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Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.

Publication ,  Journal Article
Turek, JW; Halmos, T; Sullivan, NL; Antonakis, K; Le Breton, GC
Published in: J Biol Chem
May 10, 2002

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.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 10, 2002

Volume

277

Issue

19

Start / End Page

16791 / 16797

Location

United States

Related Subject Headings

  • Trypsin
  • Spectrometry, Fluorescence
  • Signal Transduction
  • Receptors, Thromboxane
  • Protein Structure, Tertiary
  • Protein Binding
  • Precipitin Tests
  • Platelet Aggregation
  • Molecular Sequence Data
  • Models, Chemical
 

Citation

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Turek, J. W., Halmos, T., Sullivan, N. L., Antonakis, K., & Le Breton, G. C. (2002). Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. J Biol Chem, 277(19), 16791–16797. https://doi.org/10.1074/jbc.M105872200
Turek, Joseph W., Thérêse Halmos, Nora L. Sullivan, Kostas Antonakis, and Guy C. Le Breton. “Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.J Biol Chem 277, no. 19 (May 10, 2002): 16791–97. https://doi.org/10.1074/jbc.M105872200.
Turek JW, Halmos T, Sullivan NL, Antonakis K, Le Breton GC. Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. J Biol Chem. 2002 May 10;277(19):16791–7.
Turek, Joseph W., et al. “Mapping of a ligand-binding site for the human thromboxane A2 receptor protein.J Biol Chem, vol. 277, no. 19, May 2002, pp. 16791–97. Pubmed, doi:10.1074/jbc.M105872200.
Turek JW, Halmos T, Sullivan NL, Antonakis K, Le Breton GC. Mapping of a ligand-binding site for the human thromboxane A2 receptor protein. J Biol Chem. 2002 May 10;277(19):16791–16797.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

May 10, 2002

Volume

277

Issue

19

Start / End Page

16791 / 16797

Location

United States

Related Subject Headings

  • Trypsin
  • Spectrometry, Fluorescence
  • Signal Transduction
  • Receptors, Thromboxane
  • Protein Structure, Tertiary
  • Protein Binding
  • Precipitin Tests
  • Platelet Aggregation
  • Molecular Sequence Data
  • Models, Chemical