Localization of [125I]SAP-N3 binding in the human platelet thromboxane A2/prostaglandin H2 receptor by proteolytic cleavage analysis.

Photo-affinity labeling studies of purified human platelet thromboxane A2/prostaglandin H2 receptor by the ligand 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-azido-3- iodobenzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid ([125I]SAP-N3) combined with proteolytic cleavage studies were performed to initiate studies aimed at localizing the binding domain of this ligand. Two endoproteinases, endoproteinase Asp-N (Asp-N) and endoproteinase Lys-C (Lys-C), and the endoglycosidase, N-glycosidase F (endo-F), were employed to generate fragments for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. Computational analysis of the published sequence was then employed to predict cleavage products and then compared to the observed digestion results. Results of this work suggest that the majority of the binding domain of [125I]SAP-N3 includes putative transmembrane regions M-3 and M-4 (amino acids 99-192) with a minor component at the amino and carboxyl terminus.