Mutational analysis of the cytoplasmic tail of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein: effects on receptor expression and signaling.
The present studies were undertaken to examine the role of the cytoplasmic tail of the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) on receptor signaling and expression. The wild type (WT) receptor (585 amino acids) and five truncated receptors whose cytoplasmic tails terminated at residues 507, 494, 474, 466, and 458 were expressed in COS-7 cells. Based on [125I]PTHrP binding, mutants T507, T494, and T466 displayed progressively decreased levels of expression, compared with WT. The tailless mutant T458 was not expressed in a functional form, whereas T474 was expressed at a level similar to WT. Comparable results were obtained when expression levels of WT and mutated PTH/PTHrP receptors were evaluated by Western blotting. Binding affinities were similar for all mutated receptors (IC50 = 1-2 nM). Immunocytochemistry showed that WT and mutated receptors were diffusely distributed, presumably at the cell surface, except for the tailless mutant T458, which displayed striking perinuclear localization. T458 did not display an adenylyl cyclase response to PTH, while the other mutants were similar to WT both with respect to their maximal adenylyl cyclase responses to PTH and to their EC50 values. Cai2+ signaling properties of these mutants were assessed as PTH-stimulated 45Ca efflux from Xenopus oocytes that had been injected with in vitro transcribed PTH/PTHrP receptor cRNAs. The WT and mutated receptors (except for T458) responded to PTH with significant (6- to 27-fold) increases in 45Ca efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology*; D015870:Gene Expression; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010281:Parathyroid Hormone / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000235:genetics*; D015398:Signal Transduction; D014162:Transfection; D014982:Xenopus laevis
D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium