CaRs sense changes in the [Ca2+] and activate downstream effectors. Evidence indicates that the expression of adequate numbers of CaRs is necessary for precise Ca2+-sensing in tissues like the parathyroid and kidney. Patients heterozygous for 1 abnormal CaR gene have mild hypercalcemia and hypocalciuria, while homozygotes are severely affected with life-threatening hypercalcemia and hyperparathyroidism supporting a gene-dosage effect. CaR protein expression is reduced in parathyroid glands from patients with primary hyperparathyroidism. Either excessive receptor degradation or reduced biosynthesis could explain this finding. Desensitization and downregulation of receptors are mechanisms for regulating target cell responses to agonists. Internalization often accompanies these events and serves to remove active signaling receptors from the cell surface. We found that treating parathyroid cells and Xenopus oocytes expressing CaR cRNA with protein kinase C agonists desensitizes these cells to subsequent challenge with high [Ca2+] -- in terms of parathyroid hormone (PTH) release and high [Ca2+] -regulated signal transduction. We will test the hypothesis that CaR agonists promote receptor desensitization and internalization; that sequences in the CaR's carboxy-terminal tail are involved in these pathways; and that levels of CaR protein expression are critical in determining cellular responses to [Ca2+] . Three aims will address the hypothesis. (1) We will determine whether CaR agonists promote desensitization and internalization of CaRs by assessing PTH secretion and signaling responses after treating cells with agonists and by quantifying internalization of CaRs using a surface biotinylation assay. Intracellular trafficking of CaRs will be followed in HEK-293 cells expressing green fluorescent protein-tagged CaRs by confocal microscopy. (2) We will assess the role of the CaR's C-terminal tail in desensitization and internalization using tail-truncation mutants and site-directed mutagenesis to identify putative positive or negative endocytic motifs. The role of intact signaling pathways in internalization will be addressed. (3) Internalization-defective mutants will be expressed in fibroblasts and calcitonin-secreting cells and the effects on high (Ca2+] -regulated calcitonin secretion and cell proliferation will be tested. Our studies should provide insights into mechanisms underlying CaR responsiveness in secretion and growth.