Point mutation in the seventh hydrophobic domain of the human thromboxane A2 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 the full length thromboxane receptor have been isolated from human placenta mRNA by reverse transcriptase-polymerase chain reaction. An expression construct, under control of the cytomegalovirus promoter, was introduced into human embryonic kidney 293 cells. Membranes from transfected cells bound the thromboxane antagonist SQ29,548 and the agonist [15-(1 alpha,2 beta(5z)-3 alpha(1E,3S)-4 alpha)]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid) with high affinities, and significantly more receptors were expressed in these cells, compared with platelet preparations. The putative seventh transmembrane segment is highly related in all cloned members of the eicosanoid receptor family and forms a critical portion of the ligand binding pocket for G protein-coupled receptors. Several point mutations in this segment were generated. Binding of SQ29,548 was virtually abolished in cells transfected with all the variant receptor constructs. However, one receptor variant (TxR-W299L), in which a tryptophan at position 299 was substituted for a leucine residue, allowed a definite discrimination between agonist and antagonist binding sites in competition and saturation binding experiments. An antibody directed toward the third intracellular loop of the thromboxane receptor was able to immunoprecipitate native thromboxane receptor in solubilized membranes from human erythroleukemia cells and transfected cells.