Chapter Three - Structure and Function of Endoplasmic Reticulum STIM Calcium Sensors

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Abstract

Store-operated calcium (Ca2 +) entry (SOCE) is a vital Ca2 + signaling pathway in nonexcitable as well as electrically excitable cells, regulating countless physiological and pathophysiological pathways. Stromal interaction molecules (STIMs) are the principal regulating molecules of SOCE, sensing changes in sarco-/endoplasmic reticulum (S/ER) luminal Ca2 + levels and directly interacting with the Orai channel subunits to orchestrate the opening of Ca2 + release-activated Ca2 + (CRAC) channels. Recent atomic resolution structures on human STIM1 and STIM2 have illuminated critical mechanisms of STIM function in SOCE; further, the first high-resolution structure of the Drosophila melanogaster Orai channel has revealed vital data on the atomic composition of the CRAC channel pore and the assembly of individual Orai subunits. This chapter focuses on the mechanistic information garnered from these high-resolution structures and the supporting biophysical, biochemical, and live cell work that has enhanced our understanding of the relationship between STIM and Orai structural features and CRAC channel function.

Introduction

Extracellular stimuli which interact with receptors on the surface of eukaryotic cells initiate signaling cascades that control myriad cellular processes (Berridge et al., 2000, Bootman and Lipp, 2001). For example, upon T-cell receptor or G-protein-coupled receptor agonist binding, phospholipases are either indirectly or directly activated resulting in the metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2), generating inositol 1,4,5-trisphospate (IP3) and diacylglycerol (Berridge et al., 2003, Bootman et al., 2001). The IP3 molecule is a small diffusible messenger which directly binds to IP3 receptors (IP3Rs) on the cytosolic face of the endoplasmic reticulum (ER) membrane. Furthermore, IP3 binding to IP3Rs induces a conformational change on these enormous tetrameric Ca2 + release channels resulting in the opening of the IP3R pore which permeates Ca2 + ions down a large concentration gradient from the relatively high Ca2 + levels of the ER lumen (i.e., ~ 100–800 μM) into the low free Ca2 + levels of the cytosol (i.e., ~ 0.1–1 μM) (reviewed in Stathopulos et al., 2012). Due to the high Ca2 + levels of the ER lumen compared to other intracellular compartments, this cellular partition is often termed the ER Ca2 + store. This intracellular Ca2 + release channel efflux into the cytosol can be terminated by release of the IP3 molecule as well as a feedback mechanism by which Ca2 + binds to the IP3R, thereby closing the channel. Interestingly, low levels of cytosolic Ca2 + increase the open probability, whereas high Ca2 + levels decrease the open probability, generating a bell-shaped Ca2 +-dependency in channel activity (reviewed in Stathopulos et al., 2012). While IP3Rs are also found in electrically excitable cells, ryanodine receptors (RyRs) dominate intracellular Ca2 + release channel function in these cell types (reviewed in Van Petegem, 2012). Native RyRs do not bind IP3, but can be activated by Ca2 + or cyclic adenosine diphosphate ribose; however, they share a remarkable structural and functional conservation in Ca2 + release channel function. For example, RyRs demonstrate a bell-shaped Ca2 +-dependency, and key domains within IP3Rs and RyRs are interchangeable with a preservation of function suggesting a highly conserved activation mechanism exists between these two Ca2 + release channel cousins (Seo et al., 2012).

Ultimately, both RyRs and IP3Rs regulate cytosolic and luminal Ca2 + levels. The spatial and temporal changes in intracellular Ca2 + mediate myriad physiological and pathophysiological activities in cells such as memory, contraction, the immune response, and apoptosis, to name a few (Berridge et al., 2000). However, the sarco/ER (S/ER) lumen is only a limited source of Ca2 + as evidenced by S/ER Ca2+ ATPase (SERCA) pump blockers such as thapsigargin (TG) used in conjunction with fluorescent cytosolic Ca2 + indicators such as Fura-2 which demonstrate the exhaustible escape of Ca2 + from the lumen into the cytosol through leak pathways (Jackson et al., 1988, Liou et al., 2005). While local and acute increases in cytosolic Ca2 + can trigger many processes, numerous cellular activities rely on longer, sustained cytosolic Ca2 + increases to elicit the signaling response, such as in transcriptional activation (reviewed in Hogan, Chen, Nardone, & Rao, 2003). Eukaryotic cells have evolved the intercompartmental coordination of Ca2 + signals to achieve the vast array of different activities required in life and death processes.

