Abstract

Using the natural variation in histamine H₄ receptor protein sequence, we tried to identify amino acids involved in the binding of H₄ receptor agonists. To this end, we constructed a variety of chimeric human-mouse H₄ receptor proteins to localize the domain responsible for the observed pharmacological differences between human and mouse H₄ receptors in the binding of H₄ receptor agonists, such as histamine, clozapine, and VUF 8430 [S-(2-guanidylethyl)-isothiourea]. After identification of a domain between the top of transmembrane domain 4 and the top of transmembrane domain 5 as being responsible for the differences in agonist affinity between human and mouse H₄Rs, detailed site-directed mutagenesis studies were performed. These studies identified Phe¹⁶⁹ in the second extracellular loop as the single amino acid responsible for the differences in agonist affinity between the human and mouse H₄Rs. Phe¹⁶⁹ is part of a Phe-Phe motif, which is also present in the recently crystallized β₂-adrenergic receptor. These results point to an important role of the second extracellular loop in the agonist binding to the H₄ receptor and provide a molecular explanation for the species difference between human and mouse H₄ receptors.