QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.104.001487v1 66/4/880    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Capra, V. Articles by Rovati, G. E. Search for Related Content PubMed PubMed Citation Articles by Capra, V. Articles by Rovati, G. E.

Mutational Analysis of the Highly Conserved ERY Motif of the Thromboxane A₂ Receptor: Alternative Role in G Protein-Coupled Receptor Signaling

Laboratory of Molecular Pharmacology, Section of Theoretical and Receptor Pharmacology, Department of Pharmacological Sciences, University of Milan, Milan, Italy (V.C., A.V., C.F., G.E.R.); Institut National de la Sante et de la Recherche Medicale U348, IFR Circulation-Paris-Nord, Hôpital Lariboisière, Paris, France (V.C., A.H.); Departments of Biochemistry and Internal Medicine, American University of Beirut, Beirut, Lebanon (A.H.); and Department of Experimental and Environmental Medicine and Medical Biotechnologies, University of Milan Bicocca, Milan, Italy (L.C., M.P.)

The presence of highly conserved amino acid stretches in G protein-coupled receptors (GPCRs) usually predicts an important role in receptor function. Considerable attention has therefore been focused on the involvement of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif at the cytoplasmic end of transmembrane domain 3 in the regulation of GPCR conformational states and/or the mediation of G protein activation. In the present study, we investigated the role of Glu¹²⁹ and Arg¹³⁰ in the ERY of thromboxane A₂ receptor (TP) in transfected human embryonic kidney 293 cells. We show that no conservative or nonconservative substitutions of Glu¹²⁹ and Arg¹³⁰ generated a constitutively active TP mutant, but a nonconservative mutation of Arg¹³⁰ (R130V) yielded a mutant receptor with significantly impaired 9,11-dideoxy-9,11-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619)-induced accumulation of inositol phosphates (IPs). This loss-of-function phenotype seems to be caused by the uncoupling of the TP receptor from G_q, as demonstrated by the loss of high-affinity agonist binding, and not by receptor internalization, as shown by localization studies with the R130V-green fluorescent protein fusion protein. It is interesting to note that U46619-induced activation of the nonconservative E129V mutant stimulated the production of IPs with a 10-fold lower EC₅₀ and a 2-fold higher E_max than in the wild-type receptor. Collectively, these data demonstrate that, unlike other GPCRs, mutations of Glu¹²⁹ do not induce constitutive activity, whereas Arg¹³⁰ is involved in G protein coupling or recognition, and they suggest the existence within class A GPCRs of at least two different subclasses that make different uses of the highly conserved E/DRY motif.

Address correspondence to: Dr. Valérie Capra, Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: valerie.capra{at}unimi.it

This article has been cited by other articles:

				E. Peverelli, A. G. Lania, G. Mantovani, P. Beck-Peccoz, and A. Spada Characterization of Intracellular Signaling Mediated by Human Somatostatin Receptor 5: Role of the DRY Motif and the Third Intracellular Loop Endocrinology, July 1, 2009; 150(7): 3169 - 3176. [Abstract] [Full Text] [PDF]

				C. D. Proulx, B. J. Holleran, A. A. Boucard, E. Escher, G. Guillemette, and R. Leduc Mutational Analysis of the Conserved Asp2.50 and ERY Motif Reveals Signaling Bias of the Urotensin II Receptor Mol. Pharmacol., September 1, 2008; 74(3): 552 - 561. [Abstract] [Full Text] [PDF]

				R. Case, E. Sharp, T. Benned-Jensen, M. M. Rosenkilde, N. Davis-Poynter, and H. E. Farrell Functional Analysis of the Murine Cytomegalovirus Chemokine Receptor Homologue M33: Ablation of Constitutive Signaling Is Associated with an Attenuated Phenotype In Vivo J. Virol., February 15, 2008; 82(4): 1884 - 1898. [Abstract] [Full Text] [PDF]

				H. Tokunaga, B. Matsuura, M. Dong, L. J. Miller, T. Ueda, S. Furukawa, Y. Hiasa, and M. Onji Mutational analysis of predicted intracellular loop domains of human motilin receptor Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G460 - G466. [Abstract] [Full Text] [PDF]

				K. Angelova, F. Fanelli, and D. Puett Contributions of Intracellular Loops 2 and 3 of the Lutropin Receptor in Gs Coupling Mol. Endocrinol., January 1, 2008; 22(1): 126 - 138. [Abstract] [Full Text] [PDF]

				V. Binet, B. Duthey, J. Lecaillon, C. Vol, J. Quoyer, G. Labesse, J.-P. Pin, and L. Prezeau Common Structural Requirements for Heptahelical Domain Function in Class A and Class C G Protein-coupled Receptors J. Biol. Chem., April 20, 2007; 282(16): 12154 - 12163. [Abstract] [Full Text] [PDF]

				G. E. Rovati, V. Capra, and R. R. Neubig The Highly Conserved DRY Motif of Class A G Protein-Coupled Receptors: Beyond the Ground State Mol. Pharmacol., April 1, 2007; 71(4): 959 - 964. [Abstract] [Full Text] [PDF]

				C. Buccellati, A. Sala, G. Rossoni, V. Capra, G. E. Rovati, A. Di Gennaro, G. Folco, S. Colli, and C. Casagrande Pharmacological Characterization of 2NTX-99 [4-Methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a Potential Antiatherothrombotic Agent with Antithromboxane and Nitric Oxide Donor Activity in Platelet and Vascular Preparations J. Pharmacol. Exp. Ther., May 1, 2006; 317(2): 830 - 837. [Abstract] [Full Text] [PDF]

				V. Capra, S. Ravasi, M. R. Accomazzo, S. Citro, M. Grimoldi, M. P. Abbracchio, and G. E. Rovati CysLT1 receptor is a target for extracellular nucleotide-induced heterologous desensitization: a possible feedback mechanism in inflammation J. Cell Sci., December 1, 2005; 118(23): 5625 - 5636. [Abstract] [Full Text] [PDF]

				S. R. Hawtin Charged Residues of the Conserved DRY Triplet of the Vasopressin V1a Receptor Provide Molecular Determinants for Cell Surface Delivery and Internalization Mol. Pharmacol., October 1, 2005; 68(4): 1172 - 1182. [Abstract] [Full Text] [PDF]

				C. A. Flanagan A GPCR That Is Not "DRY" Mol. Pharmacol., July 1, 2005; 68(1): 1 - 3. [Abstract] [Full Text] [PDF]

				M. M. Rosenkilde, T. N. Kledal, and T. W. Schwartz High Constitutive Activity of a Virus-Encoded Seven Transmembrane Receptor in the Absence of the Conserved DRY Motif (Asp-Arg-Tyr) in Transmembrane Helix 3 Mol. Pharmacol., July 1, 2005; 68(1): 11 - 19. [Abstract] [Full Text] [PDF]