title: Ligand-selective dissociation of activation and internalization of the parathyroid hormone (PTH) receptor: conditional efficacy of PTH peptide fragments.
abstract: G protein-coupled receptors (GPCRs) mediate the action of many hormones, cytokines, and sensory and chemical signals. It is generally thought that receptor desensitization and internalization require occupancy and activation of the GPCR. PTH and PTHrP receptor (PTH1R) belongs to GPCR class B and is the major regulator of extracellular calcium homeostasis. Using kidney distal convoluted tubule cells transfected with a human PTH1R/enhanced green fluorescent protein fusion protein, quantitative, real-time fluorescence microscopy was used to analyze receptor internalization. In these cells, which are the target of the calcium-sparing action of PTH, PTH(1-34) activated adenylyl cyclase (AC) and phospholipase C (PLC) and PTH1R endocytosis. PTH(1-31), however, stimulated AC and PLC but not PTH1R endocytosis. Conversely, PTH(7-34) rapidly stimulated PTH1R internalization without activating AC or PLC. PTH(2-34) and (3-34) caused PTH1R internalization intermediate between PTH(1-34) and (7-34). PTH1R sequestration occurred in a dynamin- and clathrin-dependent manner. Directly activating AC inhibited PTH1R internalization in response to PTH(7-34). PTH1R endocytosis was sensitive to protein kinase C inhibition. PTH(1-34), (7-34), and (1-31) evoked PTH1R phosphorylation. Removal of most of the C terminus of the PTH1R eliminated receptor phosphorylation and the cAMP/protein kinase C sensitivity of internalization. PTH(1-34) and (7-34) internalized the truncated PTH1R with identical kinetics, and the response was unaffected by forskolin. Thus, the PTH1R C terminus contains regulatory sequences that are involved in, but not required for, PTH1R internalization. The results demonstrate that receptor activation and internalization can be selectively dissociated.
authors.affiliation: University of Pittsburgh School of Medicine, Department of Pharmacology, E-1347 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: W Bruce
authors.initials: WB
authors.lastname: Sneddon
authors.forename: Clara E
authors.initials: CE
authors.lastname: Magyar
authors.forename: Gordon E
authors.initials: GE
authors.lastname: Willick
authors.forename: Colin A
authors.initials: CA
authors.lastname: Syme
authors.forename: Ferruccio
authors.initials: F
authors.lastname: Galbiati
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bisello
authors.forename: Peter A
authors.initials: PA
authors.lastname: Friedman
mesh_terms: D000818:Animals; D021941:Caveolae / Q000502:physiology; D002478:Cells, Cultured; D002966:Clathrin / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism; D008024:Ligands; D051379:Mice; D010281:Parathyroid Hormone / Q000737:chemistry; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000502:physiology; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000378:metabolism / Q000502:physiology*; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction
chemical_list: D002966:Clathrin; D008024:Ligands; C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D010770:Phosphotransferases