibit homologous recombination (HR).
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36
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Wang X, Wei Z, Lan T, He Y, Cheng B, Li R, Chen H, Li F, Liu G, Jiang B, Lin Y, Lu M, Meng Z. CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation. Autophagy 2021; 18:357-374. [PMID: 34190023 PMCID: PMC8942511 DOI: 10.1080/15548627.2021.1934271] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Hepatitis B virus (HBV) particles are thought to be secreted from hepatocytes through multivesicular bodies (MVBs); however, the cellular trafficking mechanisms prior to this process remain elusive. It has been reported that CCDC88A/GIV expression, which is involved in multiple aspects of vesicular trafficking, changes dynamically at different phases of chronic HBV infection. In this study, we focused on the role of CCDC88A/GIV in HBV replication. In the liver tissues of chronically HBV-infected patients, HBV infection significantly enhanced CCDC88A/GIV expression, and increased endoplasmic reticulum (ER) stress and autophagosome formation without changing endosome formation. Additionally, colocalization of SHBsAg with early endosomes (~30.2%) far exceeded that with autophagosomes (~3.2%). In hepatoma cells, CCDC88A/GIV and its downstream proteins, DNM2 (dynamin 2; a CCDC88A/GIV effector), CLTC and RAB5A significantly enhanced HBV replication and endosome formation but inhibited autophagosome formation. Blocking endocytosis disrupted HBsAg trafficking to endosomes and caused its accumulation in the ER lumen, which triggered ER stress to initiate the unfolded protein response (UPR). Therefore, HBsAg trafficking into autophagosomes was increased, and the lysosomal activity and maturation, which was inhibited by HBV infection, were restored. Meanwhile, core particles were prevented from entering MVBs. CCDC88A/GIV and its other effector, GNAI3, decreased autophagic flux by enhancing the insulin-induced AKT-MTOR pathway, thereby inhibiting HBV antigens autophagic degradation. In conclusion, CCDC88A/GIV enhanced HBV replication by increasing endosomal trafficking and reducing autophagic degradation of HBV antigens, suggesting that CCDC88A/GIV-mediated endosomal trafficking plays an important role in HBV replication and progeny secretion.Abbreviations: ACTB: actin beta; AO: acridine orange; ATF6: activating transcription factor 6; CCDC88A/GIV: coiled-coil domain containing 88A; CLTC: clathrin heavy chain; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole; DNM2: dynamin 2; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; EIF2A: eukaryotic translation initiation factor 2A; FBS: fetal bovine serum; GNAI3: G protein subunit alpha i3; HBV: hepatitis B virus; HBV RIs: HBV replication intermediates; HBcAg: HBV core protein; HBsAg: HBV surface antigen; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MVBs: multivesicular bodies; MTOR: mechanistic target of rapamycin kinase; PDI: protein disulfide isomerase; PHH: primary human hepatocyte; pSM2: a HBV replication-competent plasmid; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; SQSTM1/p62: sequestosome 1; siRNA: small interfering RNA; SEM: standard error of the mean; UPR: unfolded protein response
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Affiliation(s)
- Xueyu Wang
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Zhiqiang Wei
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Tingyu Lan
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Yulin He
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Bin Cheng
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Ruimin Li
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Hongxia Chen
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Fahong Li
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.,Department of Infectious Diseases,Huashan Hospital, Fudan University, Shanghai, China
| | - Guohua Liu
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Bin Jiang
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Department of Hepatobiliary Pancreatic Surgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
| | - Yong Lin
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.,The Key Laboratory of Molecular Biology of Infectious Diseases Designated by the Chinese Ministry of Education, Institute for Viral Hepatitis, Department of Infectious Diseases, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
| | - Mengji Lu
- Institute of Virology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Zhongji Meng
- Institute of Biomedical Research, Hubei Clinical Research Center for Precise Diagnosis and Treatment of Liver Cancer, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China.,Hubei Key Laboratory of Embryonic Stem Cell Research, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei province, China
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Ghosh P, Mullick M. Building unconventional G protein-coupled receptors, one block at a time. Trends Pharmacol Sci 2021; 42:514-517. [PMID: 33985816 DOI: 10.1016/j.tips.2021.04.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2021] [Revised: 04/05/2021] [Accepted: 04/19/2021] [Indexed: 10/21/2022]
Abstract
The structure, function, and dynamics of canonical activation of heterotrimeric G proteins by the seven-transmembrane G protein-coupled receptors (GPCRs) have been illustrated in detail. However, emerging studies during the past decade have started to shed light on how the same G proteins may also be accessed and modulated by a diverse family of receptors that are not conventional GPCRs. Can we learn about common themes and variations in how cells assemble these atypical GPCRs?