The major Ca2 + entry pathway of nonexcitable cells such as immune cells and platelets is store-operated calcium (Ca2 +) entry (SOCE) (Shaw and Feske, 2012, Varga-Szabo et al., 2011). SOCE is the process by which an external cell stimulus results in compartmentalized S/ER luminal Ca2 + release, through the IP3-mediated pathway, for example; furthermore, this S/ER Ca2 + store release results in a communication between the Ca2 +-depleted S/ER lumen and the plasma membrane (PM). Subsequently, PM-resident Ca2 + channels open and Ca2 + enters the cytosol down a steep Ca2 + concentration gradient from the extracellular space (i.e., [Ca2 +] of the extracellular space ~ 1000 μM vs. ~ 0.1–1 μM in the cytosol) (Feske, 2007). The essentially inexhaustible supply of Ca2 + from the extracellular space has the capacity to provide a sustained Ca2 + entry required for longer term increases in cytosolic Ca2 +. Additionally, Ca2 + entering the cytosol via SOCE is an important source of Ca2 + for the SERCA pump refilling of the S/ER lumen. As critical Ca2 +-dependent processes take place within the S/ER lumen such as protein folding, chaperone quality control of protein folding, steroidogenesis, vesicle trafficking, and initiation of cell death pathways, it is essential that Ca2 + levels within the S/ER lumen do not remain chronically low (Berridge, 2002). Hence, SOCE provides the Ca2 + necessary to regulate cellular activities which require sustained elevation of cytosolic Ca2 + and to prevent Ca2 + levels of the S/ER lumen from becoming detrimentally low.

Although the SOCE model was first proposed in 1986 (Putney, 1986), almost two decades passed prior to the identification of the principal molecular players in this process. Using a systems biology approach which employed small inhibiting RNA (siRNA) knockdown of over 2300 human genes, stromal interaction molecule-1 (STIM1) and -2 (STIM2) were identified as molecular components of SOCE (Liou et al., 2005). The genes were chosen based on the presence of primary sequence-identified signaling domains, and the level of SOCE activity was assessed in siRNA-transfected mammalian cells by monitoring changes in Fura-2 fluorescence after histamine and TG treatment which empty S/ER luminal Ca2 + stores via receptor-mediated IP3 production and SERCA pump inhibition, respectively. In an independent study using an RNA interference (RNAi) screen of Drosophila melanogaster genes in S2 cells, knockdown of D. melanogaster STIM was identified to almost completely abolish an electrophysiological inward rectifying current with characteristics identical to mammalian T-cell Ca2 + release-activated Ca2 + (CRAC) current; furthermore, SOCE through CRAC channels is the principal Ca2 + entry pathway in human immune cells such as T-cells, and RNAi knockdown of the human homologue to D. melanogaster, STIM1 in human T-cells resulted in suppression of CRAC currents, thereby confirming STIM as a key molecular component of CRAC channels (Roos et al., 2005). Importantly, it was shown that mutation of key Ca2 + coordinating residues in the putative EF-hand resulted in constitutive CRAC activation, linking the a Ca2 + sensing ability of STIM to SOCE regulation (Zhang et al., 2005).

Interestingly, while STIM knockdown using inhibiting nucleic acid strategies suppressed SOCE, overexpression of STIM in mammalian cells only modestly increased SOCE activity (Liou et al., 2005, Roos et al., 2005). One year after the STIM molecular link was elucidated, a pedigree and interference RNA analysis identified another protein, Orai1 as critical in SOCE (Feske et al., 2006). A mutation in Orai1 (Arg91Trp), a predicted four-transmembrane (TM) protein, caused an inheritable form of severe combined immunodeficiency disease (SCID) in which patient T-cells showed a complete lack of CRAC entry (Feske et al., 2006). SCID was sensationalized in the late 1970s and early 1980s with reports of a SCID patient which spent the first 12 years of his life in isolation due to poor immune function (Lawrence, 1985, Stone, 1977). The identification of Orai1 as a key player in SOCE lead to studies showing that cooverexpression of STIM1 and Orai1 induces robust and dramatic increases in cytosolic Ca2 + after ER luminal Ca2 + depletion by TG (Mercer et al., 2006, Soboloff et al., 2006). Ultimately, studies confirmed that Orai1 was a subunit of the PM CRAC channel pore and a major molecular component of SOCE (Prakriya et al., 2006, Vig, Beck, et al., 2006, Vig, Peinelt, et al., 2006, Yeromin et al., 2006, Zhang et al., 2006). Prior to the identification of the PM Orai proteins, the transient receptor potential family of proteins were candidates as the PM channels mediating SOCE (Draber and Draberova, 2005, Parekh and Penner, 1997, Parekh and Putney, 2005, Varga-Szabo et al., 2009).

