LOCALIZATION OF [I-125] SAP-N-3 BINDING IN THE HUMAN PLATELET THROMBOXANE A(2) PROSTAGLANDIN H-2-RECEPTOR BY PROTEOLYTIC CLEAVAGE ANALYSIS/

Photo-affinity labeling studies of purified human platelet thromboxane A(2)/prostaglandin H-2 receptor by the ligand fonylamino)bicyclo[3.1. 1]hept-2-yl]-5(z)-heptenoic acid ([I-125]SAP-N-3) combined with proteo lytic cleavage studies were performed to initiate studies aimed at loc alizing the binding domain of this ligand. Two endoproteinases, endopr oteinase Asp-N (Asp-N) and endoproteinase Lys-C (Lys-C), and the endog lycosidase, N-glycosidase F (endo-F), were employed to generate fragme nts for analysis by sodium dodecyl sulfate-polyacrylamide gel electrop horesis (SDS-PAGE) autoradiography. Computational analysis of the publ ished sequence was then employed to predict cleavage products and then compared to the observed digestion results. Results of this work sugg est that the majority of the binding domain of [I-125]SAp-N-3 includes putative transmembrane regions M-3 and M-4 (amino acids 99-192) with a minor component at the amino and carboxyl terminus.