Molecular Endocrinology, Vol 6, 327-336, Copyright © 1992 by Endocrine Society

ARTICLES

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization

MC Rodriguez, YB Xie, H Wang, K Collison and DL Segaloff
Department of Physiology, University of Iowa College of Medicine, Iowa City 52242.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG- stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.

This article has been cited by other articles:

				D. Carrel, M. Hamon, and M. Darmon Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting J. Cell Sci., October 15, 2006; 119(20): 4276 - 4284. [Abstract] [Full Text] [PDF]

				A. M. Navratil, T. A. Farmerie, J. Bogerd, T. M. Nett, and C. M. Clay Differential Impact of Intracellular Carboxyl Terminal Domains on Lipid Raft Localization of the Murine Gonadotropin-Releasing Hormone Receptor Biol Reprod, May 1, 2006; 74(5): 788 - 797. [Abstract] [Full Text] [PDF]

				J. Robert, E. Clauser, P. X. Petit, and M. A. Ventura A Novel C-terminal Motif Is Necessary for the Export of the Vasopressin V1b/V3 Receptor to the Plasma Membrane J. Biol. Chem., January 21, 2005; 280(3): 2300 - 2308. [Abstract] [Full Text] [PDF]

				M. T. Duvernay, F. Zhou, and G. Wu A Conserved Motif for the Transport of G Protein-coupled Receptors from the Endoplasmic Reticulum to the Cell Surface J. Biol. Chem., July 16, 2004; 279(29): 30741 - 30750. [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]

				T. Okuno, H. Ago, K. Terawaki, M. Miyano, T. Shimizu, and T. Yokomizo Helix 8 of the Leukotriene B4 Receptor Is Required for the Conformational Change to the Low Affinity State after G-protein Activation J. Biol. Chem., October 17, 2003; 278(42): 41500 - 41509. [Abstract] [Full Text] [PDF]

				Y. Sun and M. C. Gershengorn Correlation between Basal Signaling and Internalization of Thyrotropin-Releasing Hormone Receptors: Evidence for Involvement of Similar Receptor Conformations Endocrinology, August 1, 2002; 143(8): 2886 - 2892. [Abstract] [Full Text] [PDF]

				Z. Chen, D. J. Dupre, C. Le Gouill, M. Rola-Pleszczynski, and J. Stankova Agonist-induced Internalization of the Platelet-activating Factor Receptor Is Dependent on Arrestins but Independent of G-protein Activation. ROLE OF THE C TERMINUS AND THE (D/N)PXXY MOTIF J. Biol. Chem., February 22, 2002; 277(9): 7356 - 7362. [Abstract] [Full Text] [PDF]

				J. C. Bermak and Q.-Y. Zhou Accessory Proteins in the Biogenesis of G Protein-Coupled Receptors Mol. Interv., December 1, 2001; 1(5): 282 - 287. [Abstract] [Full Text] [PDF]

				T. Hamalainen, M. Poutanen, and I. Huhtaniemi Promoter Function of Different Lengths of the Murine Luteinizing Hormone Receptor Gene 5'-Flanking Region in Transfected Gonadal Cells and in Transgenic Mice Endocrinology, June 1, 2001; 142(6): 2427 - 2434. [Abstract] [Full Text] [PDF]

				M. Kishi and M. Ascoli The C-Terminal Tail of the Rat Lutropin/Choriogonadotropin (CG) Receptor Independently Modulates Human (h)CG-Induced Internalization of the Cell Surface Receptor and the Lysosomal Targeting of the Internalized hCG-Receptor Complex Mol. Endocrinol., June 1, 2000; 14(6): 926 - 936. [Abstract] [Full Text]

				K. Kristiansen, W. K. Kroeze, D. L. Willins, E. I. Gelber, J. E. Savage, R. A. Glennon, and B. L. Roth A Highly Conserved Aspartic Acid (Asp-155) Anchors the Terminal Amine Moiety of Tryptamines and Is Involved in Membrane Targeting of the 5-HT2A Serotonin Receptor But Does Not Participate in Activation via a "Salt-Bridge Disruption" Mechanism J. Pharmacol. Exp. Ther., June 1, 2000; 293(3): 735 - 746. [Abstract] [Full Text]

