The lutropin/choriogonadotropin receptor (LH/CG-R) contains a relatively large extracellular domain, in addition to the seven transmembrane helices (TMH), three extracellular loops (ECL), and three intracellular loops typical of G protein-coupled receptors. While high affinity ligand binding has been attributed to the N-terminal extracellular domain, there is evidence that portions of the three ECLs may function in ligand binding and transmembrane signaling. We have investigated the role of several ionizable amino acid residues of rat LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP production. COS-7 cells were transfected with the pSVL expression vector containing cDNAs of either wild-type or mutant rat LH/CG-R. Several point mutants of Lys, located at the interface of ECL III and TMH VII, bound hCG like wild-type receptor but exhibited greatly diminished ligand-mediated signaling. Neither the point mutant, Lys Asp (ECL I), nor the double mutant, Asp Lys/Lys Asp (ECLs I and III, respectively), showed significant hCG binding to intact cells; in detergent-solubilized cells, only the double mutant bound hCG. The mutants Arg Glu (interface of the extracellular domain and TMH I) and Lys Glu (ECL II) proved to be similar to wild-type receptor in binding and signaling. Our results establish that Lys is important in signaling but not ligand binding. Its location on the opposite side of the membrane from G precludes a direct interaction, thus emphasizing the importance of a conformational change in the receptor and suggesting that ligand binding to receptor and ligand-mediated receptor activation are dissociable phenomena.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				G. Kleinau and G. Krause Thyrotropin and Homologous Glycoprotein Hormone Receptors: Structural and Functional Aspects of Extracellular Signaling Mechanisms Endocr. Rev., April 1, 2009; 30(2): 133 - 151. [Abstract] [Full Text] [PDF]

				K. D. G. Pfleger, A. J. Pawson, and R. P. Millar Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations Endocrinology, June 1, 2008; 149(6): 3118 - 3129. [Abstract] [Full Text] [PDF]

				S. M. L. Smith, Y. Lei, J. Liu, M. E. Cahill, G. M. Hagen, B. G. Barisas, and D. A. Roess Luteinizing Hormone Receptors Translocate to Plasma Membrane Microdomains after Binding of Human Chorionic Gonadotropin Endocrinology, April 1, 2006; 147(4): 1789 - 1795. [Abstract] [Full Text] [PDF]

				H. Jaeschke, S. Neumann, G. Kleinau, S. Mueller, M. Claus, G. Krause, and R. Paschke An Aromatic Environment in the Vicinity of Serine 281 Is a Structural Requirement for Thyrotropin Receptor Function Endocrinology, April 1, 2006; 147(4): 1753 - 1760. [Abstract] [Full Text] [PDF]

				M. Claus, H. Jaeschke, G. Kleinau, S. Neumann, G. Krause, and R. Paschke A Hydrophobic Cluster in the Center of the Third Extracellular Loop Is Important for Thyrotropin Receptor Signaling Endocrinology, December 1, 2005; 146(12): 5197 - 5203. [Abstract] [Full Text] [PDF]

				B. Karges, S. Gidenne, C. Aumas, F. Haddad, P. A. Kelly, E. Milgrom, and N. de Roux Zero-Length Cross-Linking Reveals that Tight Interactions between the Extracellular and Transmembrane Domains of the Luteinizing Hormone Receptor Persist during Receptor Activation Mol. Endocrinol., August 1, 2005; 19(8): 2086 - 2098. [Abstract] [Full Text] [PDF]

				D. A. Roess and S. M.L. Smith Self-Association and Raft Localization of Functional Luteinizing Hormone Receptors Biol Reprod, December 1, 2003; 69(6): 1765 - 1770. [Abstract] [Full Text] [PDF]

				M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]

				H. Shinozaki, F. Fanelli, X. Liu, J. Jaquette, K. Nakamura, and D. L. Segaloff Pleiotropic Effects of Substitutions of a Highly Conserved Leucine in Transmembrane Helix III of the Human Lutropin/Choriogonadotropin Receptor with Respect to Constitutive Activation and Hormone Responsiveness Mol. Endocrinol., June 1, 2001; 15(6): 972 - 984. [Abstract] [Full Text] [PDF]

				D. A. Roess, R. D. Horvat, H. Munnelly, and B. G. Barisas Luteinizing Hormone Receptors Are Self-Associated in the Plasma Membrane Endocrinology, December 1, 2000; 141(12): 4518 - 4523. [Abstract] [Full Text] [PDF]

				K. Angelova, P. Narayan, J. P. Simon, and D. Puett Functional Role of Transmembrane Helix 7 in the Activation of the Heptahelical Lutropin Receptor Mol. Endocrinol., April 1, 2000; 14(4): 459 - 471. [Abstract] [Full Text]

				H. Biebermann, T. Schöneberg, A. Schulz, G. Krause, A. Grüters, G. Schultz, and T. Gudermann A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling FASEB J, November 1, 1998; 12(14): 1461 - 1471. [Abstract] [Full Text]

				N. Bhowmick, P. Narayan, and D. Puett The Endothelin Subtype A Receptor Undergoes Agonist- and Antagonist-Mediated Internalization in the Absence of Signaling Endocrinology, July 1, 1998; 139(7): 3185 - 3192. [Abstract] [Full Text] [PDF]

				T. Okamoto, N. Sekiyama, M. Otsu, Y. Shimada, A. Sato, S. Nakanishi, and H. Jingami Expression and Purification of the Extracellular Ligand Binding Region of Metabotropic Glutamate Receptor Subtype 1 J. Biol. Chem., May 22, 1998; 273(21): 13089 - 13096. [Abstract] [Full Text] [PDF]

				K. Ryu, H. Lee, S. Kim, J. Beauchamp, C.-S. Tung, N. W. Isaacs, I. Ji, and T. H. Ji Modulation of High Affinity Hormone Binding. HUMAN CHORIOGONADOTROPIN BINDING TO THE EXODOMAIN OF THE RECEPTOR IS INFLUENCED BY EXOLOOP 2 OF THE RECEPTOR J. Biol. Chem., March 13, 1998; 273(11): 6285 - 6291. [Abstract] [Full Text] [PDF]

				F.-P. Zhang, A. S. Rannikko, P. R. Manna, H. M. Fraser, and I. T. Huhtaniemi Cloning and Functional Expression of the Luteinizing Hormone Receptor Complementary Deoxyribonucleic Acid from the Marmoset Monkey Testis: Absence of Sequences Encoding Exon 10 in Other Species Endocrinology, June 1, 1997; 138(6): 2481 - 2490. [Abstract] [Full Text] [PDF]

				C. Wu, P. Narayan, and D. Puett Protein Engineering of a Novel Constitutively Active Hormone-Receptor Complex J. Biol. Chem., December 6, 1996; 271(49): 31638 - 31642. [Abstract] [Full Text] [PDF]

				R. L. Gilchrist, K.-S. Ryu, I. Ji, and T. H. Ji The Luteinizing Hormone/Chorionic Gonadotropin Receptor Has Distinct Transmembrane Conductors for cAMP and Inositol Phosphate Signals J. Biol. Chem., August 9, 1996; 271(32): 19283 - 19287. [Abstract] [Full Text] [PDF]

				S. Nishi, K. Nakabayashi, B. Kobilka, and A. J. W. Hsueh The Ectodomain of the Luteinizing Hormone Receptor Interacts with Exoloop 2 to Constrain the Transmembrane Region. STUDIES USING CHIMERIC HUMAN AND FLY RECEPTORS J. Biol. Chem., February 1, 2002; 277(6): 3958 - 3964. [Abstract] [Full Text] [PDF]