Mutational analysis of the cysteines in the extracellular domain of the human Ca2+ receptor: effects on cell surface expression, dimerization and signal transduction.
Mammalian calcium receptors (CaRs) share with the metabotropic glutamate receptors (mGluRs) the relative positions of 16 cysteine residues in the amino-terminal extracellular domain. To investigate the role of these cysteines, a series of mutants in the extracellular domain of the human CaR was prepared in which each of these 16 cysteine residues and three others not conserved in the mGluRs were replaced by serines. Wild-type and mutant CaR cDNAs were expressed in HEK-293 cells, and evaluated for expression and response to extracellular calcium. Mutation of three non-conserved cysteines and of two conserved cysteines produced proteins with near wild-type phenotype. In contrast, mutation of the other conserved cysteines gave proteins that showed drastic reduction in cell surface expression and/or failed to respond to calcium. We identified 14 cysteines essential for proper trafficking and function of the receptor, two of which may be involved in formation of a disulfide-linked dimer.
Fan
G F
GF
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Ray
K
K
Zhao
X M
XM
Goldsmith
P K
PK
Spiegel
A M
AM
D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology*; D002460:Cell Line; D003545:Cysteine; D019281:Dimerization; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012694:Serine; D015398:Signal Transduction; D014162:Transfection
D010716:Phosphatidylinositols; D044169:Receptors, Calcium-Sensing; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012694:Serine; D003545:Cysteine; D002118:Calcium