Emanuel Strehler’s work on calcium pumps and calcium signaling

Abstract

Cells are equipped with mechanisms to control tightly the influx, efflux and resting level of free calcium (Ca²⁺). Inappropriate Ca²⁺ signaling and abnormal Ca²⁺ levels are involved in many clinical disorders including heart disease, Alzheimer’s disease and stroke. Ca²⁺ also plays a major role in cell growth, differentiation and motility; disturbances in these processes underlie cell transformation and the progression of cancer. Accordingly, research in the Strehler laboratory is focused on a better understanding of the molecular “toolkit” needed to ensure proper Ca²⁺ homeostasis in the cell, as well as on the mechanisms of localized Ca²⁺ signaling. A long-term focus has been on the plasma membrane calcium pumps (PMCAs), which are linked to multiple disorders including hearing loss, neurodegeneration, and heart disease. Our work over the past 20 years or more has revealed a surprising complexity of PMCA isoforms with different functional characteristics, regulation, and cellular localization. Emerging evidence shows how specific PMCAs contribute not only to setting basal intracellular Ca²⁺ levels, but also to local Ca²⁺ signaling and vectorial Ca²⁺ transport. A second major research area revolves around the calcium sensor protein calmodulin and an enigmatic calmodulin-like protein (CALML3) that is linked to epithelial differentiation. One of the cellular targets of CALML3 is the unconventional motor protein myosin-10, which raises new questions about the role of CALML3 and myosin-10 in cell adhesion and migration in normal cell differentiation and cancer.

Key Words: Calcium signaling, Calcium transport, Calmodulin, Membrane trafficking, Myosin-10, Calcium ATPase, Calcium pumps, Structure-function relationship

INTRODUCTION AND EDUCATIONAL EXPERIENCE

Dr. Ernst Emanuel Strehler (Figure 1) is a Professor of Biochemistry and Molecular Biology at the Mayo Clinic College of Medicine in Rochester, Minnesota, USA. Since 2005, he has also been an Adjunct Professor in the Department of Pharmacology, College of Graduate Studies and Research, of the University of Saskatchewan in Saskatoon, Canada. Dr. Strehler received his MS degree in Biochemistry, Cell and Molecular Biology in 1977 and his PhD in Cell Biology in 1981, both from the Swiss Federal Institute of Technology (ETHZ) in Zurich, Switzerland. He did his MS thesis under the mentorship of Professor Claus W Heizmann and his PhD thesis (on M-line muscle proteins) under Professor Hans M Eppenberger. Both his MS and his PhD thesis were recognized with a prize and the Silver Medal of the ETHZ. After a short stint with Professor Lars-Eric Thornell at the University of Umeå in Sweden (1981), Dr. Strehler pursued postdoctoral training in recombinant DNA technology and molecular biology with Professor Bernardo Nadal-Ginard at the Children’s Hospital and Harvard Medical School in Boston, USA, from 1982 to 1986. He was supported by a short-term EMBO postdoctoral fellowship (1981) and a stipend for young advanced scientists from the Swiss National Science Foundation (1982-1984). In 1986, Dr. Strehler returned to the ETHZ as an Assistant Professor in the Laboratory of Biochemistry directed by Professor Ernesto Carafoli. There, he first applied his expertise in molecular biology to the identification and characterization of the plasma membrane calcium pump (PMCA) family. In 1990, he was awarded the prestigious ETHZ Latsis prize of the International Latsis Foundation for work on the molecular biology of the PMCAs, as well as a START (Swiss Talents in Research and Teaching) Career Development Award from the Swiss NSF. He moved to his current position at the Mayo Clinic College of Medicine in 1992. Dr. Strehler served as Molecular Neuroscience Program Director in the Mayo Graduate School from 2000 to 2007. He has been an invited speaker at national and international meetings, a peer reviewer for numerous scientific journals and grant proposals, and has served on the Editorial Boards of the Journal of Biological Chemistry, Current Chemical Biology, and Biochemistry Research International. He has held grants from multiple foundations and federal organizations (including the Swiss NSF and the US NIH) and has published over 120 peer-reviewed journal articles and reviews.

Figure 1 Emanuel E Strehler, PhD, Professor of Biochemistry and Molecular Biology, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street S. W., Rochester, MN 55905, United States.

