Biochemical and functional characterization of orai proteins

Yousang Gwack, Sonal Srikanth, Stefan Feske, Fernando Cruz-Guilloty, Masatsugu Ohora, Daniel S. Neems, Patrick G. Hogan, Anjana Rao

    Research output: Contribution to journalArticle

    283 Citations (Scopus)

    Abstract

    Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

    Original languageEnglish
    Pages (from-to)16232-16243
    Number of pages12
    JournalJournal of Biological Chemistry
    Volume282
    Issue number22
    DOIs
    Publication statusPublished - Jun 1 2007

    Fingerprint

    T-cells
    Drosophila
    Cytokines
    Proteins
    T-Lymphocytes
    Centrifugation
    Asparagine
    Fibroblasts
    Cell membranes
    Glutamine
    Glycerol
    Glutamic Acid
    Substitution reactions
    Transcription Factors
    Kidney
    Genes
    RNA
    Calcium
    Messenger RNA
    RNA Interference

    All Science Journal Classification (ASJC) codes

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Gwack, Y., Srikanth, S., Feske, S., Cruz-Guilloty, F., Ohora, M., Neems, D. S., ... Rao, A. (2007). Biochemical and functional characterization of orai proteins. Journal of Biological Chemistry, 282(22), 16232-16243. https://doi.org/10.1074/jbc.M609630200

    Biochemical and functional characterization of orai proteins. / Gwack, Yousang; Srikanth, Sonal; Feske, Stefan; Cruz-Guilloty, Fernando; Ohora, Masatsugu; Neems, Daniel S.; Hogan, Patrick G.; Rao, Anjana.

    In: Journal of Biological Chemistry, Vol. 282, No. 22, 01.06.2007, p. 16232-16243.

    Research output: Contribution to journalArticle

    Gwack, Y, Srikanth, S, Feske, S, Cruz-Guilloty, F, Ohora, M, Neems, DS, Hogan, PG & Rao, A 2007, 'Biochemical and functional characterization of orai proteins', Journal of Biological Chemistry, vol. 282, no. 22, pp. 16232-16243. https://doi.org/10.1074/jbc.M609630200
    Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Ohora M, Neems DS et al. Biochemical and functional characterization of orai proteins. Journal of Biological Chemistry. 2007 Jun 1;282(22):16232-16243. https://doi.org/10.1074/jbc.M609630200
    Gwack, Yousang ; Srikanth, Sonal ; Feske, Stefan ; Cruz-Guilloty, Fernando ; Ohora, Masatsugu ; Neems, Daniel S. ; Hogan, Patrick G. ; Rao, Anjana. / Biochemical and functional characterization of orai proteins. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 22. pp. 16232-16243.
    @article{1c2eb3bb9f764a6280beac7ee916371d,
    title = "Biochemical and functional characterization of orai proteins",
    abstract = "Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.",
    author = "Yousang Gwack and Sonal Srikanth and Stefan Feske and Fernando Cruz-Guilloty and Masatsugu Ohora and Neems, {Daniel S.} and Hogan, {Patrick G.} and Anjana Rao",
    year = "2007",
    month = "6",
    day = "1",
    doi = "10.1074/jbc.M609630200",
    language = "English",
    volume = "282",
    pages = "16232--16243",
    journal = "Journal of Biological Chemistry",
    issn = "0021-9258",
    publisher = "American Society for Biochemistry and Molecular Biology Inc.",
    number = "22",

    }

    TY - JOUR

    T1 - Biochemical and functional characterization of orai proteins

    AU - Gwack, Yousang

    AU - Srikanth, Sonal

    AU - Feske, Stefan

    AU - Cruz-Guilloty, Fernando

    AU - Ohora, Masatsugu

    AU - Neems, Daniel S.

    AU - Hogan, Patrick G.

    AU - Rao, Anjana

    PY - 2007/6/1

    Y1 - 2007/6/1

    N2 - Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

    AB - Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

    UR - http://www.scopus.com/inward/record.url?scp=34247339504&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=34247339504&partnerID=8YFLogxK

    U2 - 10.1074/jbc.M609630200

    DO - 10.1074/jbc.M609630200

    M3 - Article

    C2 - 17293345

    AN - SCOPUS:34247339504

    VL - 282

    SP - 16232

    EP - 16243

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 22

    ER -