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. Clinical evidence suggests that inhibition of platelet thromboxane A 2 (TXA 2 ) 1 production provides a therapeutic basis for the treatment and/or prevention of certain thrombotic disease states (1-8). Indeed the ability of aspirin to inhibit TXA 2 synthesis is the primary rationale for the widespread use of this agent in recurrent myocardial infarction and more recently in thromboembolic stroke (9, 10). However, despite the clear importance of TXA 2 in these disease processes, the molecular interaction of TXA 2 with its receptor protein remains unknown. To date, the information available concerning the TP receptor ligand-binding domain(s) has been mostly limited to mutational analyses using receptor chimeras or expressed receptor protein containing site-specific mutations as well as ligand interactions with modified receptor peptides or molecular modeling. Collectively results from these studies have implicated transmembrane domains I, III, IV, V, VI, and VII as well as extracellular domains II and III as potential regions for ligand coordination sites (11)(12)(13)(14)(15)(16)(17)(18). Since all of the cited receptor regions would not be expected to participate in or form the ligandbinding domain, it would seem that the interpretation of some of these results may be limited by the potential for gross alterations in receptor tertiary structure. Based on this consideration, the present study used two different approaches to identify critical ligand coordination sites in the TP receptor protein.The first of these approaches utilized a novel and recently characterized bifunctional TP receptor antagonist, SQBAzide, to irreversibly label and track ligand-binding sites; the second approach utilized site-specific antibodies to probe different regions of the TP receptor protein. Our results indicate that the C-terminal portion of ED3 may form a critical ligand-binding domain for the TP receptor protein.
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