A cytosolic STIM2 preprotein created by signal peptide inefficiency activates ORAI1 in a store-independent manner.

Calcium (Ca(2+)) influx through the plasma membrane store-operated Ca(2+) channel ORAI1 is controlled by Ca(2+) sensors of the stromal interaction molecule (STIM) family. STIM1 responds to endoplasmic reticulum (ER) Ca(2+) store depletion by redistributing and activating ORAI1 from regions of the ER juxtaposed to the plasma membrane. Unlike STIM1, STIM2 can regulate ORAI1 in a store-dependent and store-independent manner, but the mechanism by which this is achieved is unknown. Here we find that STIM2 is translated from a highly conserved methionine residue and is directed to the ER by an incredibly long 101-amino acid signal peptide. We find that although the majority of the total STIM2 population resides on the ER membrane, a second population escapes ER targeting to accumulate as a full-length preprotein in the cytosol, signal peptide intact. Unlike STIM2, preSTIM2 localizes to the inner leaflet of the plasma membrane where it interacts with ORAI1 to regulate basal Ca(2+) concentration and Ca(2+)-dependent gene transcription in a store-independent manner. Furthermore, a third protein comprising a fragment of the STIM2 signal peptide is released from the ER membrane into the cytosol where it regulates gene transcription in a Ca(2+)-independent manner. This study establishes a new model for STIM2-mediated regulation of ORAI1 in which two distinct proteins, STIM2 and preSTIM2, control store-dependent and store-independent modes of ORAI1 activation.

STIM proteins: integrators of signalling pathways in development, differentiation and disease.

The stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum Ca(2+) sensors, serving to detect changes in receptor-mediated ER Ca(2+) store depletion and to relay this information to plasma membrane localized proteins, including the store-operated Ca(2+) channels of the ORAI family. The resulting Ca(2+) influx sustains the high cytosolic Ca(2+) levels required for activation of many intracellular signal transducers such as the NFAT family of transcription factors. Models of STIM protein deficiency in mice, Drosophila melanogaster and Caenorhabditis elegans, in addition to the phenotype of patients bearing mutations in STIM1 have provided great insight into the role of these proteins in cell physiology and pathology. It is now becoming clear that STIM1 and STIM2 are critical for the development and functioning of many cell types, including lymphocytes, skeletal and smooth muscle myoblasts, adipocytes and neurons, and can interact with a variety of signalling proteins and pathways in a cell- and tissue-type specific manner. This review focuses on the role of STIM proteins in development, differentiation and disease, in particular highlighting the functional differences between STIM1 and STIM2.

Stim1, an endoplasmic reticulum Ca2+ sensor, negatively regulates 3T3-L1 pre-adipocyte differentiation.

Ca(2+) plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca(2+) ions through specific plasma membrane store-operated Ca(2+) channels. Stim1 is a sensor of endoplasmic reticulum Ca(2+) store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca(2+) channels in the plasma membrane to activate these channels and induce Ca(2+) influx. To investigate the potential role of Stim1 in Ca(2+)-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca(2+) entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca(2+) entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca(2+) entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca(2+) influx through store-operated channels, to balance the level of differentiation in these cells in vitro.

Biochemical properties and cellular localisation of STIM proteins.

Human and murine STIM1 were originally discovered as candidate growth regulators in tumours and in the bone marrow stroma, and the structurally related vertebrate family members, STIM2 and the Drosophila homologue D-Stim, were subsequently identified. STIM proteins are ubiquitously expressed type I single-pass transmembrane proteins which have a unique combination of structural motifs within their polypeptide sequences. The extracellular regions contain an N-terminal unpaired EF-hand Ca(2+) binding motif adjacent to an unconventional glycosylated SAM domain, while the cytoplasmic regions contain alpha-helical coiled-coil domains within a region having homology to ERM domains adjacent to the transmembrane region, and phosphorylated proline-rich domains near the C-terminus. STIM1, STIM2 and D-Stim diverge significantly only in their structure C-terminal to the coiled-coil/ERM domains. The STIM structural domains were predicted to function in Ca(2+) binding as well as in mediating interactions between STIM proteins and other proteins, and homotypic STIM1-STIM1 and heterotypic STIM1-STIM2 interactions were demonstrated biochemically. However, the functional significance of the cellular localisation of STIM1 and its domain structure only became evident after recent breakthrough research identified STIM1 as a key regulator of store-operated calcium (SOC) entry into cells. It is now clear that STIM1 is both a sensor of Ca(2+) depletion in the endoplasmic reticulum (ER) lumen and an activator of Orai1-containing SOC channels in the plasma membrane. On the basis of recent functional studies a model can be proposed to explain how the biochemical properties of STIM1 contribute to its precise membrane localisation and its function in regulating SOC entry.

STIM2 is an inhibitor of STIM1-mediated store-operated Ca2+ Entry.

The coupling mechanism between endoplasmic reticulum (ER) Ca(2+) stores and plasma membrane (PM) store-operated channels (SOCs) remains elusive [1-3]. STIM1 was shown to play a crucial role in this coupling process [4-7]; however, the role of the closely related STIM2 protein remains undetermined. We reveal that STIM2 is a powerful SOC inhibitor when expressed in HEK293, PC12, A7r5, and Jurkat T cells. This contrasts with gain of SOC function in STIM1-expressing cells. While STIM1 is expressed in both the ER and plasma membrane, STIM2 is expressed only intracellularly. Store depletion induces redistribution of STIM1 into distinct "puncta." STIM2 translocates into puncta upon store depletion only when coexpressed with STIM1. Double labeling shows coincidence of STIM1 and STIM2 within puncta, and immunoprecipitation reveals direct interactions between STIM1 and STIM2. Independent of store depletion, STIM2 colocalizes with and blocks the function of a STIM1 EF-hand mutant that preexists in puncta and is constitutively coupled to activate SOCs. Thus, whereas STIM1 is a required mediator of SOC activation, STIM2 is a powerful inhibitor of this process, interfering with STIM1-mediated SOC activation at a point downstream of puncta formation. The opposing functions of STIM1 and STIM2 suggest they may play a coordinated role in controlling SOC-mediated Ca(2+) entry signals.