The thrombospondins

Thrombospondin-1 (TSP-1) was studied in the 1980s as a major component of platelet alpha-granules released upon platelet activation and also as a cell adhesion molecule. In 1993, we published a short review that discussed the exciting identification by molecular cloning of four additional vertebrate gene products related to TSP-1 [Current Biology 3 (1993) 188]. We put forward a structurally based classification for the newly identified proteins and discussed the functional and evolutionary implications of the new gene family. Since that time, the depth and breadth of knowledge on vertebrate TSPs and their functions in cells and tissues in health and disease has expanded into important new areas. Of particular interest is the new knowledge on the complex, domain and cell-type specific effects of TSPs on cell-signaling and cell-adhesion behaviour, the roles of TSP-1 and TSP-2 as anti-angiogenic agents, the roles of TSP-1 and TSP-2 in wound-healing, and associations of point mutations and polymorphisms in TSP-1, TSP-4 and TSP-5/COMP with human genetic diseases. The TSP family also now includes invertebrate members. In this article, we give the 2004 view on TSPs and our perspectives on the significant challenges that remain. Other articles in this issue discuss the functions of vertebrate TSPs in depth.     

Immunochemical analysis of the structure of the signature domains of thrombospondin-1 and thrombospondin-2 in low calcium concentrations

Thrombospondins (TSPs) undergo conformational changes upon removal of calcium. The eight C-type and five N-type calcium-binding repeats of TSP-2 form a circuitous wire that, in 2 mm calcium, interacts at its ends with more N-terminal epidermal growth factor (EGF)-like modules, EGF2 and EGF3, and the C-terminal lectin-like module. These components, along with the other EGF-like module(s), form the signature domain of TSPs. Characterization of conformation-sensitive epitopes of monoclonal antibodies to human TSP-2 and its TSP-1 homolog have given insights into the structure of the signature domain in the absence of calcium. The epitope for 4B6.13 anti-TSP-2 was localized to His-722 and Leu-703 in repeat 1C of the wire; recognition only occurred in constructs that included EGF3, the rest of the wire, and the lectin-like module and in the presence of calcium. The epitope for C6.7 anti-TSP-1 was localized to Glu-609 in the EGF2 module. The C6.7 epitope was preferentially recognized when EGF2 was expressed in the context of EGF1, EGF3, the wire, and the lectin-like module. Preferential recognition of the C6.7 epitope did not require calcium. Rotary shadowing electron microscopy of TSP-1 has shown elongation of the stalk and diminution of the C-terminal globule. We propose a model whereby at low calcium concentrations the lectin-like module drops away from EGF3 concomitant with changes in conformation of the wire and loss of the 4B6.13 epitope. A critical feature of the model is interaction of repeat 12N of the wire with EGF2 in both the presence and absence of calcium.     

Tumor progression: the effects of thrombospondin-1 and -2

The thrombospondins (TSPs) are a family of proteins that regulate tissue genesis and remodeling. In many tumors, down-regulation of TSPs accompanies activation of oncogenes or inactivation of tumor suppresser genes and appears to be a prerequisite for the aquisition of a pro-angiogenic phenotype. The normal suppression of angiogenesis by TSP-1 and -2 involves multiple mechanisms including direct interaction with vascular endothelial cell growth factor (VEGF), inhibition of matrix metalloproteinase 9 (MMP9) activation, inhibition of endothelial cell migration and induction of endothelial cell apoptosis. The importance of down-regulation of TSPs for tumor progression is further established by the fact that several different approaches that are designed to increase the levels of TSP-1 or -2 in tumor tissue inhibit tumor growth. These approaches include cell-based gene therapy, low dose chemotherapeutics and systemic delivery of recombinant proteins or synthetic peptides that include type 1 repeat (TSR) sequences. Initial studies indicate that these reagents, in combination with established approaches for the treatment of cancer, will offer more efficacious therapies.     

Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation

STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by F?rster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca(2+) entry. F?rster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca(2+) store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca(2+) inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.     

Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats

Thrombospondins (TSPs) are extracellular regulators of cell-matrix interactions and cell phenotype. The most highly conserved region of all TSPs are the calcium-binding type 3 (T3) repeats and the C-terminal globular domain (CTD). The crystal structure of a cell-binding TSP-1 fragment, spanning three T3 repeats and the CTD, reveals a compact assembly. The T3 repeats lack secondary structure and are organised around a core of calcium ions; two DxDxDGxxDxxD motifs per repeat each encapsulate two calcium ions in a novel arrangement. The CTD forms a lectin-like beta-sandwich and contains four strictly conserved calcium-binding sites. Disruption of the hairpin structure of T3 repeats 6 and 7 decreases protein secretion and stability. The availability for cell attachment of an RGD motif in T3 repeat 7 is modulated by calcium loading. The central architectural role of calcium explains how it is critical for the functions of the TSP C-terminal region. Mutations in the T3 repeats of TSP-5/COMP, which cause two human skeletal disorders, are predicted to disrupt the tertiary structure of the T3-CTD assembly.     

Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-activated Calcium Modulator 1 (Orai1)

Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca²⁺-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.     

The interaction of Thrombospondins with extracellular matrix proteins

The thrombospondins (TSPs) are a family of five matricellular proteins that appear to function as adapter molecules to guide extracellular matrix synthesis and tissue remodeling in a variety of normal and disease settings. Various TSPs have been shown to bind to fibronectin, laminin, matrilins, collagens and other extracellular matrix (ECM) proteins. The importance of TSP-1 in this context is underscored by the fact that it is rapidly deposited at the sites of tissue damage by platelets. An association of TSPs with collagens has been known for over 25 years. The observation that the disruption of the TSP-2 gene in mice leads to collagen fibril abnormalities provided important in vivo evidence that these interactions are physiologically important. Recent biochemical studies have shown that TSP-5 promotes collagen fibril assembly and structural studies suggest that TSPs may interact with collagens through a highly conserved potential metal ion dependent adhesion site (MIDAS). These interactions are critical for normal tissue homeostasis, tumor progression and the etiology of skeletal dysplasias.     

Biochemical and functional properties of the store-operated Ca2+ channels

Store-operated calcium entry (SOCE) is a major mechanism for Ca²⁺ entry in excitable and non-excitable cells. The best-characterised store-operated current is I_CRAC, but other currents activated by Ca²⁺ store depletion have also been reported. The recent identification of the proteins stromal interaction molecule 1 (STIM1) and Orai1 has shed new light on the nature and regulation of SOC channels. STIM1 has been presented as the endoplasmic reticulum (ER) Ca²⁺ sensor that communicates the content of the Ca²⁺ stores to the store-operated channels, a mechanism that involves redistribution of STIM1 to peripheral ER sites and co-clustering with the Ca²⁺ channel subunit, Orai1. Interestingly, TRPC1, which has long been proposed as a SOC channel candidate, associates with Orai1 and STIM1 in a ternary complex that appears to increase the variability of SOC currents available to modulate cell function.     

STIM／SOCE通路参与细胞功能调控的研究进展

钙库操作性钙离子通道（store-operated calcium entry,SOCE）是介导胞外Ca^2＋进入细胞内的重要通道之一,其核心蛋白由位于内质网上的基质相互作用分子（stromalinteractionmolecule,STIM）和位于细胞膜上的Orai蛋白构成。目前研究发现,STIM蛋白存在STIM1和STIM2两种亚型,其主要功能略有不同。当内质网内钙库中Ca^2＋消耗之后,STIM蛋白通过其特殊的结构能够感受内质网内钙库中Ca^2＋浓度的变化,发生快速的转位和聚合化等激活反应,与质膜上的Orai蛋白偶联。实现SOCE通路的功能开放,引起Ca^2＋内流。当钙库中Ca^2＋得到补充之后,STIM蛋白与Orai蛋白缓慢解离即失活,通路关闭。目前对STIM蛋白结构的研究提示,通过其激活和失活机制不仅能够参与调节SOCE通路的开放与关闭,也参与对细胞内重要的细胞增殖、分化等功能活动调控。STIM蛋白可能成为治疗多种疾病的潜在的新靶点。     

Numbers count: How STIM and Orai stoichiometry affect store-operated calcium entry

Substantial progress has been made in the past several years in establishing the stoichiometries of STIM and Orai proteins and understanding their influence on store-operated calcium entry. Depletion of ER Ca²⁺ triggers STIM1 to accumulate at ER-plasma membrane junctions where it binds and opens Ca²⁺ release-activated Ca²⁺ (CRAC) channels. STIM1 is a dimer, and release of Ca²⁺ from its two luminal domains is reported to promote their association as well as drive formation of higher-order STIM1 oligomers. The CRAC channel, originally thought to be tetrameric, is now considered to be a hexamer of Orai1 subunits based on crystallographic and electrophysiological studies. STIM1 binding activates CRAC channels in a highly nonlinear way, such that all six Orai1 binding sites must be occupied to account for the activation and signature properties of native channels. The structural basis of STIM1 engagement with the channel is currently unclear, with evidence suggesting that STIM1 dimers bind to individual or pairs of Orai1 subunits. This review examines evidence that has led to points of consensus and debate about STIM1 and Orai1 stoichiometries, and explains the importance of STIM-Orai complex stoichiometry for the regulation of store-operated calcium entry.     

