Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2α receptor

FEBS Letters

Neuschäfer-Rube, Frank¹  de Vries, Christa¹  Rehwald, Matthias¹  Püschel, Gerhard P.¹ 

[1] Institut für Biochemie und Molekulare Zellbiologie, Humboldtallee 23, D37073 Göttingen, Germany

关键词: Prostaglandin F2α receptor;    FP receptor;    Prostanoid receptor;    Site-directed mutagenesis;    Ligand binding site;    Structure-function relationship;    DEAE;    diethylaminoethyl;    EDTA;    ethylenediaminetetraacetate;    MES;    2-[N-morpholino]ethanesulfonic acid;    PBS;    phosphate-buffered saline;    PG;    prostaglandin;    PVDF;    polyvinylidene difluoride;    R;    receptor;    UTR;    untranslated region;   

DOI  :  10.1016/S0014-5793(99)00007-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

For the five principal prostanoids PGD₂, PGE₂, PGF_2α, prostacyclin and thromboxane A₂ eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF_2α receptor, which is conserved among all PGF_2α receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF_2α with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF_2α. Wild type receptor and H81Q showed a shallow pH optimum for PGF2α binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF_2α with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF_2α receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.

【 授权许可】

Unknown