Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Abstract

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca2+ entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca2+ store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1D76A, which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1D76A puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE. Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca2+ entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca2+ store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1D76A, which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1D76A puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE. Store-operated Ca2+ entry (SOCE) 3The abbreviations used are: SOCE, store-operated Ca2+ entry; LRD, lipid raft domains; MES, 4-morpholineethanesulfonic acid; MβCD, methyl-β-cyclodextrin; Tg, thapsigargin; ER, endoplasmic reticulum. is a critical Ca2+ entry mechanism that is ubiquitously present in all cell types. SOCE not only determines refilling of intracellular Ca2+ stores but also regulates a wide variety of cellular functions (1Berridge M.J. Bootman M.D. Roderick H.L. Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4256) Google Scholar, 2Parekh A.B. Putney J.W. Physiol. Rev. 2005; 85: 757-810Crossref PubMed Scopus (1807) Google Scholar, 3Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar). The molecular basis for store-operated calcium entry has long remained an enigma (3Putney Jr., J.W. Cell Calcium. 1990; 11: 611-624Crossref PubMed Scopus (1264) Google Scholar, 4Putney Jr., J.W. Cell Calcium. 2007; 42: 103-110Crossref PubMed Scopus (189) Google Scholar, 5Venkatachalam K. van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: 263-272Crossref PubMed Scopus (339) Google Scholar). Recently STIM1, an ER Ca2+ sensor protein, was suggested to be involved in coupling of ER Ca2+ store depletion to activation of plasma membrane Ca2+ entry channels (6Lewis R.S. Nature. 2007; 446: 284-287Crossref PubMed Scopus (429) Google Scholar, 7Roos J. DiGregorio P.J. Yeromin A.V. Ohlsen K. Lioudyno M. Zhang S. Safrina O. Kozak J.A. Wagner S.L. Cahalan M.D. Velicelebi G. Stauderman K.A. J. Cell Biol. 2005; 169: 435-445Crossref PubMed Scopus (1526) Google Scholar, 8Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar). Depletion of ER Ca2+ stores results in the relocation of STIM1 into puncta in the subplasma membrane region, which has been demonstrated to be the site at which Ca2+ entry occurs (8Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar, 9Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1140) Google Scholar, 10Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 29: 9301-9306Crossref Scopus (525) Google Scholar, 11Wu M.M. Buchanan J. Luik R.M. Lewis R.S. J. Cell Biol. 2006; 174: 803-813Crossref PubMed Scopus (667) Google Scholar, 12Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (539) Google Scholar). Therefore, it has been proposed that store-operated Ca2+ (SOC) channels reside in the plasma membrane juxtaposing the puncta. Indeed, Orai1 and TRPC1, which are critical components of CRAC and SOC channels, respectively, have been shown to co-localize with STIM1 puncta in stimulated cells (13Xu P. Lu J. Li Z. Yu X. Chen L. Xu T. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (202) Google Scholar, 14Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007. 2007; 282: 29678-29690Scopus (213) Google Scholar, 16Ong H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 17Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 9: 1003-1010Crossref Scopus (572) Google Scholar). Thus, the site of STIM1 clusters in the peripheral ER determines recruitment and activation of plasma membrane SOCE channels. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell are not yet known. Plasma membrane lipid rafts domains (LRD), which contain high concentrations of cholesterol and sphingolipids, are known to function as centers for the assembly of signaling complexes. Such assembly is suggested to facilitate both specificity and the rate of signaling events by positioning functionally associated molecules in close proximity to each other (18Galbiati F. Razani B. Lisanti M.P. Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 19Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5187) Google Scholar, 20Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. 2003; 4: PubMed Scopus Google Scholar). a that is involved in the of lipid has been suggested to be for SOCE M. 2003; 4: PubMed Scopus Google Scholar, I.S. Sci. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). We that TRPC1, a of SOC channels X. Singh B.B. Ambudkar I.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, X. Cheng K.T. Bandyopadhyay B.C. Pani B. A. Paria B.C. W.D. D. Singh B.B. L. Ambudkar I.S. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google is in a signaling with both the ER and plasma membrane Liu X. Singh B.B. J. S. Ambudkar I.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and that intact LRD are for activation of SOCE. have been T. Yu J. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). has been shown that of LRD of results in of TRPC1 and decreased SOCE Liu X. Singh B.B. J. S. Ambudkar I.S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Yu J. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar, Singh B.B. Liu X. W. Ambudkar I.S. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we have the of lipid raft in regulation of SOCE. The results demonstrate that intact plasma membrane lipid rafts are for clustering of STIM1 at the ER-plasma membrane junctional and regulation of SOCE. LRD facilitate the interaction of STIM1 with TRPC1 and activation of channels. Cell and and HEK293 cells in and respectively, with and in with and used of molecular cells with and for in and with and a by a of the was with an of and with of by of at for at the of the and used as of rafts was as in Li T. M. Lisanti M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in and was to by with of in and with of by of and as LRD and as in O. J. 2003; Full Text Full Text PDF PubMed Scopus Google membrane rafts by cholesterol HSG cells for with at in and to cholesterol following cells with of cholesterol with was by the and cholesterol was by a cholesterol the and and to rafts and respectively, and to with and with HSG cells stimulated with for at in with and in LRD as and in with and and concentrations to and with with of and in by as H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). store cells stimulated for with in and in with for a of each the with and with B. of was the of used is as cells and with for and used for cells stimulated with for with and with following for the with the was an as H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). cells in a was by a The was into an to the of the and a to a was a a the with for in at to as H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, X. Cheng K.T. Bandyopadhyay B.C. Pani B. A. Paria B.C. W.D. D. Singh B.B. L. Ambudkar I.S. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google Scholar). an at cells. to the and with an as in X. Cheng K.T. Bandyopadhyay B.C. Pani B. A. Paria B.C. W.D. D. Singh B.B. L. Ambudkar I.S. Proc. Natl. Acad. Sci. U. S. A. 2007; PubMed Scopus Google in the cell at an of the was by the at a of high in to to a of was and at a rate of the the was used for of the in the was used to the cells. was of are as in the to be at of STIM1 and TRPC1 in the rafts cell and and with the and used as for LRD, was used to and cholesterol and also in these to the lipid HSG cell a of STIM1 with lipid raft in However, a of the STIM1 was in TRPC1, and also into both lipid raft and and in the The also relatively higher cholesterol to the This that these contain LRD. of in the and was in HEK293 cells as well a to the raft and partitioning of STIM1, TRPC1, and other in and was Thus, the of in raft and shown in is not to by the of The of LRD in the association of TRPC1 and STIM1 H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 17Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 9: 1003-1010Crossref Scopus (572) Google Scholar, J.P. Zeng W. G.N. Worley P.F. Muallem S. Nat. Cell Biol. 2007; 9: PubMed Scopus Google was by the raft and the shown in the interaction TRPC1 and STIM1 was in with TRPC1 is associated with in this of the in the are shown in these results that STIM1 and TRPC1 into LRD and with each other within this Recruitment of STIM1 and TRPC1 into of Ca2+ store depletion STIM1 partitioning into LRD was by its in raft and and cells with and for the of STIM1 and TRPC1. STIM1 was in both and with its in the in as compared with the in unstimulated cells, the of STIM1 in rafts was relatively increased and in cells with a in the TRPC1 also displayed in the raft that of STIM1 with lipid raft was increased in cells stimulated with and and not the of peripheral STIM1 puncta, but not the with determine whether the of cells the interaction STIM1 and TRPC1 in LRD, STIM1 TRPC1 lipid raft stimulated and unstimulated cells. that increased regulation of interaction was by raft cells where ER stores of Ca2+ with for in in an in However, stores to be by by of Ca2+ was a in the STIM1 interaction with TRPC1 with Thus, these data demonstrate that store depletion induces recruitment of STIM1 and TRPC1 into lipid rafts and that the status of ER Ca2+ stores the of STIM1 and TRPC1 TRPC1 is for of STIM1 into lipid we TRPC1 as H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). recruitment of STIM1 into LRD was independent of TRPC1 demonstrate this we used and STIM1 in cells. Peripheral clustering of STIM1 was in cells in to and this of STIM1 was in cells that this TRPC1 and SOCE in HSG cells H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). these data that STIM1 and puncta in lipid raft domains is independent of TRPC1 but is regulated by intracellular Ca2+ store depletion. Disruption of STIM1 and of lipid rafts in targeting STIM1 clusters to the subplasma membrane region was by the of LRD which membrane