Thrombospondins function as regulators of angiogenesis

Thrombospondins (TSPs) -1 and -2 were among the first protein inhibitors of angiogenesis to be identified, a property that was subsequently attributed to the interactions of sequences in their type I repeats with endothelial cell-surface receptors. The interactions of TSPs-1 and -2 with cell-surface receptors, proteases, growth factors, and other bioactive molecules, coupled with the absence of direct structural functions that can be attributed to these matrix proteins, qualify them for inclusion in the category of ‘matricellular proteins’. The phenotypes of TSP-1, TSP-2, and double TSP-1/2-null mice confirm the roles that these proteins play in the regulation of angiogenesis, and provide clues to some of the other important functions of these multi-domain proteins. One of these functions is the ability of TSP-1 to activate the latent TGFβ1 complex, a property that is not shared by TSP-2. A major pathway by which TSP1 or TSP2 inhibits angiogenesis involves an interaction with CD 36 on endothelial cells, which leads to apoptosis of both the liganded and adjacent cells. However a homeostatic mechanism, which inhibits endothelial cell proliferation, and may be physiologically preferable under some circumstances, has also been elucidated, and involves interaction with the very low density lipoprotein receptor (VLDLR). The interaction of TSP1with its receptor, CD47, further inhibits angiogenesis by antagonizing nitric oxide signaling in endothelial and vascular smooth muscle cells. Paradoxically, there is also evidence that TSP-1 can function to promote angiogenesis. This apparent contradiction can be explained by the presence of sequences in different domains of the protein that interact with different receptors on endothelial cells. The anti-angiogenic function of TSPs has spurred interest in their use as anti-tumor agents. Currently, peptide mimetics, based on sequences in the type I repeats of TSPs that have been shown to have anti-angiogenic properties, are undergoing clinical testing.     

Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase

Voltage-gated calcium channels are multiprotein complexes that regulate calcium influx and are important contributors to cardiac excitability and contractility. The auxiliary beta-subunit (CaV beta) binds a conserved domain (the alpha-interaction domain (AID)) of the pore-forming CaV alpha1 subunit to modulate channel gating properties and promote cell surface trafficking. Recently, members of the RGK family of small GTPases (Rem, Rem2, Rad, Gem/Kir) have been identified as novel contributors to the regulation of L-type calcium channel activity. Here, we describe the Rem-association domain within CaV beta2a. The Rem interaction module is located in a approximately 130-residue region within the highly conserved guanylate kinase domain that also directs AID binding. Importantly, CaV beta mutants were identified that lost the ability to bind AID but retained their association with Rem, indicating that the AID and Rem association sites of CaV beta2a are structurally distinct. In vitro binding studies indicate that the affinity of Rem for CaV beta2a interaction is lower than that of AID for CaV beta2a. Furthermore, in vitro binding studies indicate that Rem association does not inhibit the interaction of CaV beta2a with AID. Instead, CaV beta can simultaneously associate with both Rem and CaV alpha1-AID. Previous studies had suggested that RGK proteins may regulate Ca2+ channel activity by blocking the association of CaV beta subunits with CaV alpha1 to inhibit plasma membrane trafficking. However, surface biotinylation studies in HIT-T15 cells indicate that Rem can acutely modulate channel function without decreasing the density of L-type channels at the plasma membrane. Together these data suggest that Rem-dependent Ca2+ channel modulation involves formation of a Rem x CaV beta x AID regulatory complex without the need to disrupt CaV alpha1 x CaV beta association or alter CaV alpha1 expression at the plasma membrane.     

