Point mutations in the second extracellular loop of the histamine H2 receptor do not affect the species-selective activity of guanidine-type agonists

Abstract

Residues in the second extracellular loop (e2) play a role in ligand binding in certain aminergic G protein coupled receptors (GPCRs). N-[3-(1H-Imidazol-4-yl)propyl)]guanidines and N (G)-acylated derivatives are more efficacious and potent agonists at fusion proteins of the guinea pig histamine H(2) receptor and the short splice variant of G(salpha), G(salphaS) (gpH(2)R-G(salphaS)) than at the human isoform (hH(2)R-G(salphaS)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(salphaS) fusion protein with the four e2 residues differing in both species isoforms mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(salphaS) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, efficacies and potencies of guanidine-type agonists were similar at mutant and wild-type receptors indicating that e2 does not contribute to the species-selectivity. In several class 1 GPCRs, amino acids in the vicinity of a highly conserved cysteine in e2 participate in ligand binding. A three-dimensional homology model of the hH(2)R predicted Lys-173 and Lys-175, adjacent to Cys-174 in e2, to be in close proximity to the binding pocket of guanidine-type agonists. To elucidate the putative role of both residues for interactions with the agonists, two hH(2)R-G(salphaS) fusion proteins, with single-point mutations of Lys-173-->Ala-173 and Lys-175-->Ala-175 respectively, were generated. With these mutants, the efficacies and potencies of small and bulky H(2)R agonists did not significantly change. However, increases in GTPase activity upon agonist stimulation were reduced, suggesting an impact of both residues on the efficiency of receptor coupling to G(salphaS). In conclusion, none of the point mutations generated within this study substantially altered the efficacies and potencies of guanidine-type agonists relative to the wild-type receptors, suggesting that these residues do not directly face the H(2)R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.

Interactions of Histamine H1-Receptor Agonists and Antagonists with the Human Histamine H4-Receptor

“…Ovalbumin (purity Ͼ98%; EndoGrade) was obtained from Hyglos (Regensburg, Germany) and dissolved at a concentration of 1 mg/ml in PBS, pH 7.4. [␥-32 P]GTP was synthesized as described previously (Preuss et al, 2007). […”

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

“…32 P]GTP was synthesized as described previously (Preuss et al, 2007b). [ 3 H]Mepyramine (30.0 Ci/mmol) was from PerkinElmer Life and Analytical Sciences (Walatham, MA).…”

Ligand-Specific Contribution of the N Terminus and E2-Loop to Pharmacological Properties of the Histamine H1-Receptor

“…For the muscarinic M 3 receptor, several amino acids in the E2-loop are important for efficient agonist-induced activation of the muscarinic M 3 receptor (Scarselli et al, 2007). In contrast to these results, the E2-loop does not contribute to the species selectivity of guanidine-type agonists at human and guinea pig histamine H 2 receptor (Preuss et al, 2007). Our present study shows that the E2-loop and the E2-loop in combination with the N terminus have ligand-specific influence on the pharmacology of the H 1 R. Based on these data, it can be concluded that the extracellular loop E2 as well as the N terminus contribute to ligand binding, receptor activation, and selectivity in biogenic amine and nucleoside GPCRs.…”

“…[␥-32 P]GTP was synthesized as described previously (Preuss et al, 2007), and [ 3 H]mepyramine (30.0 Ci/mmol) was obtained from PerkinElmer Life and Analytical Sciences (Waltham, MA). A Rotiszint ecoplus (Roth, Karlsruhe, Germany) liquid scintillation cocktail was used.…”

“…Several studies have analyzed the contribution of the E2-loop to agonist or antagonist binding or activation of GPCRs, and the same analysis was performed with the adenosine A 1 , A 2a , A 3 receptors (Olah et al, 1994;Kim et al, 1996), the dopamine D 2 receptor (Shi and Javitch, 2004), the muscarinic acetylcholine M 3 receptor (Scarselli et al, 2007), the ␣ 1 -adrenergic receptor (Zhao et al, 1996), the ␣ 2A -adrenergic receptor (Laurila et al, 2007), and the histamine H 2 receptor (Preuss et al, 2007). To study the influence of the species differences in N terminus and E2-loop between hH 1 R and gpH 1 R, we constructed chimeric hH 1 R with gp E2-loop (h gpE2 H 1 R) and hH 1 R with gp N terminus and gp E2-loop (h gpNgpE2 H 1 R) (Fig.…”