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Affiliation(s)
- Pradipta Ghosh
- Department of Medicine, University of California, San Diego, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, CA 92093, USA.
| | - Madhubanti Mullick
- Department of Cellular and Molecular Medicine, University of California, San Diego, CA 92093, USA
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38
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Garcia-Marcos M. Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators. eLife 2021; 10:65620. [PMID: 33787494 PMCID: PMC8034979 DOI: 10.7554/elife.65620] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Accepted: 03/30/2021] [Indexed: 01/14/2023] Open
Abstract
It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.
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Affiliation(s)
- Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
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39
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Stolerman LM, Ghosh P, Rangamani P. Stability Analysis of a Signaling Circuit with Dual Species of GTPase Switches. Bull Math Biol 2021; 83:34. [PMID: 33609194 PMCID: PMC8378325 DOI: 10.1007/s11538-021-00864-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 01/25/2021] [Indexed: 10/22/2022]
Abstract
GTPases are molecular switches that regulate a wide range of cellular processes, such as organelle biogenesis, position, shape, function, vesicular transport between organelles, and signal transduction. These hydrolase enzymes operate by toggling between an active ("ON") guanosine triphosphate (GTP)-bound state and an inactive ("OFF") guanosine diphosphate (GDP)-bound state; such a toggle is regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins). Here we propose a model for a network motif between monomeric (m) and trimeric (t) GTPases assembled exclusively in eukaryotic cells of multicellular organisms. We develop a system of ordinary differential equations in which these two classes of GTPases are interlinked conditional to their ON/OFF states within a motif through coupling and feedback loops. We provide explicit formulae for the steady states of the system and perform classical local stability analysis to systematically investigate the role of the different connections between the GTPase switches. Interestingly, a coupling of the active mGTPase to the GEF of the tGTPase was sufficient to provide two locally stable states: one where both active/inactive forms of the mGTPase can be interpreted as having low concentrations and the other where both m- and tGTPase have high concentrations. Moreover, when a feedback loop from the GEF of the tGTPase to the GAP of the mGTPase was added to the coupled system, two other locally stable states emerged. In both states the tGTPase is inactivated and active tGTPase concentrations are low. Finally, the addition of a second feedback loop, from the active tGTPase to the GAP of the mGTPase, gives rise to a family of steady states that can be parametrized by a range of inactive tGTPase concentrations. Our findings reveal that the coupling of these two different GTPase motifs can dramatically change their steady-state behaviors and shed light on how such coupling may impact signaling mechanisms in eukaryotic cells.
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Affiliation(s)
- Lucas M Stolerman
- Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA, 92093, USA
| | - Pradipta Ghosh
- Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA.
- Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, 92093, USA.
- Moores Comprehensive Cancer Center, University of California, San Diego, La Jolla, CA, 92093, USA.
| | - Padmini Rangamani
- Department of Mechanical and Aerospace Engineering, University of California, San Diego, La Jolla, CA, 92093, USA.
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Ham D, Ahn D, Ashim J, Cho Y, Kim HR, Yu W, Chung KY. Conformational switch that induces GDP release from Gi. J Struct Biol 2021; 213:107694. [PMID: 33418033 DOI: 10.1016/j.jsb.2020.107694] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Revised: 12/14/2020] [Accepted: 12/24/2020] [Indexed: 12/26/2022]
Abstract
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gβγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.
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Affiliation(s)
- Donghee Ham
- School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea
| | - Donghoon Ahn
- School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea
| | - Janbolat Ashim
- Department of Brain and Cognitive Sciences, DGIST, 333 Techno jungang-daero, Daegu 42988, Republic of Korea
| | - Yejin Cho
- School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea
| | - Hee Ryung Kim
- School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea
| | - Wookyung Yu
- Department of Brain and Cognitive Sciences, DGIST, 333 Techno jungang-daero, Daegu 42988, Republic of Korea; Core Protein Resources Center, DGIST, 333 Techno jungang-daero, Daegu 42988, Republic of Korea.
| | - Ka Young Chung
- School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 16419, Republic of Korea.