ACADEMIC STRATEGIES AND GOALS

Dr. Strehler’s research is focused on the biochemistry, molecular cell biology, and structure/function analysis of the PMCA family as it contributes to intracellular calcium signaling in health and disease. Using a wide range of techniques, including protein and Ca²⁺ transport biochemistry, recombinant DNA technology, and cell biology methods such as fluorescence microscopy and immunocytochemistry, his research group has been studying the human PMCA gene family and its multiple encoded isoforms and alternative splice variants. Helped by productive and long-standing collaborations, his group has contributed to a better understanding of the mechanism of regulation of different PMCAs, their tissue expression and cellular targeting, their interactions with scaffolding and signaling proteins, and their different contribution to handling cellular Ca²⁺ spikes. The ultimate goal of these studies is to comprehend how the diverse PMCAs are integrated in the complex Ca²⁺ signaling networks that control physiological and pathophysiological cell function. Dr. Strehler’s research has also uncovered a surprising complexity of the human calmodulin gene family in which multiple genes code for an identical protein, and has led to the characterization of a calmodulin-like protein with a putative role in cell differentiation and migration. The overarching goal of Dr. Strehler’s research is to understand the molecular underpinnings and regulation of intracellular Ca²⁺ signaling as it relates to normal cell physiology and disease.

ACADEMIC ACHIEVEMENTS

The following sections provide brief vignettes of discoveries and research accomplishments of Dr. Strehler’s laboratory in the field of the calcium pumps and calcium sensor proteins.

Characterization of the human PMCA gene family and discovery of alternative splicing as a mechanism to generate isoform complexity

PMCAs are generally low-abundance membrane proteins, therefore, they are difficult to isolate and purify. The first cloning of a PMCA therefore required immuno-isolation of clones from λgt11 bacteriophage human cDNA expression libraries and low stringency hybridization cloning using degenerate oligonucleotides. After the breakthrough cloning of the first human PMCA cDNA[1], additional PMCA isoforms were rapidly isolated and sequenced. Dr. Strehler was involved in the cloning of all four human PMCAs, and his team also reported the first human PMCA gene structure[2]. The molecular cloning work revealed an (at the time) unexpected complexity of alternative RNA splicing in the PMCA genes[3]. Work over several years has shown that all human PMCA genes are subject to alternative splicing, which affects two major sites of functional importance: one in the first cytosolic loop of the pump (site A) and one in the C-terminal tail (site C)[4]. In total, over 30 different human PMCA variants can be generated from the four genes and through combinatorial alternative splicing. A schematic overview of the PMCA and its major splice options is shown in Figure 2. This body of work has provided a comprehensive description of the human PMCA family, and has opened the door to detailed molecular, biochemical, and functional studies on this important calcium extrusion system.

Figure 2 Scheme of the plasma membrane calcium pump and of the alternative splice options generated from the four plasma membrane calcium pump genes. Top: Scheme of the plasma membrane calcium pump (PMCA). The transmembrane regions are numbered 1-10, and arrows indicate the sites affected by alternative splicing. Splicing at site C affects the calmodulin-binding domain (CBD) and can result in different C tails due to a shift in reading frame (indicated as separate lines). Only splice variants of the “b-type” carry a PDZ domain recognition sequence (PDZ) at the C terminus. N: N terminus; C: C terminus; Bottom: Exon structure of the regions involved in alternative splicing in the four human PMCA genes. Constitutively spliced exons are shown as gray boxes. The sizes of alternatively spliced exons (colored) are given in nucleotides, the alternative splicing options are indicated by connecting lines, and the resultant splice products are labeled by their lower case symbol. Combinatorial use of the splice options at sites A and C yields over 30 possible PMCA splice variants.

Discovery of rapidly regulated alternative splicing in the PMCA

The discovery of a large family of PMCA isoforms and splice variants immediately raises the question about the specific roles of these isoforms. In 1996, Dr. Strehler’s laboratory showed for the first time that a specific alternative splice shift in PMCA2 occurs as a consequence of a transient increase in intracellular Ca^2+[5]. The isoform switch was independent of new protein synthesis and was observed within 30 min or less of the Ca²⁺ stimulus. This work has shown that rapid changes in alternative RNA splicing may be induced by a change in [Ca²⁺], which has given rise to new hypotheses concerning the molecular mechanisms that are responsible for second-messenger-induced reprogramming of the splicing machinery. The results also suggested a dynamic role for specific PMCA isoforms in Ca²⁺ regulation rather than the static function originally assigned to these pumps.

