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(s alpha), G(s alpha S) (gpH(2)R-G(s alpha S)) than at the human isoform (hH(2)R-G(s alpha S)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(s alpha S) fusion protein with the four e2 residues differing in both species isoforrns mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(s alpha S) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, effcacies 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(s alpha S) 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 H2R 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(s alpha S). 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 H2R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.