				S. Li, X. Liu, and M. Ascoli p38JAB1 Binds to the Intracellular Precursor of the Lutropin/Choriogonadotropin Receptor and Promotes Its Degradation J. Biol. Chem., April 28, 2000; 275(18): 13386 - 13393. [Abstract] [Full Text] [PDF]

				G. Ferguson, K. R. Watterson, and T. M. Palmer Subtype-Specific Kinetics of Inhibitory Adenosine Receptor Internalization Are Determined by Sensitivity to Phosphorylation by G Protein-Coupled Receptor Kinases Mol. Pharmacol., March 1, 2000; 57(3): 546 - 552. [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]

				K. Nakamura and M. Ascoli A Dileucine-Based Motif in the C-Terminal Tail of the Lutropin/Choriogonadotropin Receptor Inhibits Endocytosis of the Agonist-Receptor Complex Mol. Pharmacol., October 1, 1999; 56(4): 728 - 736. [Abstract] [Full Text]

				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]

				K. Nakamura, M. d. F. M. Lazari, S. Li, C. Korgaonkar, and M. Ascoli Role of the Rate of Internalization of the Agonist-Receptor Complex on the Agonist-Induced Down-Regulation of the Lutropin/ Choriogonadotropin Receptor Mol. Endocrinol., August 1, 1999; 13(8): 1295 - 1304. [Abstract] [Full Text]

				J.-L. Parent, P. Labrecque, M. J. Orsini, and J. L. Benovic Internalization of the TXA2 Receptor alpha  and beta  Isoforms. ROLE OF THE DIFFERENTIALLY SPLICED COOH TERMINUS IN AGONIST-PROMOTED RECEPTOR INTERNALIZATION J. Biol. Chem., March 26, 1999; 274(13): 8941 - 8948. [Abstract] [Full Text] [PDF]

				R. Schülein, R. Hermosilla, A. Oksche, M. Dehe, B. Wiesner, G. Krause, and W. Rosenthal A Dileucine Sequence and an Upstream Glutamate Residue in the Intracellular Carboxyl Terminus of the Vasopressin V2 Receptor Are Essential for Cell Surface Transport in COS.M6 Cells Mol. Pharmacol., September 1, 1998; 54(3): 525 - 535. [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]

				E. Fire, C. M. Brown, M. G. Roth, Y. I. Henis, and N. O. Petersen Partitioning of Proteins into Plasma Membrane Microdomains. CLUSTERING OF MUTANT INFLUENZA VIRUS HEMAGGLUTININS INTO COATED PITS DEPENDS ON THE STRENGTH OF THE INTERNALIZATION SIGNAL J. Biol. Chem., November 21, 1997; 272(47): 29538 - 29545. [Abstract] [Full Text] [PDF]

				C. Le Gouill, J.-L. Parent, M. Rola-Pleszczynski, and J. Stankova Structural and Functional Requirements for Agonist-induced Internalization of the Human Platelet-activating Factor Receptor J. Biol. Chem., August 22, 1997; 272(34): 21289 - 21295. [Abstract] [Full Text] [PDF]

				D. Bayle, D. Weeks, and G. Sachs Identification of Membrane Insertion Sequences of the Rabbit Gastric Cholecystokinin-A Receptor by in Vitro Translation J. Biol. Chem., August 8, 1997; 272(32): 19697 - 19707. [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]

				D. P. Davis, T. G. Rozell, X. Liu, and D. L. Segaloff The Six N-linked Carbohydrates of the Lutropin/Choriogonadotropin Receptor Are Not Absolutely Required for Correct Folding, Cell Surface Expression, Hormone Binding, or Signal Transduction Mol. Endocrinol., May 1, 1997; 11(5): 550 - 562. [Abstract] [Full Text]

				Z. Wang, X. Liu, and M. Ascoli Phosphorylation of the Lutropin/Choriogonadotropin Receptor Facilitates Uncoupling of the Receptor from Adenylyl Cyclase and Endocytosis of the Bound Hormone Mol. Endocrinol., February 1, 1997; 11(2): 183 - 192. [Abstract] [Full Text]