PMCA splice variants are functionally different - implications for Ca²⁺ handling

PMCA isoforms and splice variants are expressed in tissue- and developmental stage-specific patterns, which suggests functional specialization among different pumps. Much work has therefore been dedicated to determination of the regulatory and kinetic differences between PMCA isoforms. The PMCAs are regulated (activated) by Ca²⁺-calmodulin, which binds to a specific region in the C tail of the pump[6-8]. The discovery that alternative splicing at site C affects the calmodulin-binding region of the pump immediately suggests a mechanism for differential regulation of the different splice variants. Work by different groups, including that of Dr. Strehler, has shown that different C-terminal splice variants indeed differ in calmodulin affinity and, of particular interest, in their activation and inactivation kinetics[9-11]. This work is highly significant because it demonstrates that different PMCA isoforms and splice variants handle Ca²⁺ signals differently. Tissues and cells with a need for fast and efficient expulsion of Ca²⁺ therefore express “fast” PMCAs, whereas cells and tissues with more modest (slow-tonic) changes in [Ca²⁺]i are well served by “slow” PMCAs such as PMCA4b[12].

Elucidation of the activated complex of calmodulin with the calmodulin-binding domain of the PMCA

Detailed kinetic work has shown that calmodulin binds to the slow PMCA4b in a multistep process, and has tentatively identified important anchor residues in the PMCA that tightly interact with Ca²⁺ calmodulin[13,14]. In recent collaborative work, the Strehler group has determined the NMR structure of the full-length calmodulin binding domain of PMCA4b in complex with holo-calmodulin[15]. This structure represents a novel (18-1 motif) calmodulin-target peptide complex, and confirms the identity of anchor residues proposed in earlier kinetic studies of the interaction of calmodulin with the PMCA. This work is of further significance because it suggests the possibility of obtaining more extensive structural information on the regulatory C tail of this difficult-to-study membrane protein.

Discovery of PDZ proteins as partners of specific PMCAs

Using yeast two-hybrid technology, Dr. Strehler and his group were the first to identify PDZ domain proteins as binding partners of the b-splice variants of the PMCAs[16]. Specifically, members of the membrane-associated guanylate kinase family, including PSD95, SAP97 and chapsyn-110, have been found to interact strongly with PMCA4b and 2b[17]. All PMCA b-splice forms contain a PDZ-binding motif at the C terminus, and work by Dr. Strehler and others has uncovered different PDZ proteins that interact promiscuously or selectively with PMCA isoforms[18]. These PDZ proteins represent scaffolding and clustering as well as signaling proteins. The identification of these novel protein interactions is a major finding because it reveals an additional role for alternative splicing in the PMCA family, and suggests that isoform-specific interactions with PDZ proteins are responsible for the incorporation of the Ca²⁺ pumps into multiprotein complexes involved in local Ca²⁺ signaling. This work lends strong support to the emerging hypothesis that the PMCAs are dynamic components of local Ca²⁺ control at the plasma membrane[18].

Alternative splicing controls the membrane targeting of PMCAs in polarized cells

When searching for a role of alternative splicing at site A (Figure 2), Dr. Strehler and his co-workers discovered that the size of the splice insert in the first cytosolic loop of PMCA2 affects the apical vs basolateral targeting of the pump. The large w-insert is responsible for directing the PMCA2 to the apical stereocilia in cochlear hair cells or the apical membrane in polarized kidney epithelial cells[20]. Recent work has shown that interaction with PDZ proteins, such as the apical scaffolding protein NHERF2[21], further enhances the recruitment of PMCA2w/b to the apical membrane and leads to reduced lateral mobility and decreased endocytosis of the pump[22]. These studies suggest that an interplay of effects of altered loop structure and specific protein-protein interactions of the C tail determine the outcome of the membrane targeting and final localization of different PMCAs in the cell. Ongoing work in the Strehler laboratory aims to understand the molecular mechanism of the specific membrane targeting of the PMCAs, and the functional consequences of the selective deployment of PMCA isoforms for local Ca²⁺ signaling.

