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CELLULAR AND MOLECULAR

Phenylalanine 169 in the Second Extracellular Loop of the Human Histamine H₄ Receptor Is Responsible for the Difference in Agonist Binding between Human and Mouse H₄ Receptors

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands (H.D.L., A.J., R.A.B., E.H., I.J.P.d.E., R.L.); and Boehringer Ingelheim Austria GmbH, Vienna, Austria (E.H.)

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.

Address correspondence to: Prof. Dr. Rob Leurs, Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: r.leurs{at}few.vu.nl