Abstract
Previous studies revealed pharmacological differences between human and guinea pig histamine H2 receptors (H2Rs) with respect to the interaction with guanidine-type agonists. Because H2R species variants are structurally very similar, comparative studies are suited to relate different properties of H2R species isoforms to few molecular determinants. Therefore, we systematically compared H2Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH2R, gpH2R, rH2R, and cH2R, respectively, and the short splice variant of Gsα, GsαS, were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH2R-GsαS but neither gpH2R-GsαS nor rH2R-GsαS showed the hallmarks of increased constitutive activity compared with hH2R-GsαS, i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH2R-GsαS, increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H2Rs without or together with mammalian GsαS or H2R-Gsα fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH2R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH2R-GsαS, gpH2R-GsαS, and rH2R-GsαS in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH2R-GsαS. In conclusion, the cH2R exhibits increased constitutive activity compared with hH2R, gpH2R, and rH2R, and there is evidence for ligand-specific conformations in H2R species isoforms.
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.107.120014.
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ABBREVIATIONS: H2R, histamine H2 receptor; TM, transmembrane domain of a G protein-coupled receptor; H1R, histamine H1 receptor; gpH2R, guinea pig histamine H2 receptor; gpH2R-GsαS, fusion protein of the guinea pig histamine H2 receptor and the short splice variant of Gsα; hH2R, human histamine H2 receptor; hH2R-GsαS, fusion protein of the human histamine H2 receptor and the short splice variant of Gsα; Gsα, α-subunit of the Gs, protein that mediates adenylyl cyclase activation; GsαS, short splice variant of the Gs protein Gsα; HA, histamine; DIM, dimaprit; AMT, amthamine; BET, betahistine; IMP, impromidine; ARP, arpromidine; CIM, cimetidine; RAN, ranitidine; FAM, famotidine; APT, aminopotentidine; IAPT, iodoaminopotentidine; AC, adenylyl cyclase; GPCR, G protein-coupled receptor; rH2R, rat histamine H2 receptor; rH2R-GsαS, fusion protein of the rat histamine H2 receptor and the short splice variant of Gsα; S, signal peptide from influenza hemagglutinin; F, FLAG epitope; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; cH2R, canine histamine H2 receptor; cH2R-GsαS, fusion protein of the canine histamine H2 receptor and the short splice variant of Gsα; AR, adrenoceptor; ANOVA, analysis of variance.
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↵1 Current affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.
- Received January 16, 2007.
- Accepted February 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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