The present studies were done to evaluate the role of the cytoplasmic tail of the G-protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) in the endocytosis of agonist-occupied receptors. PTH/PTHrP receptor mutants progressively truncated from the C terminus were expressed in COS-7 cells, and their ability to internalize I-PTHrP(1-34)amide was determined. Most of the C-terminal tail (91 of 127 residues) could be deleted without affecting internalization. However, further truncation removing residues 475-494 resulted in a 50-60% decrease in ligand internalization. A mutant with an internal deletion of these 20 amino acids showed a similar reduction in internalization, confirming the presence of a positive endocytic signal. No additional positive signals were found in the membrane-proximal region of the tail. However, alanine mutagenesis of the membrane-proximal residues 459-461 (EVQ AAA) resulted in a mutant PTH/PTHrP receptor displaying a 40% increase in ligand endocytosis, indicating that EVQ functions as a negative signal. Treatment of COS-7 cells with hypertonic sucrose (to disrupt clathrin lattices) markedly suppressed (by >80%) PTH/PTHrP receptor internalization. These results demonstrate the presence of both positive and negative endocytic signals in the membrane-proximal cytoplasmic tail of the PTH/PTHrP receptor and suggest that these signals regulate the ability of the receptor to accumulate in clathrin-coated pits.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. H. Turner and J. R. Raymond Interaction of Calmodulin with the Serotonin 5-Hydroxytryptamine2A Receptor: A PUTATIVE REGULATOR OF G PROTEIN COUPLING AND RECEPTOR PHOSPHORYLATION BY PROTEIN KINASE C J. Biol. Chem., September 2, 2005; 280(35): 30741 - 30750. [Abstract] [Full Text] [PDF]

				T. Seck, M. Pellegrini, A. M. Florea, V. Grignoux, R. Baron, D. F. Mierke, and W. C. Horne The {Delta}e13 Isoform of the Calcitonin Receptor Forms a Six-Transmembrane Domain Receptor with Dominant-Negative Effects on Receptor Surface Expression and Signaling Mol. Endocrinol., August 1, 2005; 19(8): 2132 - 2144. [Abstract] [Full Text] [PDF]

				P. Vazquez, I. Roncero, E. Blazquez, and E. Alvarez The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation J. Endocrinol., July 1, 2005; 186(1): 221 - 231. [Abstract] [Full Text] [PDF]

				S. P. Singh, D. McDonald, T. J. Hope, and B. S. Prabhakar Upon Thyrotropin Binding the Thyrotropin Receptor Is Internalized and Localized to Endosome Endocrinology, February 1, 2004; 145(2): 1003 - 1010. [Abstract] [Full Text] [PDF]

				N. M. Fiaschi-Taesch and A. F. Stewart Minireview: Parathyroid Hormone-Related Protein as an Intracrine Factor--Trafficking Mechanisms and Functional Consequences Endocrinology, February 1, 2003; 144(2): 407 - 411. [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]

				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]

				H. A. W. Tawfeek, J. Che, F. Qian, and A. B. Abou-Samra Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E545 - E557. [Abstract] [Full Text] [PDF]

				D. A. McCulloch, E. M. Lutz, M. S. Johnson, D. N. Robertson, C. J. MacKenzie, P. J. Holland, and R. Mitchell ADP-Ribosylation Factor-Dependent Phospholipase D Activation by VPAC Receptors and a PAC1 Receptor Splice Variant Mol. Pharmacol., June 1, 2001; 59(6): 1523 - 1532. [Abstract] [Full Text]

				S. S. G. Ferguson Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling Pharmacol. Rev., March 1, 2001; 53(1): 1 - 24. [Abstract] [Full Text] [PDF]

				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]

				K. N. Pandey, R. Kumar, M. Li, and H. Nguyen Functional Domains and Expression of Truncated Atrial Natriuretic Peptide Receptor-A: The Carboxyl-Terminal Regions Direct the Receptor Internalization and Sequestration in COS-7 Cells Mol. Pharmacol., February 1, 2000; 57(2): 259 - 267. [Abstract] [Full Text]

				K. Nakamura, X. Liu, and M. Ascoli Seven Non-contiguous Intracellular Residues of the Lutropin/Choriogonadotropin Receptor Dictate the Rate of Agonist-induced Internalization and Its Sensitivity to Non-visual Arrestins J. Biol. Chem., January 7, 2000; 275(1): 241 - 247. [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]

				C. Baratti-Elbaz, N. Ghinea, O. Lahuna, H. Loosfelt, C. Pichon, and E. Milgrom Internalization and Recycling Pathways of the Thyrotropin Receptor Mol. Endocrinol., October 1, 1999; 13(10): 1751 - 1765. [Abstract] [Full Text]

				S. Hoare, J. A. Copland, Z. Strakova, K. Ives, Y.-J. Jeng, M. R. Hellmich, and M. S. Soloff The Proximal Portion of the COOH Terminus of the Oxytocin Receptor Is Required for Coupling to Gq, but Not Gi. INDEPENDENT MECHANISMS FOR ELEVATING INTRACELLULAR CALCIUM CONCENTRATIONS FROM INTRACELLULAR STORES J. Biol. Chem., October 1, 1999; 274(40): 28682 - 28689. [Abstract] [Full Text] [PDF]

