The human thromboxane A2 (TP) receptor, a member of the G protein-coupled receptor superfamily, consists of seven transmembrane segments. Attempts to elucidate the specific segment(s) that define the receptor ligand-binding pocket have produced less than definitive and sometimes conflicting results. On this basis, the present work identified an amino acid sequence of the TP receptor that is directly involved in ligand binding. Mapping of this domain was confirmed by two separate approaches: photoaffinity labeling and site-specific antibodies. The newly synthesized, biotinylated photoaffinity probe, SQBAzide, was first shown to specifically label TP receptor protein. Sequential digestion of this protein with CNBr/trypsin revealed photolabeling of a 2.9-kDa peptide. Using anti-peptide antibodies directed against different regions of the receptor protein, it was established that this peptide represents the predicted cleavage product for CNBr/trypsin and corresponds to amino acids Arg174–Met202 of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys183–Asp193 was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly172–Cys183 was not. Collectively these findings provide evidence that ligands interact with amino acids contained within the C-terminal portion of the third extracellular domain (ED3) of the receptor protein. This information should be of significant value in the study of TP receptor structure and signaling.
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Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M105872200
This work was supported by National Institutes of Health Grant HL24530 (to G. C. L.), the North Atlantic Treaty Organization (to G. C. L. and K. A.), the Pharmaceutical Research and Manufacturers of America Foundation (to J. W. T.), and the Chateaubriand Fellowship Program offered by the Office for Science and Technology of the Embassy of France in the United States (to J. W. T.) and was conducted under the auspices of the Association for United States-French Biomedical Cooperation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
