Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F-2 alpha receptor

For the five principal prostanoids PGD(2), PGE(2), PGF(2 alpha), prostacycl in and thromboxane A(2) eight receptors have been identified that belong to the family of G-protein-coupled receptors, They display an overall homolog y of merely 30%. However, single amino acids in the transmembrane domains s uch as an Arg in the seventh transmembrane domain are highly conserved, Thi s Arg has been identified as part of the ligand binding pocket. It interact s with the carboxyl group of the prostanoid. The aim of the current study w as to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF(2 alpha) receptor, which is conserved a mong all PGF(2 alpha) receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand bin ding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exch anged by site-directed mutagenesis to Gin (Q), ASp (D), Arg (R), Ala (A) an d Gly (G), The receptor molecules were N-terminally extended by a Flag epit ope for immunological detection. All mutant proteins were expressed at leve ls between 50% and 80% of the wild type construct, The H81Q and H81D recept or bound PGF(2 alpha) 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(2 alpha). Wild type receptor and H81Q showed a shallow pH optimum for PGF2 alpha binding aroun d pH 5.5 with almost no reduction of binding at higher pH, In contrast the H81D mutant bound PGF(2 alpha) with a sharp optimum at pH 4.5, a pH at,whic h the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pll values, The data indicate that the His-81 in the second transmembrane domain of the PGF(2 alpha) receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not bg ionic interaction with the prostagla ndin's carboxyl group but rather as a hydrogen bond donor. (C) 1999 Federat ion of European Biochemical Societies.