Elucidation of three-dimensional (3D), atomic resolution protein structure is vital to understanding the precise mechanisms by which proteins function. In recent years, tremendous progress has been made in revealing high-resolution structural information on important conserved regions of STIM1 and Orai; further, combined with live cell experiments assessing SOCE in mammalian cells, great strides have been made in understanding the mechanisms of CRAC channel regulation and function. This chapter discusses the current known structural information of the CRAC components, with a particular emphasis on the mechanisms by which STIM molecules regulate Orai1 channel formation. Additionally, known structural differences between human STIM1 and STIM2 and how the distinctions and similarities relate to the discrete role of these homologues in mammalian cell signaling are discussed.

Section snippets

STIM and Orai Domain Architectures

STIMs are single-pass TM proteins (Cai, 2007a). A small fraction of these proteins is localized to the PM after glycosylation of Asn131 and Asn171, while the vast majority is localized to the ER membrane where the function of these regulatory molecules is best understood (Manji et al., 2000, Williams et al., 2001, Williams et al., 2002, Zhang et al., 2005). Vertebrates express two homologues, STIM1 and STIM2. The sequence-identifiable ER luminal domains consist of an EF-hand and sterile α-motif

STIM1 and Orai1 in the Activation in SOCE

The sequence of cellular events leading to SOCE through the STIM and Orai pathway is a multistep process. Within the luminal region, STIMs contain the machinery required to sense Ca2 + changes and initiate SOCE. After ER Ca2 + store depletion, through the agonist-induced IP3-mediated pathway, for example, STIM1 self-associates. This oligomerization is prerequisite to the subsequent translocation of STIM1 molecules from a pervasive distribution on the ER to sites which are in close apposition to

Human STIM1 and STIM2 EF–SAM Biophysical Features

Mobilization of the molecular components involved in SOCE is initiated in the ER lumen after Ca2 + store depletion. The EF-hand together with SAM domain of STIMs (i.e., EF–SAM) is highly conserved from lower to higher order eukaryotes. In vitro studies show that EF–SAM can be recombinantly expressed and isolated with high purity from Escherichia coli (Stathopulos, Li, Plevin, Ames, & Ikura, 2006). Remarkably, the two domains fold cooperatively indicative of the mutual dependency of the two

Human STIM1 EF–SAM Structure

The modular architecture of STIM proteins has facilitated a fragmentary approach to elucidating atomic resolution structural information on these proteins. The first atomic resolution structure solved on any component of the CRAC complex was Ca2 +-loaded STIM1 EF–SAM (Stathopulos et al., 2008). This ER lumen-residing region of STIM1 encompassing residues 58–201 folds into a primarily α-helical protein consisting of 10 helices (Fig. 3.4A). Remarkably, the STIM1 EF–SAM structure revealed that

Human STIM2 EF–SAM Structure

Structurally, human STIM2 EF–SAM is highly homologous to STIM1, as the two human homologues share 85% sequence identity through the EF–SAM region (Zheng et al., 2011). Despite this high sequence similarity, STIM2 EF–SAM has distinct biophysical characteristics and the full-length molecule exhibits a unique role in basal Ca2 + homeostasis (see above). In the presence of Ca2 +, STIM2 EF–SAM folds into a 10-helix globular structure (Fig. 3.4D). A noncanonical EF-hand is located adjacent to the

Human STIM1 and STIM2 Cytosolic Domains

While the STIM luminal domains regulate the initiation of SOCE, the cytosolic domains play a crucial role in further STIM oligomerization, stabilization of the oligomers, targeting oligomerized STIM molecules to ER–PM junctions, coupling to and recruitment of the Orai subunits, gating of the CRAC channel pore, and channel inactivation (Covington et al., 2010, Derler et al., 2009, Muik et al., 2009, Park et al., 2009). Several independent studies showed that the cytosolic domains within STIM1,

STIM Coupling to Orai

Orai proteins have an N-terminal region, C-terminal region, and an intracellular loop oriented in the cytosol (Fig. 3.2B). A yeast-two-hybrid approach was employed to show that human STIM1 CAD interacts with the N- and C-termini of Orai1, but not the sole intracellular Orai1 loop (Park et al., 2009). Further, Orai1 truncation studies demonstrated that removal of Orai1 residues 1–91 (i.e., cytosolic N-terminus) or 257–301 (i.e., cytosolic C-terminus) abolished CRAC current measurements (Park et

Concluding Remarks

The high-resolution structural data on the EF–SAM region of STIM1 and STIM2, the SOAR domain of STIM1 and C. elegans STIM, and the D. melanogaster Orai hexamer channel structure have revealed a remarkable breadth of mechanistic data on how STIM and Orai proteins create CRAC channels. In the case of the EF–SAM domains, the intimate interaction between the EF-hand and SAM domains keeps STIM molecules in a quiescent state and maintains SOCE in an OFF state. Moreover, the “open” EF-hand

Acknowledgments

This work was made possible through CIHR, HSFC, NSERC, and CFI funding to M. I. M. I. holds the Canada Research Chain in Cancer structural biology.

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