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41
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Receptor tyrosine kinases activate heterotrimeric G proteins via phosphorylation within the interdomain cleft of Gαi. Proc Natl Acad Sci U S A 2020; 117:28763-28774. [PMID: 33139573 DOI: 10.1073/pnas.2004699117] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
The molecular mechanisms by which receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major signaling hubs in eukaryotes, independently relay signals across the plasma membrane have been extensively characterized. How these hubs cross-talk has been a long-standing question, but answers remain elusive. Using linear ion-trap mass spectrometry in combination with biochemical, cellular, and computational approaches, we unravel a mechanism of activation of heterotrimeric G proteins by RTKs and chart the key steps that mediate such activation. Upon growth factor stimulation, the guanine-nucleotide exchange modulator dissociates Gαi•βγ trimers, scaffolds monomeric Gαi with RTKs, and facilitates the phosphorylation on two tyrosines located within the interdomain cleft of Gαi. Phosphorylation triggers the activation of Gαi and inhibits second messengers (cAMP). Tumor-associated mutants reveal how constitutive activation of this pathway impacts cell's decision to "go" vs. "grow." These insights define a tyrosine-based G protein signaling paradigm and reveal its importance in eukaryotes.
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Garcia-Marcos M, Parag-Sharma K, Marivin A, Maziarz M, Luebbers A, Nguyen LT. Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein. eLife 2020; 9:60155. [PMID: 32936073 PMCID: PMC7515630 DOI: 10.7554/elife.60155] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Accepted: 09/15/2020] [Indexed: 12/24/2022] Open
Abstract
Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.
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Affiliation(s)
- Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
| | - Kshitij Parag-Sharma
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
| | - Arthur Marivin
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
| | - Marcin Maziarz
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
| | - Alex Luebbers
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
| | - Lien T Nguyen
- Department of Biochemistry, Boston University School of Medicine, Boston, United States
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Rohena C, Rajapakse N, Lo IC, Novick P, Sahoo D, Ghosh P. GIV/Girdin and Exo70 Collaboratively Regulate the Mammalian Polarized Exocytic Machinery. iScience 2020; 23:101246. [PMID: 32590327 PMCID: PMC7322189 DOI: 10.1016/j.isci.2020.101246] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2019] [Revised: 04/20/2020] [Accepted: 06/03/2020] [Indexed: 11/17/2022] Open
Abstract
Polarized exocytosis is a fundamental process by which membranes and cargo proteins are delivered to the cell surface with precise spatial control. Although the need for the octameric exocyst complex is conserved from yeast to humans, what imparts spatial control is known only in yeast, i.e., a polarity scaffold called Bem1p. We demonstrate here that the mammalian scaffold protein, GIV/Girdin, fulfills the key criteria and functions of its yeast counterpart Bem1p; both bind Exo70 proteins via similar short-linear interaction motifs, and each prefers its evolutionary counterpart. Selective disruption of the GIV⋅Exo-70 interaction derails the delivery of the metalloprotease MT1-MMP to invadosomes and impairs collagen degradation and haptotaxis through basement membrane matrix. GIV's interacting partners reveal other components of polarized exocytosis in mammals. Findings expose how the exocytic functions aid GIV's pro-metastatic functions and how signal integration via GIV may represent an evolutionary advancement of the exocytic process in mammals.
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Affiliation(s)
- Cristina Rohena
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 232, 239, La Jolla, CA 92093, USA
| | - Navin Rajapakse
- Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, CA 92093, USA
| | - I-Chung Lo
- Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, CA 92093, USA
| | - Peter Novick
- Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, CA 92093, USA
| | - Debashis Sahoo
- Department of Pediatrics, University of California San Diego, San Diego, CA 92093, USA; Department of Computer Science and Engineering, Jacob's School of Engineering, University of California San Diego, San Diego, CA 92093, USA; Rebecca and John Moore Comprehensive Cancer Center, University of California San Diego, San Diego, CA 92093, USA
| | - Pradipta Ghosh
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 232, 239, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California San Diego, San Diego, CA 92093, USA; Rebecca and John Moore Comprehensive Cancer Center, University of California San Diego, San Diego, CA 92093, USA; Veterans Affairs Medical Center, 3350 La Jolla Village Dr, San Diego, CA 92161, USA.
