Amino acids in the cytoplasmic C terminus of the parathyroid Ca2+-sensing receptor mediate efficient cell-surface expression and phospholipase C activation

The C-terminal tail of the calcium receptor (CaR) regulates the affinity of the receptor for ligand, desensitization, and membrane localization. To de termine the role of specific amino acids in the bovine parathyroid CaR in m ediating signal transduction and cell-surface expression, we transfected tr uncated and mutated Call cDNAs into HEK-293 cells. The ability of high extr acellular [Ca2+] ([Ca2+](o)) to increase total inositol phosphate (InsP) pr oduction, an index of phospholipase C (PLC) activation, was determined. Rec eptor 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 [Ca 2+](o) increased InsPs to levels comparable with those of cells expressing wild-type Calls. 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 scan ned the residues between Ser(866) and Val(895) using tandem-Ala and single- site mutagenesis. Two point mutants (His(880) --> Ala and Phe(882) --> Ala CaR) showed 50-70% reductions in high [Ca2+](o)-induced InsP production. Th e levels of expression and glycosylation of these mutants were comparable w ith wild-type Calls, but both receptors were profoundly retained in intrace llular organelles and co-localized with the endoplasmic reticulum marker Bi P. This suggested that the signaling defects of these receptors were likely because of defective trafficking of receptors to the cell surface. Modelin g of the C-terminal domain of the CaR indicated that His(880) and Phe(882) are situated in a putative alpha -helical structure of 15 amino acids betwe en residues 877 and 891 in the C-terminal tail. Our studies support the ide a that specific amino acids, and possibly a unique secondary structure in t he C-terminal tail, are required for the efficient targeting of the CaR to the cell surface required for PLC activation.