This Article Full Text Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malecz, N. Articles by Nissenson, R. A. Search for Related Content PubMed PubMed Citation Articles by Malecz, N. Articles by Nissenson, R. A.

Identification of Phosphorylation Sites in the G Protein-Coupled Receptor for Parathyroid Hormone. Receptor Phosphorylation Is Not Required for Agonist-Induced Internalization

Endocrine Research Unit Veterans Administration Medical Center and the Departments of Medicine and Physiology University of California San Francisco, California 94121

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.

This article has been cited by other articles:

				S. U. Miedlich and A. B. Abou-Samra Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E665 - E671. [Abstract] [Full Text] [PDF]

				F. Wang, X. Chen, X. Zhang, and L. Ma Phosphorylation State of {micro}-Opioid Receptor Determines the Alternative Recycling of Receptor via Rab4 or Rab11 Pathway Mol. Endocrinol., August 1, 2008; 22(8): 1881 - 1892. [Abstract] [Full Text] [PDF]

				B. Wang, A. Bisello, Y. Yang, G. G. Romero, and P. A. Friedman NHERF1 Regulates Parathyroid Hormone Receptor Membrane Retention without Affecting Recycling J. Biol. Chem., December 14, 2007; 282(50): 36214 - 36222. [Abstract] [Full Text] [PDF]

				G. S. Bounoutas, H. Tawfeek, L. F. Frohlich, U.-i. Chung, and A. B. Abou-Samra Impact of Impaired Receptor Internalization on Calcium Homeostasis in Knock-In Mice Expressing a Phosphorylation-Deficient Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Endocrinology, October 1, 2006; 147(10): 4674 - 4679. [Abstract] [Full Text] [PDF]

				Y. Ueda, N. F. Neel, E. Schutyser, D. Raman, and A. Richmond Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells Cancer Res., June 1, 2006; 66(11): 5665 - 5675. [Abstract] [Full Text] [PDF]

				K. A. Neve Novel Features of G Protein-Coupled Receptor Kinase 4 Mol. Pharmacol., March 1, 2006; 69(3): 673 - 676. [Abstract] [Full Text] [PDF]

				G. Barthet, F. Gaven, B. Framery, K. Shinjo, T. Nakamura, S. Claeysen, J. Bockaert, and A. Dumuis Uncoupling and Endocytosis of 5-Hydroxytryptamine 4 Receptors: DISTINCT MOLECULAR EVENTS WITH DIFFERENT GRK2 REQUIREMENTS J. Biol. Chem., July 29, 2005; 280(30): 27924 - 27934. [Abstract] [Full Text] [PDF]

				P.-Q. Liu, S. Tan, M. C. Mendel, R. J. Murrills, B. M. Bhat, B. Schlag, R. Samuel, J. J. Matteo, R. de la Rosa, K. Howes, et al. Isogenic Human Cell Lines for Drug Discovery: Regulation of Target Gene Expression by Engineered Zinc-Finger Protein Transcription Factors J Biomol Screen, June 1, 2005; 10(4): 304 - 313. [Abstract] [PDF]

				L. Wang, S. Liu, L. D. Quarles, and R. F. Spurney Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E826 - E834. [Abstract] [Full Text] [PDF]

				R. Jorgensen, L. Martini, T. W. Schwartz, and C. E. Elling Characterization of Glucagon-Like Peptide-1 Receptor {beta}-Arrestin 2 Interaction: A High-Affinity Receptor Phenotype Mol. Endocrinol., March 1, 2005; 19(3): 812 - 823. [Abstract] [Full Text] [PDF]

				T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]

				J. L. Estall, B. Yusta, and D. J. Drucker Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization Mol. Biol. Cell, August 1, 2004; 15(8): 3673 - 3687. [Abstract] [Full Text] [PDF]

				W. B. Sneddon, C. E. Magyar, G. E. Willick, C. A. Syme, F. Galbiati, A. Bisello, and P. A. Friedman Ligand-Selective Dissociation of Activation and Internalization of the Parathyroid Hormone (PTH) Receptor: Conditional Efficacy of PTH Peptide Fragments Endocrinology, June 1, 2004; 145(6): 2815 - 2823. [Abstract] [Full Text] [PDF]

