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.
Footnotes
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R.L. is recipient of a PIONIER award of the Technology Foundation (Stichting voor de Technische Wetenschapppen) of the Netherlands Foundation of Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.140343.
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ABBREVIATIONS: GPCR, G-protein coupled receptor; β2AR, β2-adrenergic receptor; EL2, second extracellular loop; H1R, histamine H1 receptor; H2R, histamine H2 receptor; H3R, histamine H3 receptor; H4R, histamine H4 receptor; VUF 8430, S-(2-guanidylethyl)-isothiourea; TM, transmembrane; JNJ 7777120, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; NFAT, nuclear factor of activated T cells; IL, interleukin.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received April 24, 2008.
- Accepted July 16, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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