				K. Nakamura, X. Liu, and M. Ascoli The Rate of Internalization of the Gonadotropin Receptors Is Greatly Affected by the Origin of the Extracellular Domain J. Biol. Chem., September 3, 1999; 274(36): 25426 - 25432. [Abstract] [Full Text] [PDF]

				M. B. Wheeler, R. W. Gelling, S. A. Hinke, B. Tu, R. A. Pederson, F. Lynn, J. Ehses, and C. H. S. McIntosh Characterization of the Carboxyl-terminal Domain of the Rat Glucose-dependent Insulinotropic Polypeptide (GIP) Receptor. A ROLE FOR SERINES 426 AND 427 IN REGULATING THE RATE OF INTERNALIZATION J. Biol. Chem., August 27, 1999; 274(35): 24593 - 24601. [Abstract] [Full Text] [PDF]

				A. M. Cypess, C. G. Unson, C.-R. Wu, and T. P. Sakmar Two Cytoplasmic Loops of the Glucagon Receptor Are Required to Elevate cAMP or Intracellular Calcium J. Biol. Chem., July 2, 1999; 274(27): 19455 - 19464. [Abstract] [Full Text] [PDF]

				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]

				J. Hilpert, A. Nykjaer, C. Jacobsen, G. Wallukat, R. Nielsen, S. K. Moestrup, H. Haller, F. C. Luft, E. I. Christensen, and T. E. Willnow Megalin Antagonizes Activation of the Parathyroid Hormone Receptor J. Biol. Chem., February 26, 1999; 274(9): 5620 - 5625. [Abstract] [Full Text] [PDF]

				G. N. Prado, D. F. Mierke, M. Pellegrini, L. Taylor, and P. Polgar Motif Mutation of Bradykinin B2 Receptor Second Intracellular Loop and Proximal C Terminus Is Critical for Signal Transduction, Internalization, and Resensitization J. Biol. Chem., December 11, 1998; 273(50): 33548 - 33555. [Abstract] [Full Text] [PDF]

				N. Malecz, T. Bambino, M. Bencsik, and R. A. Nissenson Identification of Phosphorylation Sites in the G Protein-Coupled Receptor for Parathyroid Hormone. Receptor Phosphorylation Is Not Required for Agonist-Induced Internalization Mol. Endocrinol., December 1, 1998; 12(12): 1846 - 1856. [Abstract] [Full Text]

				N. Hukovic, R. Panetta, U. Kumar, M. Rocheville, and Y. C. Patel The Cytoplasmic Tail of the Human Somatostatin Receptor Type 5 Is Crucial for Interaction with Adenylyl Cyclase and in Mediating Desensitization and Internalization J. Biol. Chem., August 14, 1998; 273(33): 21416 - 21422. [Abstract] [Full Text] [PDF]

				I. Ishii, E. Saito, T. Izumi, M. Ui, and T. Shimizu Agonist-induced Sequestration, Recycling, and Resensitization of Platelet-activating Factor Receptor. ROLE OF CYTOPLASMIC TAIL PHOSPHORYLATION IN EACH PROCESS J. Biol. Chem., April 17, 1998; 273(16): 9878 - 9885. [Abstract] [Full Text] [PDF]

				X. Lin, J. A. Janovick, S. Brothers, M. Blömenrohr, J. Bogerd, and P. M. Conn Addition of Catfish Gonadotropin-Releasing Hormone (GnRH) Receptor Intracellular Carboxyl-Terminal Tail to Rat GnRH Receptor Alters Receptor Expression and Regulation Mol. Endocrinol., February 1, 1998; 12(2): 161 - 171. [Abstract] [Full Text]

				A. Faussner, D. Proud, M. Towns, and J. M. Bathon Influence of the Cytosolic Carboxyl Termini of Human B1 and B2 Kinin Receptors on Receptor Sequestration, Ligand Internalization, and Signal Transduction J. Biol. Chem., January 30, 1998; 273(5): 2617 - 2623. [Abstract] [Full Text] [PDF]

				A. M. Gabilondo, J. Hegler, C. Krasel, V. Boivin-Jahns, L. Hein, and M. J. Lohse A dileucine motif in the C terminus of the beta 2-adrenergic receptor is involved in receptor internalization PNAS, November 11, 1997; 94(23): 12285 - 12290. [Abstract] [Full Text] [PDF]

				K. K. Arora, Z. Cheng, and K. J. Catt Mutations of the Conserved DRS Motif in the Second Intracellular Loop of the Gonadotropin-Releasing Hormone Receptor Affect Expression, Activation, and Internalization Mol. Endocrinol., August 1, 1997; 11(9): 1203 - 1212. [Abstract] [Full Text]

