The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions.
Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca(2+)](o))-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys(129) and Cys(131) in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys(129) and Cys(131), showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.
Zhang
Z
Z
Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Sun
S
S
Quinn
S J
SJ
Brown
E M
EM
Bai
M
M
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D014162:Transfection
D004220:Disulfides; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D012694:Serine; D003545:Cysteine; D002118:Calcium