Regulation of calcium channels by RGK proteins

The RGK family of proteins, small GTPases of the Ras superfamily, are known to regulate calcium currents. It is commonly thought that this is due to an interaction with the Cavβ subunit, however, the mechanism of this inhibition is unclear. There have been conflicting reports of whether RGK proteins can affect channel trafficking or whether they reduce calcium currents by interacting with channels at the membrane. In the last year, several studies have emerged which explore the intricacies of RGK protein interaction with the channel itself and the importance of the Cavβ subunit for this interaction, in addition to providing some tantalizing suggestions for the mechanism by which RGK proteins reduce or eliminate calcium currents. In this review, we present an overview of these recent advances and suggest a model that may synthesize these latest works.     

STIM1 and STIM2 Proteins Differently Regulate Endogenous Store-operated Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of I_min channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of I_min channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca²⁺ influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; I_min channels are regulated by STIM2, TRPC3-containing I_NS channels are induced by STIM1, and TRPC1-composed I_max channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.     

MURC/CAVIN-4 facilitates store-operated calcium entry in neonatal cardiomyocytes

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca²⁺ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca²⁺ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies.     

Regulation of calcium channels by RGK proteins

The RGK family of proteins, small GTPases of the Ras superfamily, are known to regulate calcium currents. Iit is commonly thought that this is due to an interaction with the Cavβ subunit, however, the mechanism of this inhibition is unclear. There have been conflicting reports of whether RGK proteins can affect channel trafficking or whether they reduce calcium currents by interacting with channels at the membrane. In the last year, several studies have emerged which explore the intricacies of RGK protein interaction with the channel itself and the importance of the Cavβ subunit for this interaction, in addition to providing some tantalizing suggestions for the mechanism by which RGK proteins reduce or eliminate calcium currents. In this review, we present an overview of these recent advances and suggest a model that may synthesize these latest works.     

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

Calcium flux through store-operated calcium entry is a major regulator of intracellular calcium homeostasis and various calcium signaling pathways. Two key components of the store-operated calcium release-activated calcium channel are the Ca²⁺-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. Following calcium depletion from the endoplasmic reticulum, STIM1 undergoes conformational changes that unmask an Orai1-activating domain called CAD. CAD binds to two sites in Orai1, one in the N terminal and one in the C terminal. Most previous studies suggested that gating is initiated by STIM1 binding at the Orai1 N-terminal site, just proximal to the TM1 pore-lining segment, and that binding at the C terminal simply anchors STIM1 within reach of the N terminal. However, a recent study had challenged this view and suggested that the Orai1 C-terminal region is more than a simple STIM1-anchoring site. In this study, we establish that the Orai1 C-terminal domain plays a direct role in gating. We identify a linker region between TM4 and the C-terminal STIM1-binding segment of Orai1 as a key determinant that couples STIM1 binding to gating. We further find that Proline 245 in TM4 of Orai1 is essential for stabilizing the closed state of the channel. Taken together with previous studies, our results suggest a dual-trigger mechanism of Orai1 activation in which binding of STIM1 at the N- and C-terminal domains of Orai1 induces rearrangements in proximal membrane segments to open the channel.     

Visualizing the store-operated channel complex assembly in real time: Identification of SERCA2 as a new member

Depletion of intracellular calcium stores leads to the activation of calcium influx via the so-called store-operated channels (SOCs). Recent evidence positions Orai proteins as the putative channels responsible for this process. The stromal interacting molecule (STIM1) has been recently identified as the calcium sensor located at the endoplasmic reticulum (ER), and responsible for communicating the deplete state of calcium stores to Orai at the plasma membrane (PM). However, recent experimental findings suggest that Orai and STIM1 are only part of a larger molecular complex required to modulate store-operated calcium entry (SOCE). In the present study we describe the assembly of the several of the components from the SOC complex in real-time, utilizing a novel imaging method. Using FRET imaging we show that under resting conditions (with calcium stores replenished) STIM1 travels continuously through the ER associated to the microtubule tracking protein, EB1. Upon depletion of the ER STIM1 dissociates from EB1 and aggregates into macromolecular complexes at the ER which includes the microsomal calcium ATPase. This association follows the assembly of Orai into macromolecular aggregates at the PM. We show that STIM1–Orai association follows a similar time course as that of Orai aggregation at the PM. During this last step of the process, calcium-selective, whole-cell inward currents developed, simultaneously. We show that this process is fully reversible. Replenishing intracellular calcium stores induces STIM1–Orai complex dissociation and shuts down inward currents. Under these conditions STIM1 re-associates to EB1, and reinitiates its travel through the ER.