				W. B. Sneddon, C. A. Syme, A. Bisello, C. E. Magyar, M. D. Rochdi, J.-L. Parent, E. J. Weinman, A. B. Abou-Samra, and P. A. Friedman Activation-independent Parathyroid Hormone Receptor Internalization Is Regulated by NHERF1 (EBP50) J. Biol. Chem., October 31, 2003; 278(44): 43787 - 43796. [Abstract] [Full Text] [PDF]

				K. Tumova, R. M. Iwasiow, and M. Tiberi Insight into the Mechanism of Dopamine D1-like Receptor Activation. EVIDENCE FOR A MOLECULAR INTERPLAY BETWEEN THE THIRD EXTRACELLULAR LOOP AND THE CYTOPLASMIC TAIL J. Biol. Chem., February 28, 2003; 278(10): 8146 - 8153. [Abstract] [Full Text] [PDF]

				J. Humrich, C. Bermel, T. Grubel, U. Quitterer, and M. J. Lohse Regulation of Phosducin-like Protein by Casein Kinase 2 and N-terminal Splicing J. Biol. Chem., February 7, 2003; 278(7): 4474 - 4481. [Abstract] [Full Text] [PDF]

				S. Chauvin, M. Bencsik, T. Bambino, and R. A. Nissenson Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and {beta}-Arrestin Mol. Endocrinol., December 1, 2002; 16(12): 2720 - 2732. [Abstract] [Full Text] [PDF]

				X. Chen, J.-C. Dai, and E. M. Greenfield Termination of immediate-early gene expression after stimulation by parathyroid hormone or isoproterenol Am J Physiol Cell Physiol, November 1, 2002; 283(5): C1432 - C1440. [Abstract] [Full Text] [PDF]

				A. Bisello, M. Chorev, M. Rosenblatt, L. Monticelli, D. F. Mierke, and S. L. Ferrari Selective Ligand-induced Stabilization of Active and Desensitized Parathyroid Hormone Type 1 Receptor Conformations J. Biol. Chem., October 4, 2002; 277(41): 38524 - 38530. [Abstract] [Full Text] [PDF]

				M. Castro, F. Dicker, J.-P. Vilardaga, C. Krasel, M. Bernhardt, and M. J. Lohse Dual Regulation of the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Signaling by Protein Kinase C and {beta}-Arrestins Endocrinology, October 1, 2002; 143(10): 3854 - 3865. [Abstract] [Full Text] [PDF]

				J. N. Mason, L. B. Kozell, and K. A. Neve Regulation of Dopamine D1 Receptor Trafficking by Protein Kinase A-Dependent Phosphorylation Mol. Pharmacol., April 1, 2002; 61(4): 806 - 816. [Abstract] [Full Text] [PDF]

				J.-P. Vilardaga, C. Krasel, S. Chauvin, T. Bambino, M. J. Lohse, and R. A. Nissenson Internalization Determinants of the Parathyroid Hormone Receptor Differentially Regulate beta -Arrestin/Receptor Association J. Biol. Chem., March 1, 2002; 277(10): 8121 - 8129. [Abstract] [Full Text] [PDF]

				H. A. W. Tawfeek, F. Qian, and A. B. Abou-Samra Phosphorylation of the Receptor for PTH and PTHrP Is Required for Internalization and Regulates Receptor Signaling Mol. Endocrinol., January 1, 2002; 16(1): 1 - 13. [Abstract] [Full Text] [PDF]

				R. Horikawa, B. D. Gaylinn, C. E. Lyons Jr., and M. O. Thorner Molecular Cloning of Ovine and Bovine Growth Hormone-Releasing Hormone Receptors: The Ovine Receptor Is C-Terminally Truncated Endocrinology, June 1, 2001; 142(6): 2660 - 2668. [Abstract] [Full Text] [PDF]

				S. Desai, H. April, C. Nwaneshiudu, and B. Ashby Comparison of Agonist-Induced Internalization of the Human EP2 and EP4 Prostaglandin Receptors: Role of the Carboxyl Terminus in EP4 Receptor Sequestration Mol. Pharmacol., April 13, 2001; 58(6): 1279 - 1286. [Abstract] [Full Text]

				P. H. Carter, B. D. Petroni, R. C. Gensure, E. Schipani, J. T. Potts Jr., and T. J. Gardella Selective and Nonselective Inverse Agonists for Constitutively Active Type-1 Parathyroid Hormone Receptors: Evidence for Altered Receptor Conformations Endocrinology, April 1, 2001; 142(4): 1534 - 1545. [Abstract] [Full Text]

