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 [I-125]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 EC(50) values. Ca-I(2+) signaling properties of these mutants were assessed as PTH-stimulated Ca-45 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 Ca-45 efflux. These results demonstrate that the cytoplasmic tail of the PTH/PTHrP receptor is not required for ligand binding or adenylyl cyclase or Ca-I(2+) signaling but is essential for the proper cell surface localization and steady state expression of the receptor.