POINT MUTATION IN THE 7TH HYDROPHOBIC DOMAIN OF THE HUMAN THROMBOXANE-A(2) RECEPTOR ALLOWS DISCRIMINATION BETWEEN AGONIST AND ANTAGONIST BINDING-SITES

Thromboxane A2, a potent platelet agonist and vasoconstrictor, exerts its actions via specific G protein-coupled receptors. cDNAs encoding t he full length thromboxane receptor have been isolated from human plac enta mRNA by reverse transcriptase-polymerase chain reaction. An expre ssion construct, under control of the cytomegalovirus promoter, was in troduced into human embryonic kidney 293 cells. Membranes from transfe cted cells bound the thromboxane antagonist SQ29,548 and the agonist b utenyl)-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid) with high affi nities, and significantly more receptors were expressed in these cells , compared with platelet preparations. The putative seventh transmembr ane segment is highly related in all cloned members of the eicosanoid receptor family and forms a critical portion of the ligand binding poc ket for G protein-coupled receptors. Several point mutations in this s egment were generated. Binding of SQ29,548 was virtually abolished in cells transfected with all the variant receptor constructs. However, o ne receptor variant (TxR-W299L), in which a tryptophan at position 299 was substituted for a leucine residue, allowed a definite discriminat ion between agonist and antagonist binding sites in competition and sa turation binding experiments. An antibody directed toward the third in tracellular loop of the thromboxane receptor was able to immunoprecipi tate native thromboxane receptor in solubilized membranes from human e rythroleukemia cells and transfected cells.