				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]

				C. A. McArdle, G. B. Willars, R. C. Fowkes, S. R. Nahorski, J. S. Davidson, and W. Forrest-Owen Desensitization of Gonadotropin-releasing Hormone Action in alpha T3-1 Cells Due to Uncoupling of Inositol 1,4,5-Trisphosphate Generation and Ca2+ Mobilization J. Biol. Chem., September 27, 1996; 271(39): 23711 - 23717. [Abstract] [Full Text] [PDF]

				R. T. Premont, A. D. Macrae, R. H. Stoffel, N. Chung, J. A. Pitcher, C. Ambrose, J. Inglese, M. E. MacDonald, and R. J. Lefkowitz Characterization of the G Protein-coupled Receptor Kinase GRK4 J. Biol. Chem., March 15, 1996; 271(11): 6403 - 6410. [Abstract] [Full Text] [PDF]

				A. Abell, X. Liu, and D. L. Segaloff Deletions of Portions of the Extracellular Loops of the Lutropin/Choriogonadotropin Receptor Decrease the Binding Affinity for Ovine Luteinizing Hormone, but Not Human Choriogonadotropin, by Preventing the Formation of Mature Cell Surface Receptor J. Biol. Chem., February 23, 1996; 271(8): 4518 - 4527. [Abstract] [Full Text] [PDF]

				A. C. Latronico, J. Anasti, I. J.P. Arnhold, R. Rapaport, B. B. Mendonca, W. Bloise, M. Castro, C. Tsigos, and G. P. Chrousos Testicular and Ovarian Resistance to Luteinizing Hormone Caused by Inactivating Mutations of the Luteinizing Hormone-Receptor Gene N. Engl. J. Med., February 22, 1996; 334(8): 507 - 512. [Full Text] [PDF]

				R. W. Hipkin, X. Liu, and M. Ascoli Truncation of the C-terminal Tail of the Follitropin Receptor Does Not Impair the Agonist- or Phorbol Ester-induced Receptor Phosphorylation and Uncoupling J. Biol. Chem., November 3, 1995; 270(44): 26683 - 26689. [Abstract] [Full Text] [PDF]

				K. K. Arora, A. Sakai, and K. J. Catt Effects of Second Intracellular Loop Mutations on Signal Transduction and Internalization of the Gonadotropin-releasing Hormone Receptor J. Biol. Chem., September 29, 1995; 270(39): 22820 - 22826. [Abstract] [Full Text] [PDF]

				M.-J. Tseng, K. Detjen, V. Struk, and C. D. Logsdon Carboxyl-terminal Domains Determine Internalization and Recycling Characteristics of Bombesin Receptor Chimeras J. Biol. Chem., August 11, 1995; 270(32): 18858 - 18864. [Abstract] [Full Text] [PDF]

				M.-J. Tseng, S. Coon, E. Stuenkel, V. Struk, and C. D. Logsdon Influence of Second and Third Cytoplasmic Loops on Binding, Internalization, and Coupling of Chimeric Bombesin/m3 Muscarinic Receptors J. Biol. Chem., July 28, 1995; 270(30): 17884 - 17891. [Abstract] [Full Text] [PDF]

				N. Matus-Leibovitch, D. R. Nussenzveig, M. C. Gershengorn, and Y. Oron Truncation of the Thyrotropin-releasing Hormone Receptor Carboxyl Tail Causes Constitutive Activity and Leads to Impaired Responsiveness in Xenopus Oocytes and AtT20 Cells J. Biol. Chem., January 20, 1995; 270(3): 1041 - 1047. [Abstract] [Full Text] [PDF]

				Z. Huang, Y. Chen, and R. A. Nissenson The Cytoplasmic Tail of the G-protein-coupled Receptor for Parathyroid Hormone and Parathyroid Hormone-related Protein Contains Positive and Negative Signals for Endocytosis J. Biol. Chem., January 6, 1995; 270(1): 151 - 156. [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]