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CELLULAR AND MOLECULAR

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

Departments of Pharmaceutical/Medicinal Chemistry II (H.P., P.G., A.K., S.D., A.B.) and Pharmacology and Toxicology (R.S.), Institute of Pharmacy, University of Regensburg, Regensburg, Germany

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 (gpH₂R-G_sS) than human (hH₂R-G_sS) histamine H₂ receptor, coupled to the short splice variant of G_s, G_sS. 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-G_sS with Cys-17 Tyr-17/Ala-271 Asp-271 exchanges (hH₂RgpH₂R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH₂R-G_sS and gpH₂R-G_sS. Potencies and efficacies of guanidines and N^G-acylguanidines were increased at this mutant receptor compared with hH₂R-G_sS, but they were still lower than at gpH₂R-G_sS, suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH₂R-G_sS mutant with a Cys-17 Tyr-17 exchange showed inefficient coupling to G_sS 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-G_sS fusion proteins.

Address correspondence to: Dr. Roland Seifert, Department of Pharmacology and Toxicology, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany. E-mail: roland.seifert{at}chemie.uni-regensburg.de

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