Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells.
The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
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Bai
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Trivedi
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Brown
D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry*; D003432:Cross-Linking Reagents; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000196:embryology; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011233:Precipitin Tests; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D002118:Calcium; D007460:Iodoacetamide