title: 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.
abstract: In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl)]guanidines and N(G)-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH(2)R-G(salphaS)) than human (hH(2)R-G(salphaS)) histamine H(2) receptor, coupled to the short splice variant of G(salpha), G(salphaS). Whereas Ala-271 (hH(2)R) and Asp-271 (gpH(2)R) 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 gpH(2)R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH(2)R. In the present study, we generated a mutant hH(2)R-G(salphaS) with Cys-17--> Tyr-17/Ala-271--> Asp-271 exchanges (hH(2)R-->gpH(2)R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH(2)R-G(salphaS) and gpH(2)R-G(salphaS). Potencies and efficacies of guanidines and N(G)-acylguanidines were increased at this mutant receptor compared with hH(2)R-G(salphaS), but they were still lower than at gpH(2)R-G(salphaS), suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH(2)R-G(salphaS) mutant with a Cys-17--> Tyr-17 exchange showed inefficient coupling to G(salphaS) 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 H(2)R-G(salphaS) fusion proteins.
authors.affiliation: Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, Regensburg, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Preuss
authors.forename: Prasanta
authors.initials: P
authors.lastname: Ghorai
authors.forename: Anja
authors.initials: A
authors.lastname: Kraus
authors.forename: Stefan
authors.initials: S
authors.lastname: Dove
authors.forename: Armin
authors.initials: A
authors.lastname: Buschauer
authors.forename: Roland
authors.initials: R
authors.lastname: Seifert
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D006632:Histamine / Q000494:pharmacology; D017442:Histamine Agonists / Q000737:chemistry / Q000494:pharmacology*; D006635:Histamine H2 Antagonists / Q000494:pharmacology; D006801:Humans; D020125:Mutation, Missense; D011970:Receptors, Histamine H2 / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D018411:Spodoptera; D014162:Transfection
chemical_list: D017442:Histamine Agonists; D006635:Histamine H2 Antagonists; D011970:Receptors, Histamine H2; D011993:Recombinant Fusion Proteins; D006632:Histamine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases; D019791:Guanidine; D003545:Cysteine; D000409:Alanine