Discovery of a calmodulin multigene family in humans and the unusual concept of multiple genes - one protein

Dr. Strehler was first to realize that the human genome contains multiple genes that code for an identical calmodulin protein[23]. This finding has introduced the unusual concept of multiple genes - one protein, and has raised interesting questions concerning the evolution and functional significance of the CALM gene family. In subsequent work, the Strehler group has characterized all three human CALM genes[24,25] (before the advent of the human genome sequence), and in the process, has discovered multiple CALM pseudogenes[26], as well as an intriguing intronless calmodulin-like gene[27] (see below). Dr. Strehler’s group also has provided the first analysis of all three CALM gene promoters and a direct comparison of their relative transcriptional strengths[25]. This work is significant in that it has opened the way for future studies on the differential regulation of the human CALM gene family in normal and pathological situations. The discovery of a seemingly redundant calmodulin gene family also generates new hypotheses about the levels of regulation of calmodulin in time and space[28].

Identification of human calmodulin-like protein (CALML3) as a tumor-sensitive protein involved in epithelial differentiation

The serendipitous discovery of an intronless gene for a human calmodulin-like protein[27] gained interest when it became clear that calmodulin-like protein (CLP) (human genome nomenclature CALML3) is a tumor-sensitive protein specifically expressed in normal epithelial cells but downregulated in tumorigenesis[29]. Dr. Strehler’s group has characterized the biochemical properties of CLP[30,31], provided the first description of the gene and its regulation[32], and has shown that downregulation of the protein is an early event in breast cancer development[33]. However, the causal role of CLP in epithelial differentiation[34] and malignant transformation remains to be determined.

Discovery of myosin-10 as a specific target of CLP/CALML3: role in filopodial motility and wound healing

One of the most pressing questions raised by the discovery of CLP/CALML3 is that of its potential targets. Although it is 85% identical to human calmodulin, the distinct properties of CLP suggest that it has specific targets or targets that only partially overlap with those of calmodulin. Research in Dr. Strehler’s group has identified the unconventional myosin-10 (Myo10) as a specific target of CLP[35]. This work also has shown that CLP functions as a light chain for Myo10 by binding to one or several of the three IQ domains in the neck region of the myosin (Figure 3). CLP competes effectively with calmodulin for Myo10 binding[36], and upregulation of CLP results in increased Myo10 expression and function[37]. CLP promotes Myo10 function in filopodial extension and cell migration, and thereby promotes epidermal wound healing[38]. The discovery of Myo10 as a specific target of CLP is highly significant and suggests multiple lines of further research such as investigations of the Ca²⁺ regulation of Myo10 and the role of the loss of CLP in epithelial differentiation, adhesion, and cancer.

Figure 3 Calmodulin-like protein serves as light chain for Myo10 and increases its expression and function. Myo10 consists of two heavy chains with an N-terminal motor domain, a “neck” region of three IQ domains involved in light chain binding, a putative coiled-coil region, and a tail region that contains pleckstrin homology (PH) domains, a myosin tail homology (MyTH) domain, and a C-terminal FERM (4.1, ezrin, radixin, moesin) domain. The IQ motifs bind calmodulin (CaM) or calmodulin-like light chains, which regulate Myo10 activity and Ca²⁺ sensitivity. Upon expression of calmodulin-like protein (CLP) (in the presence of Ca²⁺), it competes successfully with calmodulin and binds tightly to IQ3 and possibly IQ1 and IQ2 of Myo10. CLP binding results in increased Myo10 expression and function, as demonstrated by increased filopodial extension and enhanced directional cell migration.

CONCLUSION

Cellular processes ranging from fertilization to programmed cell death are regulated by precisely timed changes in the intracellular free Ca²⁺ concentration. Cells therefore must be equipped with mechanisms to control tightly the influx, efflux and resting level of Ca²⁺. Dr. Strehler’s research has made major contributions to the understanding of the biochemical, structural, regulatory, and functional properties of the human PMCAs. These pumps represent the primary high-affinity Ca²⁺ efflux system of all eukaryotic cells and are now recognized as integral and dynamic players in cellular Ca²⁺ signaling. Dr. Strehler’s research has also shed light on the organization and function of the human calmodulin gene family and has shown that calmodulin-like isoforms may assume unique roles in the transmission of the Ca²⁺ signal to specific cellular targets, and thereby have an impact on crucial processes such as cell differentiation, motility, and adhesion.