RT Journal Article SR Electronic T1 Mutations of Cys-17 and Ala-271 in the Human Histamine H2 Receptor Determine the Species Selectivity of Guanidine-Type Agonists and Increase Constitutive Activity JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 975 OP 982 DO 10.1124/jpet.107.120519 VO 321 IS 3 A1 Hendrik Preuss A1 Prasanta Ghorai A1 Anja Kraus A1 Stefan Dove A1 Armin Buschauer A1 Roland Seifert YR 2007 UL http://jpet.aspetjournals.org/content/321/3/975.abstract AB 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. The American Society for Pharmacology and Experimental Therapeutics