Mutations of Cys-17 and Ala-271 in the Human Histamine H₂Receptor 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 s␣S ) than human (hH 2 R-G s␣S ) histamine H 2 receptor, coupled to the short splice variant of G s␣ , G s␣S . 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 s␣S with Cys-173 Tyr-17/Ala-2713 Asp-271 exchanges (hH 2 R3gpH 2 R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH 2 R-G s␣S and gpH 2 R-G s␣S . Potencies and efficacies of guanidines and N G -acylguanidines were increased at this mutant receptor compared with hH 2 R-G s␣S , but they were still lower than at gpH 2 R-G s␣S , 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 s␣S mutant with a Cys-173 Tyr-17 exchange showed inefficient coupling to G s␣S 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 s␣S fusion proteins.The histamine H 2 receptor (H 2 R) is a biogenic amine receptor that belongs to the class A of the family of GPCRs. After stimulation by histamine (HA; Fig. 1, 1), the H 2 R couples to G s proteins to activate adenylyl cyclase (AC). H 2 Rs mediate regulation of gastric acid secretion in parietal cells, cardiac contractility, and myeloid cell differentiation (Del Valle and Gantz, 1997).N- propyl]guanidines are the most potent agonists at the H 2 R known so far (up to 400 times more active than HA at the guinea pig right atrium), and they are possibly useful as positive inotropic drugs for the treatment of severe congestive heart failure, as agents inducing cell differentiation in acute myelogenous leukemia, and as anti-inflammatory drugs (Dove et al., 2004). Guanidines are less potent and efficient agonists at the Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.120519.ABBREVIATIONS: H 2 R, histamine H 2 receptor; GPCR, G protein-coupled receptor; HA, histamine; AC, adenylyl cyclase; G s␣ , ␣-subunit of the G s protein that mediates adenylyl cyclase activation; G s␣S , short splice variant of the G s protein G s␣ ; gpH 2 R, guinea pig histamine H 2 receptor; ...

Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H2Receptors

“…The control data for hH 2 R-G S␣S and gpH 2 R-G S␣S are identical with the control data for these constructs in Fig. 4 of Preuss et al (2007).…”

“…K B values and inverse agonist efficacies, respectively, of antagonists at hH 2 R-G s␣S were compared with the corresponding parameters at gpH 2 R-G s␣S , rH 2 R-G s␣S , and cH 2 R-G s␣S , respectively, using one-way ANOVA. The control data for hH 2 R-G S␣S and gpH 2 R-G S␣S are identical with the control data for these constructs in Table 2 of Preuss et al (2007 G s␣S (ϳ29% reduction) and gpH 2 R plus G s␣S (ϳ23% reduction). Taken together, among H 2 R species isoforms coexpressed with G s␣S , cH 2 R was the most constitutively active GPCR.…”

“…GTP Effect), the effect of GTP (10 M) was referred to the effect of GTP (10 M) plus HA (100 M). The control data for hH 2 R-G S␣S and gpH 2 R-G S␣S are identical with the control data for these constructs in Table 3 of Preuss et al (2007 C terminus on the constitutive activities of various GPCRs was described previously (Prezeau et al, 1996;WenzelSeifert and Seifert, 2003). Ligand-Specific Interactions at H 2 R Species Isoforms.…”

Molecular Basis for the Selective Interaction of Synthetic Agonists with the Human Histamine H1-Receptor Compared with the Guinea Pig H1-Receptor

“…This technique allows us to obtain useful information about the interactions of amino acids with the ligand in the binding pocket. Beyond the H 1 R, the analysis of receptor species isoforms has also been most valuable for the H 2 R (Preuss et al, 2007a), H 3 R (Ligneau et al, 2000), and H 4 R (Thurmond et al, 2004).…”

Comparison of the pharmacological properties of human and rat histamine H3-receptors

“…The H 4 -receptor possesses very high constitutive activity as well. Similar constitutive activity renders the system well suited for the analysis of species-specific ligand effects, since differences in constitutive activity between GPCRs can alter their pharmacological profiles and lead to a further complication of data interpretation [40,41].…”