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J. Biol. Chem., Vol. 276, Issue 47, 44129-44136, November 23, 2001

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Amino Acids in the Cytoplasmic C Terminus of the Parathyroid Ca²⁺-sensing Receptor Mediate Efficient Cell-surface Expression and Phospholipase C Activation^*

Wenhan Chang, Stacy Pratt, Tsui-Hua Chen, Lilly Bourguignon, and Dolores Shoback

From the Endocrine Research Unit, Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To determine the role of specific amino acids in the bovine parathyroid CaR in mediating signal transduction and cell-surface expression, we transfected truncated and mutated CaR cDNAs into HEK-293 cells. The ability of high extracellular [Ca²⁺] ([Ca²⁺]_o) to increase total inositol phosphate (InsP) production, an index of phospholipase C (PLC) activation, was determined. Receptor expression was assessed by immunoblotting and immunocytochemistry. In cells transiently or stably expressing receptors with the C-terminal tail truncated after residue 895 (CaR-(1-895)) or 929 (CaR-(1-929)), raising [Ca²⁺]_o increased InsPs to levels comparable with those of cells expressing wild-type CaRs. There were no PLC responses to high [Ca²⁺]_o (up to 30 mM) in cells expressing CaRs with C-terminal tails of only 3 residues (CaR-(1-866)), even though these receptors were expressed in the membrane. We scanned the residues between Ser⁸⁶⁶ and Val⁸⁹⁵ using tandem-Ala and single-site mutagenesis. Two point mutants (His⁸⁸⁰  Ala and Phe⁸⁸²  Ala CaR) showed 50-70% reductions in high [Ca²⁺]_o-induced InsP production. The levels of expression and glycosylation of these mutants were comparable with wild-type CaRs, but both receptors were profoundly retained in intracellular organelles and co-localized with the endoplasmic reticulum marker BiP. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modeling of the C-terminal domain of the CaR indicated that His⁸⁸⁰ and Phe⁸⁸² are situated in a putative -helical structure of 15 amino acids between residues 877 and 891 in the C-terminal tail. Our studies support the idea that specific amino acids, and possibly a unique secondary structure in the C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.

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