Abstract

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H₂-receptor (H₂R) agonists are lower at the H₂R of human neutrophils than at the H₂R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H₂R (hH₂R) and guinea pig H₂R (gpH₂R), respectively, and the short splice variant of G_sα(G_sαS) expressed in Sf9 cells. The potencies and efficacies of small H₂R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH₂R-G_sαS and gpH₂R-G_sαS were similar. In contrast, the potencies and efficacies of guanidines were lower at hH₂R-G_sαS than at gpH₂R-G_sαS. Guanidines bound to hH₂R-G_sαS with lower affinity than to gpH₂R-G_sαS, and high-affinity binding of guanidines at gpH₂R-G_sαS was more resistant to disruption by GTPγS than binding at hH₂R-G_sαS. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH₂R (Ala-271 in hH₂R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271→Asp-271 mutation in hH₂R-G_sαS. Intriguingly, the efficacies of guanidines at the Ala-271→Asp-271 mutant and at hH₂R/gpH₂R chimeras were lower than at gpH₂R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH₂R-G_sαS but not the other constructs studied, stabilizes the active guanidine-H₂R state. Collectively, our data show 1) distinct interaction of H₂R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy.