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The Role of Cysteines and Charged Amino Acids in Extracellular Loops of the Human Ca²⁺ Receptor in Cell Surface Expression and Receptor Activation Processes

Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Kausik Ray, Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, 5 Research Court, Room 2A11, Rockville, Maryland 20850. E-mail: rayk{at}nidcd.nih.gov.

The Ca²⁺ receptor is a plasma-membrane bound G protein-coupled receptor stimulated by extracellular calcium [Ca²⁺]_o and other di- and poly-cations. We investigated the role in receptor activation of all the charged amino acid residues and cysteines in the three extracellular loops (EL1, 2, and 3) of the human Ca²⁺ receptor by alanine-scanning mutagenesis. The mutant receptors were transiently expressed in HEK-293 cells, and cell surface expression patterns were analyzed by endoglycosidase-H digestion, immunoblotting, intact cell ELISA, and hydrolysis of phosphoinositides (PI) induced by [Ca²⁺]_o. The mutation of Cys677 and Cys765 located in EL1 and EL2, respectively, ablated PI hydrolysis completely, showed less than 5% cell surface expression of the wild-type receptor, and were not properly glycosylated. Replacement of the charged residues by using a single mutation or multiple alanine mutations in EL1, 2, and 3 produced only minor changes in receptor activation, except for Glu767 and Lys831. The E767A and K831A mutations in EL2 and EL3, respectively, showed gain-of-function by significantly enhancing apparent [Ca²⁺]_o affinity. E767A and K831A exhibited EC₅₀ values of 2.1 and 2.8 mM, respectively, for [Ca²⁺]_o-stimulated PI hydrolysis as opposed to EC₅₀ value of 4.2 mM for the wild-type receptor. Like E767A, substitutions of Glu767 with Gln and Lys was similarly activating, whereas Asp substitution displayed wild-type [Ca²⁺]_o sensitivity. Substitution of Lys831 with Glu but not with Gln showed similar activating effect as Ala replacement. A double-mutant E767K/K831E in which charged residues were switched positions showed impaired cell surface expression and failed to respond to [Ca²⁺]_o. Taken together, these results suggest that in ELs, two cysteines form critical disulfide links, and the side chains of Glu767 and Lys831 are probably involved in ionic interactions with other prospective oppositely charged residues. Some of these interactions could be important for receptor folding and also may contribute to keep the Ca²⁺ receptor transmembrane helix bundle in an inactive conformation.

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