Receptor-induced Ca 2؉ signals are key to the function of all cells and involve release of Ca 2؉ from endoplasmic reticulum (ER) stores, triggering Ca 2؉ entry through plasma membrane (PM) ''storeoperated channels'' (SOCs). The identity of SOCs and their coupling to store depletion remain molecular and mechanistic mysteries. The single transmembrane-spanning Ca 2؉ -binding protein, STIM1, is necessary in this coupling process and is proposed to function as an ER Ca 2؉ sensor to provide the trigger for SOC activation. Here we reveal that, in addition to being an ER Ca 2؉ sensor, STIM1 functions within the PM to control operation of the Ca 2؉ entry channel itself. Increased expression levels of STIM1 correlate with a gain in function of Ca 2؉ release-activated Ca 2؉ (CRAC) channel activity. Point mutation of the N-terminal EF hand transforms the CRAC channel current (I CRAC) into a constitutively active, Ca 2؉ store-independent mode. Mutants in the EF hand and cytoplasmic C terminus of STIM1 alter operational parameters of CRAC channels, including pharmacological profile and inactivation properties. Last, Ab externally applied to the STIM1 N-terminal EF hand blocks both I CRAC in hematopoietic cells and SOC-mediated Ca 2؉ entry in HEK293 cells, revealing that STIM1 has an important functional presence within the PM. The results reveal that, in addition to being an ER Ca 2؉ sensor, STIM1 functions within the PM to exert control over the operation of SOCs. As a cell surface signaling protein, STIM1 represents a key pharmacological target to control fundamental Ca 2؉ -regulated processes including secretion, contraction, metabolism, cell division, and apoptosis.calcium signaling ͉ calcium channel ͉ patch-clamp ͉ mast cells ͉ T lymphocytes

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.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

Transforming growth factor-beta (TGF-beta) and the renin-angiotensin system (RAS) have both been implicated in the pathogenesis of chronic renal disease. The present experiment investigated the chronology of TGF-beta 1 gene expression following subtotal nephrectomy (STNx) in the rat and the effect of blocking the RAS by angiotensin converting enzyme (ACE) inhibition or by angiotensin II receptor (AT1) antagonism. Rats that had undergone subtotal nephrectomy developed hypertension, proteinuria, renal impairement, glomerulosclerosis, tubulointerstitial fibrosis and mononuclear cell infiltration. These changes were associated with a 2.5-fold increase in TGF-beta 1 gene expression during a 16-week time course. In situ hybridization localized TGF-beta 1 mRNA to sclerotic glomeruli, areas of tubuloin-terstitial injury and sites of mononuclear cell infiltration. Administration of the ACE inhibitor ramipril and the AT1 receptor blocker valsartan blunted the increase in TGF-beta 1 mRNA, and attenuated the structural and functional manifestations of injury. These data suggest an interaction between the intrarenal RAS and TGF-beta in the pathogenesis of the glomerular and tubulointerstitial fibrosis that follow a major reduction in renal mass.