decreased TRPC1 and STIM1 in lipid rafts and with a in cholesterol it attenuated recruitment of STIM1 and TRPC1 into the raft domains and the of in the for LRD as a of in the membrane cholesterol following LRD and also was of membrane cholesterol in HSG cells the of and STIM1 demonstrated that STIM1 puncta in the cell was decreased in cells with However, the sequestration of membrane cholesterol not have the of the STIM1 puncta, following store depletion in of TRPC1 and STIM1 was decreased in cells with to compared with cells data that intact LRD are for clustering of STIM1 in the subplasma membrane region of the cells, and its increased association with TRPC1 in the ER-plasma membrane junctional region in to Ca2+ store depletion. The of LRD SOCE was by HSG cells with both of which membrane decreased SOCE stores data are shown in Ca2+ in HSG cells also by with which not the of the and Additionally we the of SOCE in HEK293 cells. This cell has been used to the of STIM1 in the regulation of SOCE both CRAC and SOC channels G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 9: 1003-1010Crossref Scopus (572) Google Scholar, M. A. A. S. D.L. Soboloff J. Gill D.L. A. J.P. Curr. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of both of channels has been shown to peripheral with HSG cells of HEK293 cells also decreased peripheral clustering of STIM1 and SOCE and data demonstrate that lipid rafts also determine activation of SOCE in other cell types. for of STIM1D76A in Peripheral STIM1D76A has a in the that it to it is in the plasma membrane region of the cell in a constitutively SOCE in cells this Jr., J.W. Cell Calcium. 2007; 42: 103-110Crossref PubMed Scopus (189) Google Scholar, R.S. Nature. 2007; 446: 284-287Crossref PubMed Scopus (429) Google Scholar). has been that is localized in the ER where it with plasma membrane SOCE channels J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 29: 9301-9306Crossref Scopus (525) Google Scholar, 11Wu M.M. Buchanan J. Luik R.M. Lewis R.S. J. Cell Biol. 2006; 174: 803-813Crossref PubMed Scopus (667) Google Scholar). with STIM1D76A was as clusters in the plasma membrane region of unstimulated cells these with the LRD a of STIM1D76A was to be a raft associated in unstimulated cells cells STIM1D76A a high of constitutive Ca2+ which was with and demonstrated that decreased the constitutive STIM1D76A clustering in the plasma membrane region However, was not by with the constitutive Ca2+ entry in these cells, STIM1D76A relatively high association with TRPC1 in unstimulated cells. Additionally interaction was in cells with the of plasma membrane LRD in targeting STIM1 to specific ER-PM junctional sites and in SOCE by the functional have STIM1 as a critical for SOCE Jr., J.W. Cell Calcium. 2007; 42: 103-110Crossref PubMed Scopus (189) Google Scholar, R.S. Nature. 2007; 446: 284-287Crossref PubMed Scopus (429) Google Scholar). STIM1, a localized in the ER, clustering and to the subplasma membrane of the cells, where it (8Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar, 9Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1140) Google Scholar, 10Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 29: 9301-9306Crossref Scopus (525) Google Scholar). data that STIM1 puncta in the peripheral region of the cells the location where the functionally interacts with plasma membrane SOCE channels and regulates Ca2+ SOCE has been shown to at sites with the STIM1 peripheral clusters M.M. Buchanan J. Luik R.M. Lewis R.S. J. Cell Biol. 2006; 174: 803-813Crossref PubMed Scopus (667) Google Scholar, 12Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (539) Google of these puncta. clustering of CRAC in the plasma membrane STIM1 puncta and is with the location of the puncta (13Xu P. Lu J. Li Z. Yu X. Chen L. Xu T. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (202) Google Scholar, 14Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). SOC TRPC1, is also with STIM1 clusters H.L. Cheng K.T. Liu X. Bandyopadhyay B.C. Paria B.C. Soboloff J. Pani B. Gwack Y. Srikanth S. Singh B.B. Gill D.L. Ambudkar I.S. J. Biol. Chem. 2007; 282: 9105-9116Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 17Huang G.N. Zeng W. Kim J.Y. Yuan J.P. Han L. Muallem S. Worley P.F. Nat. Cell Biol. 2006; 9: 1003-1010Crossref Scopus (572) Google Scholar). results that in to mediate SOCE, STIM1 to be to specific of the where the of its interaction with plasma membrane SOC be The that determine the site of STIM1 clusters in the cell as well as its functional interaction with and regulation of SOCE are not yet known. data that that plasma membrane LRD determine the peripheral clustering of STIM1 and regulation of SOCE. data demonstrate that Ca2+ store depletion the association of STIM1 with LRD. this association to be critical for the of at the ER-plasma membrane junctional region of the cells. Disruption of the LRD by membrane cholesterol in of STIM1 clustering the plasma STIM1 partitioning into LRD in to of cells was also by cholesterol depletion. the LRD association of STIM1 was by the with cells displayed SOCE and STIM1D76A was constitutively in the cell and was present at relatively higher in LRD compared with STIM1. membrane of STIM1D76A was also determined by LRD the depletion of plasma membrane cholesterol by not only STIM1D76A but also its interaction with TRPC1 in a of the constitutive Ca2+ Together, these data a critical for LRD in the peripheral clustering of STIM1 in the subplasma membrane region that occurs as a of store depletion and in regulation of SOCE. have that are components of CRAC channels M. S. Gwack Y. Srikanth S. A. Nature. 