title: Location and function of STIM1 in the activation of Ca2+ entry signals.
abstract: Store-operated channels (SOCs) mediate Ca(2+) entry signals in response to endoplasmic reticulum (ER) Ca(2+) depletion in most cells. STIM1 senses decreased ER luminal Ca(2+) through its EF-hand Ca(2+)-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca(2+) binding. EF20 cells were viable despite constitutive Ca(2+) entry, allowing study of SOC activation without depleting ER Ca(2+). STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIM(EF)-expressing cells had normal ER Ca(2+) levels but substantially reduced ER Ca(2+) leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca(2+) leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca(2+) binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1(WT), is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM(EF)-mediated Ca(2+) entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIM(WT). This suggests that PM-STIM1 may play a regulatory role in SOC activation.
authors.affiliation: Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Thamara
authors.initials: T
authors.lastname: Hewavitharana
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Deng
authors.forename: Youjun
authors.initials: Y
authors.lastname: Wang
authors.forename: Michael F
authors.initials: MF
authors.lastname: Ritchie
authors.forename: Gannareddy V
authors.initials: GV
authors.lastname: Girish
authors.forename: Jonathan
authors.initials: J
authors.lastname: Soboloff
authors.forename: Donald L
authors.initials: DL
authors.lastname: Gill
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D017209:Apoptosis; D019921:Biotinylation; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000502:physiology; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000502:physiology; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D000071737:Stromal Interaction Molecule 1; D013499:Surface Properties
chemical_list: D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D002118:Calcium