				Y. Fukushima, T. Asano, K. Takata, M. Funaki, T. Ogihara, M. Anai, K. Tsukuda, T. Saitoh, H. Katagiri, M. Aihara, et al. Role of the C Terminus in Histamine H2 Receptor Signaling, Desensitization, and Agonist-induced Internalization J. Biol. Chem., August 1, 1997; 272(31): 19464 - 19470. [Abstract] [Full Text] [PDF]

				M. Pohl, S. Silvente-Poirot, J. R. Pisegna, N. I. Tarasova, and S. A. Wank Ligand-induced Internalization of Cholecystokinin Receptors. DEMONSTRATION OF THE IMPORTANCE OF THE CARBOXYL TERMINUS FOR LIGAND-INDUCED INTERNALIZATION OF THE RAT CHOLECYSTOKININ TYPE B RECEPTOR BUT NOT THE TYPE A RECEPTOR J. Biol. Chem., July 18, 1997; 272(29): 18179 - 18184. [Abstract] [Full Text] [PDF]

				S. G. Mueller, J. R. White, W. P. Schraw, V. Lam, and A. Richmond Ligand-induced Desensitization of the Human CXC Chemokine Receptor-2 Is Modulated by Multiple Serine Residues in the Carboxyl-terminal Domain of the Receptor J. Biol. Chem., March 28, 1997; 272(13): 8207 - 8214. [Abstract] [Full Text] [PDF]

				A. Subtil, M. Delepierre, and A. Dautry-Varsat An alpha -Helical Signal in the Cytosolic Domain of the Interleukin 2 Receptor beta  Chain Mediates Sorting Towards Degradation after Endocytosis J. Cell Biol., February 10, 1997; 136(3): 583 - 595. [Abstract] [Full Text] [PDF]

				S. K. Bohm, L. M. Khitin, S. P. Smeekens, E. F. Grady, D. G. Payan, and N. W. Bunnett Identification of Potential Tyrosine-containing Endocytic Motifs in the Carboxyl-tail and Seventh Transmembrane Domain of the Neurokinin 1Receptor J. Biol. Chem., January 24, 1997; 272(4): 2363 - 2372. [Abstract] [Full Text] [PDF]

				G. N. Prado, H. Suzuki, N. Wilkinson, B. Cousins, and J. Navarro Role of the C Terminus of the Interleukin 8Receptor in Signal Transduction and Internalization J. Biol. Chem., August 9, 1996; 271(32): 19186 - 19190. [Abstract] [Full Text] [PDF]

				M. J. Smit, E. Roovers, H. Timmerman, Y. van de Vrede, A. E. Alewijnse, and R. Leurs Two Distinct Pathways for Histamine H(2) Receptor Down-regulation J. Biol. Chem., March 29, 1996; 271(13): 7574 - 7582. [Abstract] [Full Text] [PDF]

				R. Pals-Rylaarsdam, Y. Xu, P. Witt-Enderby, J. L. Benovic, and M. M. Hosey Desensitization and Internalization of the m2 Muscarinic Acetylcholine Receptor Are Directed by Independent Mechanisms J. Biol. Chem., December 1, 1995; 270(48): 29004 - 29011. [Abstract] [Full Text] [PDF]

				S. S. G. Ferguson, L. Ménard, L. S. Barak, W. J. Koch, A.-M. Colapietro, and M. G. Caron Role of Phosphorylation in Agonist-promoted beta(2)-Adrenergic Receptor Sequestration J. Biol. Chem., October 20, 1995; 270(42): 24782 - 24789. [Abstract] [Full Text] [PDF]

				W. G. Thomas, K. M. Baker, T. J. Motel, and T. J. Thekkumkara Angiotensin II Receptor Endocytosis Involves Two Distinct Regions of the Cytoplasmic Tail J. Biol. Chem., September 22, 1995; 270(38): 22153 - 22159. [Abstract] [Full Text] [PDF]

				Lás. Hunyady, Már. Bor, A. J. Baukal, T.ás Balla, and K. J. Catt A Conserved NPLFY Sequence Contributes to Agonist Binding and Signal Transduction but Is Not an Internalization Signal for the Type 1 Angiotensin II Receptor J. Biol. Chem., July 14, 1995; 270(28): 16602 - 16609. [Abstract] [Full Text] [PDF]

				R.-M. Lyu, P. M. Germano, J. K. Choi, S. V. Le, and J. R. Pisegna Identification of an Essential Amino Acid Motif within the C Terminus of the Pituitary Adenylate Cyclase-activating Polypeptide Type I Receptor That Is Critical for Signal Transduction but Not for Receptor Internalization J. Biol. Chem., November 10, 2000; 275(46): 36134 - 36142. [Abstract] [Full Text] [PDF]

				R. El Kouhen, A. L. Burd, L. J. Erickson-Herbrandson, C.-Y. Chang, P.-Y. Law, and H. H. Loh Phosphorylation of Ser363, Thr370, and Ser375 Residues within the Carboxyl Tail Differentially Regulates {micro}-Opioid Receptor Internalization J. Biol. Chem., April 13, 2001; 276(16): 12774 - 12780. [Abstract] [Full Text] [PDF]