title: Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.
abstract: The human thromboxane A(2) (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 Arg(174)-Met(202) of the receptor protein. Furthermore, antibody screening revealed that inhibition of the amino acid region Cys(183)-Asp(193) was critical for radioligand binding and platelet aggregation, whereas inhibition of Gly(172)-Cys(183) 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.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Turek
authors.forename: Thérêse
authors.initials: T
authors.lastname: Halmos
authors.forename: Nora L
authors.initials: NL
authors.lastname: Sullivan
authors.forename: Kostas
authors.initials: K
authors.lastname: Antonakis
authors.forename: Guy C
authors.initials: GC
authors.lastname: Le Breton
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017482:Receptors, Thromboxane / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; D017482:Receptors, Thromboxane; D001224:Aspartic Acid; D014357:Trypsin; D003545:Cysteine; D005998:Glycine