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J. Biol. Chem., Vol. 283, Issue 38, 26252-26262, September 19, 2008

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Location and Function of STIM1 in the Activation of Ca²⁺ Entry Signals^*

From the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Store-operated channels (SOCs) mediate Ca²⁺ entry signals in response to endoplasmic reticulum (ER) Ca²⁺ depletion in most cells. STIM1 senses decreased ER luminal Ca²⁺ through its EF-hand Ca²⁺-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 Ca²⁺ binding. EF20 cells were viable despite constitutive Ca²⁺ entry, allowing study of SOC activation without depleting ER Ca²⁺. 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 Ca²⁺ levels but substantially reduced ER Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.

Received for publication, March 20, 2008 , and in revised form, July 1, 2008.

^* This work was supported in whole or in part, by National Institutes of Health Grants HL55426 (to D. L. G.) AI058173 (to D. L. G.). This work was also supported by the American Heart Association (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104.

² To whom correspondence may be addressed: Dept. of Biochemistry, Temple University School of Medicine, 3400 N. Broad St., Philadelphia PA 19140. Tel.: 215-707-6567; Fax: 215-707-7536; E-mail: soboloff{at}temple.edu.

³ To whom correspondence may be addressed: Dept. of Biochemistry, Temple University School of Medicine, 3400 N. Broad St., Philadelphia PA 19140. Tel.: 215-707-2501; Fax: 215-707-3263; E-mail: dgill{at}temple.edu.

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