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Maziarz M, Park JC, Leyme A, Marivin A, Garcia-Lopez A, Patel PP, Garcia-Marcos M. Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design. Cell 2020; 182:770-785.e16. [PMID: 32634377 DOI: 10.1016/j.cell.2020.06.020] [Citation(s) in RCA: 73] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Revised: 04/21/2020] [Accepted: 06/08/2020] [Indexed: 12/28/2022]
Abstract
Heterotrimeric G-proteins (Gαβγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gβγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.
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Affiliation(s)
- Marcin Maziarz
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Jong-Chan Park
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Anthony Leyme
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Arthur Marivin
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Alberto Garcia-Lopez
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Prachi P Patel
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.
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Rohena C, Kalogriopoulos N, Rajapakse N, Roy S, Lopez-Sanchez I, Ablack J, Sahoo D, Ghosh P. GIV•Kindlin Interaction Is Required for Kindlin-Mediated Integrin Recognition and Activation. iScience 2020; 23:101209. [PMID: 32535026 PMCID: PMC7300163 DOI: 10.1016/j.isci.2020.101209] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2020] [Revised: 04/15/2020] [Accepted: 05/24/2020] [Indexed: 11/16/2022] Open
Abstract
Cells perceive and respond to the extracellular matrix via integrin receptors; their dysregulation has been implicated in inflammation and cancer metastasis. Here we show that a guanine nucleotide-exchange modulator of trimeric-GTPase Gαi, GIV (a.k.a Girdin), directly binds the integrin adaptor Kindlin-2. A non-canonical short linear motif within the C terminus of GIV binds Kindlin-2-FERM3 domain at a site that is distinct from the binding site for the canonical NPxY motif on the -integrin tail. Binding of GIV to Kindlin-2 allosterically enhances Kindlin-2's affinity for β1-integrin. Consequently, integrin activation and clustering are maximized, which augments cell adhesion, spreading, and invasion. Findings elucidate how the GIV•Kindlin-2 complex has a 2-fold impact: it allosterically synergizes integrin activation and enables β1-integrins to indirectly access and modulate trimeric GTPases via the complex. Furthermore, Cox proportional-hazard models on tumor transcriptomics provide trans-scale evidence of synergistic interactions between GIV•Kindlin-2•β1-integrin on time to progression to metastasis.
GIV and Kindlin (K2), two integrin adaptors that promote metastasis, bind each other Binding of GIV or integrin to K2 allosterically enhances GIV•K2•integrin complexes Binding is required for the maximal recruitment of GIV and K2 to active integrins Binding facilitates integrin clustering, activation, tumor cell adhesion, invasion
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Affiliation(s)
- Cristina Rohena
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 239, La Jolla, CA 92093, USA
| | - Nicholas Kalogriopoulos
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 239, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Navin Rajapakse
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Suchismita Roy
- Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA
| | - Inmaculada Lopez-Sanchez
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 239, La Jolla, CA 92093, USA
| | - Jailal Ablack
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 239, La Jolla, CA 92093, USA
| | - Debashis Sahoo
- Department of Pediatrics, University of California San Diego, CA 92093, USA; Department of Computer Science and Engineering, Jacob's School of Engineering, University of California San Diego, CA 92093, USA; Rebecca and John Moore Comprehensive Cancer Center, University of California San Diego, CA 92093, USA
| | - Pradipta Ghosh
- Department of Medicine, University of California San Diego, 9500 Gilman Drive (MC 0651), George E. Palade Bldg, Rm 239, La Jolla, CA 92093, USA; Department of Cellular and Molecular Medicine, University of California San Diego, CA 92093, USA; Rebecca and John Moore Comprehensive Cancer Center, University of California San Diego, CA 92093, USA; Veterans Affairs Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.
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Biehler C, Wang LT, Sévigny M, Jetté A, Gamblin CL, Catterall R, Houssin E, McCaffrey L, Laprise P. Girdin is a component of the lateral polarity protein network restricting cell dissemination. PLoS Genet 2020; 16:e1008674. [PMID: 32196494 PMCID: PMC7112241 DOI: 10.1371/journal.pgen.1008674] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 04/01/2020] [Accepted: 02/14/2020] [Indexed: 01/07/2023] Open
Abstract
Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. Here we show that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, we observed cell dissemination from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, we discovered a core mechanism contributing to epithelial cell polarization from flies to humans. Our data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.
