Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H1R

Numerous competitive radioligand binding studies revealed significant differences between human and guinea pig histamine H1-receptors (hH1R and gpH1R), e.g. for the partial H1R agonist phenoprodifen. But until now, there are only few studies with regard to binding kinetics at H1R. Previous studies from our group revealed an influence of the exchange of N-terminus and E2-loop between hH1R and gpH1R onto affinity of phenoprodifen to H1R (Strasser A, Wittmann HJ, Seifert R, J Pharmacol Exp Ther 326:783–791, 2008). The aim of this study was, therefore, to examine the impact of the N-terminus and the E2-loop on binding kinetics of the H1R. The wild type hH1R and gpH1R and the chimeric hgpE2H1R (E2-loogp from guinea pig) and hgpNgpE2H1R (N-terminus and E2-loop from guinea pig) were co-expressed with regulator of G-protein signaling protein RGS4 in Sf9 insect cells and kinetic binding studies were performed using the antagonist [3H]mepyramine as radioligand. The rate constants for association and dissociation were, in dependence of the ligand, different between hH1R and gpH1R. Furthermore, the rate constants for association at hgpNgpE2H1R were significantly different compared to hH1R and gpH1R. Molecular dynamic simulation studies detected different interactions of amino acid side chains on the extracellular surface of the receptor. Based on these findings, the influence of extracellular surface onto binding kinetics and binding affinity can be explained. Thus, the extracellular surface of G protein-coupled receptors for biogenic amines, exhibits influence onto kinetics of ligand binding, onto ligand recognition and ligand guiding into the binding pocket.

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