Location and function of STIM1 in the activation of Ca2+ entry signals

Thamara Hewavitharana, Xiaoxiang Deng, Youjun Wang, Michael F. Ritchie, Gannareddy V. Girish, Jonathan Soboloff, Donald L. Gill

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 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 (STIM1EF) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1EF was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1WT.STIMEF-expressing cells had normal ER Ca 2+ 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 STIM1WT or STIM1 EF revealed strong PM interactions of both proteins. Although surface expression of STIM1WT was clearly detectable, STIM1EF was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1EF mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1WT and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.

Original languageEnglish (US)
Pages (from-to)26252-26262
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number38
DOIs
StatePublished - Sep 19 2008

Fingerprint

Cell membranes
Chemical activation
EF Hand Motifs
Endoplasmic Reticulum
Cell Membrane
Cells
Biotinylation
Streptavidin
HEK293 Cells
Cell Survival
Proteins
Cell Line
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Hewavitharana, T., Deng, X., Wang, Y., Ritchie, M. F., Girish, G. V., Soboloff, J., & Gill, D. L. (2008). Location and function of STIM1 in the activation of Ca2+ entry signals. Journal of Biological Chemistry, 283(38), 26252-26262. https://doi.org/10.1074/jbc.M802239200
Hewavitharana, Thamara ; Deng, Xiaoxiang ; Wang, Youjun ; Ritchie, Michael F. ; Girish, Gannareddy V. ; Soboloff, Jonathan ; Gill, Donald L. / Location and function of STIM1 in the activation of Ca2+ entry signals. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 38. pp. 26252-26262.
@article{f8c6c2e50f78403493b8b3b0b4ffe177,
title = "Location and function of STIM1 in the activation of Ca2+ entry signals",
abstract = "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 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 (STIM1EF) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1EF was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1WT.STIMEF-expressing cells had normal ER Ca 2+ 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 STIM1WT or STIM1 EF revealed strong PM interactions of both proteins. Although surface expression of STIM1WT was clearly detectable, STIM1EF was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1EF mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1WT and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.",
author = "Thamara Hewavitharana and Xiaoxiang Deng and Youjun Wang and Ritchie, {Michael F.} and Girish, {Gannareddy V.} and Jonathan Soboloff and Gill, {Donald L.}",
year = "2008",
month = "9",
day = "19",
doi = "10.1074/jbc.M802239200",
language = "English (US)",
volume = "283",
pages = "26252--26262",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "38",

}

Hewavitharana, T, Deng, X, Wang, Y, Ritchie, MF, Girish, GV, Soboloff, J & Gill, DL 2008, 'Location and function of STIM1 in the activation of Ca2+ entry signals', Journal of Biological Chemistry, vol. 283, no. 38, pp. 26252-26262. https://doi.org/10.1074/jbc.M802239200

Location and function of STIM1 in the activation of Ca2+ entry signals. / Hewavitharana, Thamara; Deng, Xiaoxiang; Wang, Youjun; Ritchie, Michael F.; Girish, Gannareddy V.; Soboloff, Jonathan; Gill, Donald L.

In: Journal of Biological Chemistry, Vol. 283, No. 38, 19.09.2008, p. 26252-26262.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Location and function of STIM1 in the activation of Ca2+ entry signals

AU - Hewavitharana, Thamara

AU - Deng, Xiaoxiang

AU - Wang, Youjun

AU - Ritchie, Michael F.

AU - Girish, Gannareddy V.

AU - Soboloff, Jonathan

AU - Gill, Donald L.

PY - 2008/9/19

Y1 - 2008/9/19

N2 - 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 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 (STIM1EF) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1EF was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1WT.STIMEF-expressing cells had normal ER Ca 2+ 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 STIM1WT or STIM1 EF revealed strong PM interactions of both proteins. Although surface expression of STIM1WT was clearly detectable, STIM1EF was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1EF mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1WT and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.

AB - 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 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 (STIM1EF) deficient in Ca2+ binding. EF20 cells were viable despite constitutive Ca2+ entry, allowing study of SOC activation without depleting ER Ca2+. STIM1EF was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1WT.STIMEF-expressing cells had normal ER Ca 2+ 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 STIM1WT or STIM1 EF revealed strong PM interactions of both proteins. Although surface expression of STIM1WT was clearly detectable, STIM1EF was undetectable at the cell surface. Thus, the Ca2+ binding-defective STIM1EF mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1WT, is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIMEF-mediated Ca2+ entry, but only in cells expressing endogenous STIM1WT and not in DT40 STIM1 knock-out cells devoid of STIMWT. This suggests that PM-STIM1 may play a regulatory role in SOC activation.

UR - http://www.scopus.com/inward/record.url?scp=54449096217&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=54449096217&partnerID=8YFLogxK

U2 - 10.1074/jbc.M802239200

DO - 10.1074/jbc.M802239200

M3 - Article

C2 - 18635545

AN - SCOPUS:54449096217

VL - 283

SP - 26252

EP - 26262

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 38

ER -

Hewavitharana T, Deng X, Wang Y, Ritchie MF, Girish GV, Soboloff J et al. Location and function of STIM1 in the activation of Ca2+ entry signals. Journal of Biological Chemistry. 2008 Sep 19;283(38):26252-26262. https://doi.org/10.1074/jbc.M802239200