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Affiliation(s)
- Cornélia Biehler
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
| | - Li-Ting Wang
- Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Canada
| | - Myriam Sévigny
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
| | - Alexandra Jetté
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
| | - Clémence L. Gamblin
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
| | - Rachel Catterall
- Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Canada
| | - Elise Houssin
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
| | - Luke McCaffrey
- Rosalind and Morris Goodman Cancer Research Centre, McGill University, Montreal, Canada
- Gerald Bronfman Department of Oncology, McGill University, Montreal, Canada
| | - Patrick Laprise
- Centre de Recherche sur le Cancer, Université Laval, Québec, Canada
- axe oncologie du Centre de Recherche du Centre Hospitalier, Universitaire de Québec-UL, Québec, Canada
- * E-mail:
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Srivastava D, Artemyev NO. Ric-8A, a GEF, and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface. Bioessays 2020; 42:e1900208. [PMID: 31967346 PMCID: PMC7034654 DOI: 10.1002/bies.201900208] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 12/30/2019] [Indexed: 12/21/2022]
Abstract
Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.
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Affiliation(s)
- Dhiraj Srivastava
- Department of Molecular Physiology and Biophysics, The University of Iowa Carver College of Medicine, Iowa City, IA 52242
| | - Nikolai O. Artemyev
- Department of Molecular Physiology and Biophysics, The University of Iowa Carver College of Medicine, Iowa City, IA 52242
- Department of Ophthalmology and Visual Sciences, The University of Iowa Carver College of Medicine, Iowa City, IA 52242
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Tai JW, Yu H, Chilukuri AT, Bhargava R, Deshpande R, Li W, Ongkeko WM, Bhargava V, Rajasekaran MR. Characterization of urethral fibrosis in a rabbit model: Potential roles of Wnt-β catenin pathway and epithelial to mesenchymal transition. Neurourol Urodyn 2020; 39:625-632. [PMID: 31961960 DOI: 10.1002/nau.24281] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2019] [Accepted: 12/23/2019] [Indexed: 12/24/2022]
Abstract
AIM To elucidate the precise cellular and molecular mechanisms that underlie urethral fibrogenesis. METHODS Endoluminal electrocautery injury (using Karl Storz 10 Fr. Pediatric urethroscope) was employed in male rabbits (n = 6) to create mucosal injury. Retrograde urethrogram (RUG) and endoluminal ultrasound techniques were used to assess severity and changes in luminal cross-sectional area. Six control rabbits were subjected to sham injury, in which the electrocautery was inserted but not powered. Urethral tissues were harvested 30 days postinjury and subjected to RNA sequencing and quantitative polymerase chain reaction (qPCR) to determine changes in gene expression. Histological, immunostaining, and Western blot studies were used to determine changes in protein expression of known markers of fibrosis (eg, collagen, Integrinαv, GIV/Girdin, transforming growth factor-β (TGF-β), and pSMAD1,2,3). RESULTS Trichrome staining confirmed increased connective tissue in urethral scar tissues. Immunostaining revealed a potential role for epithelial to mesenchymal cell transition (EMT) and positive labeling for all fibrotic markers (eg, collagen-1, Integrin αv, GIV/Girdin, transforming growth factor-β (TGF-β), and SMAD1,2,3). Western blot analysis confirmed increased protein levels of these fibrotic markers. CONCLUSION Our RNA sequencing and qPCR studies, in conjunction with our protein data, suggest that urethral mucosal fibrogenesis may be mediated by novel fibrogenic signaling pathways involving Wnt-β catenin, TGF-β, GIV/Girdin, and EMT which lead to increased collagen deposition. Therapeutic strategies targeting these pathways may be beneficial in attenuating fibrogenesis and stricture progression.
