Abstract
In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl)]guanidines and NG-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH2R-GsαS) than human (hH2R-GsαS) histamine H2 receptor, coupled to the short splice variant of Gsα, GsαS. Whereas Ala-271 (hH2R) and Asp-271 (gpH2R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH2R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH2R. In the present study, we generated a mutant hH2R-GsαS with Cys-17→ Tyr-17/Ala-271→ Asp-271 exchanges (hH2R→gpH2R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH2R-GsαS and gpH2R-GsαS. Potencies and efficacies of guanidines and NG-acylguanidines were increased at this mutant receptor compared with hH2R-GsαS, but they were still lower than at gpH2R-GsαS, suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH2R-GsαS mutant with a Cys-17→ Tyr-17 exchange showed inefficient coupling to GsαS as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H2R-GsαS fusion proteins.
Footnotes
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This work was supported by the Research Training Program (Graduiertenkolleg) GRK 760 “Medicinal Chemistry: Molecular Recognition–Ligand-Receptor Interactions” of the Deutsche Forschungsgemeinschaft.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.120519.
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ABBREVIATIONS: H2R, histamine H2 receptor; GPCR, G protein-coupled receptor; HA, histamine; AC, adenylyl cyclase; Gsα, α-subunit of the Gs protein that mediates adenylyl cyclase activation; GsαS, short splice variant of the Gs protein Gsα; gpH2R, guinea pig histamine H2 receptor; gpH2R-GsαS, fusion protein of the guinea pig histamine H2 receptor and the short splice variant of Gsα; H1R, histamine H1 receptor; hH2R, human histamine H2 receptor; hH2R-GsαS, fusion protein of the human histamine H2 receptor and the short splice variant of Gsα; hH2R-C17Y-GsαS, fusion protein of the human histamine H2 receptor bearing a Cys→ Tyr mutation at position 17 and the short splice variant of Gsα; hH2R-C17Y-A271D-GsαS, fusion protein of the human histamine H2 receptor bearing a Cys→ Tyr mutation at position 17 and an Ala→ Asp mutation at position 271 and the short splice variant of Gsα; DIM, dimaprit; AMT, amthamine; TM, transmembrane domain of a G protein-coupled receptor; IMP, impromidine; ARP, arpromidine; CIM, cimetidine; RAN, ranitidine; FAM, famotidine; APT, aminopotentidine; IAPT, iodoaminopotentidine; PCR, polymerase chain reaction; S, signal peptide from influenza hemagglutinin; F, FLAG epitope; PAGE, polyacrylamide gel electrophoresis; AR, adrenoceptor; β2AR-Gsα, fusion protein of the β2-adrenoceptor and Gsα.
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↵1 Current affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.
- Received January 25, 2007.
- Accepted March 2, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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