2006; PubMed Scopus Google Scholar, M. A. A. S. D.L. Soboloff J. Gill D.L. A. J.P. Curr. Biol. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Biochem. 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The data show that functional interaction STIM1 and TRPC1 occurs within LRD and is dynamically regulated by ER Ca2+ store depletion and store is Thus, data an into the mechanism that is involved in the regulation of channels by STIM1. we that LRD in the plasma membrane a for clustering and interaction of STIM1 and TRPC1 in the ER-plasma membrane junctions in we not whether STIM1 is for TRPC1 clustering and recruitment into LRD, TRPC1 not STIM1 We that of STIM1 with LRD results in the in close proximity to each which facilitates the critical association STIM1 in the ER and TRPC1 in the plasma membrane that is involved in the activation of SOCE. we have not yet determined whether also within LRD, have that STIM1 puncta determine Orai1 clustering and activation in HEK293 cells (13Xu P. Lu J. Li Z. Yu X. Chen L. Xu T. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (202) Google Scholar, 14Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Várnai P. Tóth B. Tóth D.J. Hunyady L. Balla T. J. Biol. Chem. 2007. 2007; 282: 29678-29690Scopus (213) Google Scholar). data show that LRD also determine STIM1 puncta in HEK293 cells and also have a in regulation of channels. However, be to The demonstrate that STIM1 clusters in peripheral ER that are in to are with LRD in the plasma We that of STIM1 by plasma membrane LRD results in relatively ER-plasma membrane junctions that SOCE. Ca2+ store depletion induces of STIM1, which has been to to puncta in the cell (8Liou J. Kim M.L. Heo W.D. Jones J.T. Myers J.W. Ferrell Jr., J.E. Meyer T. Curr. Biol. 2005; 15: 1235-1241Abstract Full Text Full Text PDF PubMed Scopus (1765) Google Scholar, 9Zhang S.L. Yu Y. Roos J. Kozak J.A. Deerinck T.J. Ellisman M.H. Stauderman K.A. Cahalan M.D. Nature. 2005; 437: 902-905Crossref PubMed Scopus (1140) Google Scholar, 10Liou J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 29: 9301-9306Crossref Scopus (525) Google Scholar). The for and targeting of STIM1 to specific ER-plasma membrane junctional where STIM1 with SOCE channels in the membrane J. Fivaz M. Inoue T. Meyer T. Proc. Natl. Acad. Sci. U. S. A. 2007; 29: 9301-9306Crossref Scopus (525) Google Scholar, 11Wu M.M. Buchanan J. Luik R.M. Lewis R.S. J. Cell Biol. 2006; 174: 803-813Crossref PubMed Scopus (667) Google Scholar, 12Luik R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (539) Google Scholar). The in the of STIM1 is to be for its which contain a region, to be for the targeting of the STIM1 to the cell calcium store depletion (13Xu P. Lu J. Li Z. Yu X. Chen L. Xu T. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (202) Google Scholar, 14Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The region in the of STIM1 also to STIM1 targeting to region but is not for Ca2+ store depletion R.M. Wu M.M. Buchanan J. Lewis R.S. J. Cell Biol. 2006; 174: 815-825Crossref PubMed Scopus (539) Google Scholar, P. Lu J. Li Z. Yu X. Chen L. Xu T. Biochem. Biophys. Res. Commun. 2006; 350: 969-976Crossref PubMed Scopus (202) Google Scholar, 14Li Z. Lu J. Xu P. Xie X. Chen L. Xu T. J. Biol. Chem. 2007; 282: 29448-29456Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Thus, of STIM1 that occurs in to a in and its to the subplasma membrane region be the is the data demonstrate that clustering of STIM1 in the cell but not its plasma membrane lipid we have not the of STIM1 that is involved in its interaction with LRD, we that the of STIM1 with lipid components of the LRD, and that this interaction to STIM1 clusters in specific of the cell where it with and SOCE. we report that the clustering of STIM1 in the plasma membrane region, but not its that occurs in to ER Ca2+ store depletion is determined by plasma membrane LRD. We show that STIM1 association with LRD store depletion. This is by that the constitutively STIM1D76A increased partitioning into LRD. The of LRD in peripheral STIM1 clustering as well as SOCE. with of LRD also attenuated recruitment of store-operated TRPC1 channels into LRD, and its functional association with STIM1. these data demonstrate that LRD STIM1, and determines its in specific ER-plasma membrane junctions where it functionally with plasma membrane channels and SOCE. We the at for We Meyer for the STIM1 D. and of for and with