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Affiliation(s)
- Jesse W Tai
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
| | - Hosong Yu
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
| | - Abinav T Chilukuri
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
| | - Raag Bhargava
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
| | - Rucha Deshpande
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
| | - Wei Li
- Department of Surgery, San Diego VA Health Care System and University of California, San Diego, California
| | - Weg M Ongkeko
- Department of Surgery, San Diego VA Health Care System and University of California, San Diego, California
| | - Valmik Bhargava
- Division of Cardiology, San Diego VA Health Care System and University of California, San Diego, California
| | - Mahadevan Raj Rajasekaran
- Department of Urology, San Diego VA Health Care System and University of California, San Diego, California
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49
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Marivin A, Maziarz M, Zhao J, DiGiacomo V, Olmos Calvo I, Mann EA, Ear J, Blanco-Canosa JB, Ross EM, Ghosh P, Garcia-Marcos M. DAPLE protein inhibits nucleotide exchange on Gα s and Gα q via the same motif that activates Gαi. J Biol Chem 2020; 295:2270-2284. [PMID: 31949046 DOI: 10.1074/jbc.ra119.011648] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2019] [Revised: 01/08/2020] [Indexed: 01/03/2023] Open
Abstract
Besides being regulated by G-protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE (Dvl-associating protein with a high frequency of leucine) are the best-characterized members of a group of cytoplasmic regulators that contain a Gα-binding and -activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11, or G12/13), and promoting nucleotide exchange in vitro However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G-protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remain to be investigated. Here, we show that DAPLE's GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq Unlike the nucleotide-exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq These findings indicate that GBA motifs have versatility in their G-protein-modulating effect, i.e. they can bind to Gα subunits of different classes and either stimulate or inhibit nucleotide exchange depending on the G-protein subtype.
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Affiliation(s)
- Arthur Marivin
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Marcin Maziarz
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Jingyi Zhao
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Vincent DiGiacomo
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Isabel Olmos Calvo
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Emily A Mann
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
| | - Jason Ear
- Department of Medicine and Cellular and Molecular Medicine, University of California, San Diego, California 92093
| | - Juan B Blanco-Canosa
- Department of Biological Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 08034
| | - Elliott M Ross
- Department of Pharmacology, Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390
| | - Pradipta Ghosh
- Department of Medicine and Cellular and Molecular Medicine, University of California, San Diego, California 92093
| | - Mikel Garcia-Marcos
- Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118.
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50
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Ear J, Dunkel Y, Mittal Y, Lim BBC, Liu L, Holda MK, Nitsche U, Barbazán J, Goel A, Janssen KP, Aznar N, Ghosh P. Two Isoforms of the Guanine Nucleotide Exchange Factor, Daple/CCDC88C Cooperate as Tumor Suppressors. Sci Rep 2019; 9:12124. [PMID: 31431650 PMCID: PMC6702192 DOI: 10.1038/s41598-019-48420-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2018] [Accepted: 08/01/2019] [Indexed: 01/27/2023] Open
Abstract
Previously, Aznar et al., showed that Daple/CCDC88C enables Wnt receptors to transactivate trimeric G-proteins during non-canonical Wnt signaling via a novel G-protein binding and activating (GBA) motif. By doing so, Daple serves two opposing roles; earlier during oncogenesis it suppresses neoplastic transformation and tumor growth, but later it triggers epithelial-to-mesenchymal-transition (EMT). We have identified and characterized two isoforms of the human Daple gene. While both isoforms cooperatively suppress tumor growth via their GBA motif, only the full-length transcript triggers EMT and invasion. Both isoforms are suppressed during colon cancer progression, and their reduced expression carries additive prognostic significance. These findings provide insights into the opposing roles of Daple during cancer progression and define the G-protein regulatory GBA motif as one of the minimal modules essential for Daple’s role as a tumor suppressor.
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Affiliation(s)
- Jason Ear
- Department of Medicine, University of California, San Diego, La Jolla, California, USA.,Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA
| | - Ying Dunkel
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Yash Mittal
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Blaze B C Lim
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Lawrence Liu
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Magda K Holda
- Department of Medicine, University of California, San Diego, La Jolla, California, USA
| | - Ulrich Nitsche
- Department of Surgery, Klinikumrechts der Isar, TechnischeUniversitätMünchen, Munich, Germany
| | - Jorge Barbazán
- Translational Medical Oncology Laboratory, Health Research Institute of Santiago (IDIS), SERGAS., Santiago de Compostela, Spain
| | - Ajay Goel
- Division of Gastroenterology, Department of Internal Medicine and Charles A. Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Center, Dallas, Texas, USA
| | - Klaus-Peter Janssen
- Department of Surgery, Klinikumrechts der Isar, TechnischeUniversitätMünchen, Munich, Germany
| | - Nicolas Aznar
- Department of Medicine, University of California, San Diego, La Jolla, California, USA. .,Cancer Research Center of Lyon, Centre Léon Bérard, Lyon, France.
| | - Pradipta Ghosh
- Department of Medicine, University of California, San Diego, La Jolla, California, USA. .,Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA. .,Moores Cancer Center, University of California, San Diego, La Jolla, California, USA.
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