				S. L. Ferrari and A. Bisello Cellular Distribution of Constitutively Active Mutant Parathyroid Hormone (PTH)/PTH-Related Protein Receptors and Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling by {beta}-Arrestin2 Mol. Endocrinol., January 1, 2001; 15(1): 149 - 163. [Abstract] [Full Text]

				L. Min and M. Ascoli Effect of Activating and Inactivating Mutations on the Phosphorylation and Trafficking of the Human Lutropin/Choriogonadotropin Receptor Mol. Endocrinol., November 1, 2000; 14(11): 1797 - 1810. [Abstract] [Full Text]

				J. A. Olivares-Reyes, S. Jayadev, L. Hunyady, K. J. Catt, and R. D. Smith Homologous and Heterologous Phosphorylation of the AT2 Angiotensin Receptor by Protein Kinase C Mol. Pharmacol., November 1, 2000; 58(5): 1156 - 1161. [Abstract] [Full Text]

				A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]

				C.-C. Tseng and X.-Y. Zhang Role of G Protein-Coupled Receptor Kinases in Glucose-Dependent Insulinotropic Polypeptide Receptor Signaling Endocrinology, March 1, 2000; 141(3): 947 - 952. [Abstract] [Full Text] [PDF]

				M. Mannstadt, H. Juppner, and T. J. Gardella Receptors for PTH and PTHrP: their biological importance and functional properties Am J Physiol Renal Physiol, November 1, 1999; 277(5): F665 - F675. [Abstract] [Full Text] [PDF]

				S. L. Ferrari, V. Behar, M. Chorev, M. Rosenblatt, and A. Bisello Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY J. Biol. Chem., October 15, 1999; 274(42): 29968 - 29975. [Abstract] [Full Text] [PDF]

				M. d. F. M. Lazari, X. Liu, K. Nakamura, J. L. Benovic, and M. Ascoli Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor Mol. Endocrinol., June 1, 1999; 13(6): 866 - 878. [Abstract] [Full Text]

				Z. Huang, T. Bambino, Y. Chen, J. Lameh, and R. A. Nissenson Role of Signal Transduction in Internalization of the G Protein-Coupled Receptor for Parathyroid Hormone (PTH) and PTH-Related Protein Endocrinology, March 1, 1999; 140(3): 1294 - 1300. [Abstract] [Full Text]

				K. Berrada, C. L. Plesnicher, X. Luo, and M. Thibonnier Dynamic Interaction of Human Vasopressin/Oxytocin Receptor Subtypes with G Protein-coupled Receptor Kinases and Protein Kinase C after Agonist Stimulation J. Biol. Chem., August 25, 2000; 275(35): 27229 - 27237. [Abstract] [Full Text] [PDF]

				A. C. Hanyaloglu, M. Vrecl, K. M. Kroeger, L. E. C. Miles, H. Qian, W. G. Thomas, and K. A. Eidne Casein Kinase II Sites in the Intracellular C-terminal Domain of the Thyrotropin-releasing Hormone Receptor and Chimeric Gonadotropin-releasing Hormone Receptors Contribute to beta -Arrestin-dependent Internalization J. Biol. Chem., May 18, 2001; 276(21): 18066 - 18074. [Abstract] [Full Text] [PDF]

				S. Li, X. Liu, L. Min, and M. Ascoli Mutations of the Second Extracellular Loop of the Human Lutropin Receptor Emphasize the Importance of Receptor Activation and De-emphasize the Importance of Receptor Phosphorylation in Agonist-induced Internalization J. Biol. Chem., March 9, 2001; 276(11): 7968 - 7973. [Abstract] [Full Text] [PDF]

				J.-P. Vilardaga, M. Frank, C. Krasel, C. Dees, R. A. Nissenson, and M. J. Lohse Differential Conformational Requirements for Activation of G Proteins and the Regulatory Proteins Arrestin and G Protein-coupled Receptor Kinase in the G Protein-coupled Receptor for Parathyroid Hormone (PTH)/PTH-related Protein J. Biol. Chem., August 31, 2001; 276(36): 33435 - 33443. [Abstract] [Full Text] [PDF]

				J. A. Olivares-Reyes, R. D. Smith, L. Hunyady, B. H. Shah, and K. J. Catt Agonist-induced Signaling, Desensitization, and Internalization of a Phosphorylation-deficient AT1A Angiotensin Receptor J. Biol. Chem., October 5, 2001; 276(41): 37761 - 37768. [Abstract] [Full Text] [PDF]