Characterization by photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor: evidence for N-linked glycosylation

Abstract Thromboxane A 2 (TXA 2 ) and prostaglandin H 2 (PGH 2 ) are potent proaggregatory and vasoconstrictor lipids acting through a receptor referred to as the TXA 2 /PGH 2 receptor. The receptor was purified using a modification of a previously described method from human platelet membranes solubilized using the detergent (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and a combination of affinity chromatography and wheat germ lectin chromatography. This procedure resulted in a 1075 ± 375-fold purification and a specific activity of 1.45 ± 0.55 nmol/mg protein ( n = 5). Repeating these chromatography steps on this partially purified receptor resulted in a preparation with a specific activity of 21 ± 3 nmol/mg protein ( n = 5). This represents the theoretical specific activity if one assumes a molecular weight of 50,000 for the receptor. The fold purification was 11,750 ± 1250 based on crude membranes and an overall yield of 24%. To further the characterization of this receptor, we synthesized a new radioiodinated photoaffinity probe, 7-[(1R,2S,3S,5R)-6, 6-dimethyl-3-(4-azido-3-iodobenzene-sulfonylamino)-bicyclo[3.1.1] hept-2-yl]-5(Z)-heptenoic acid (I-SAP-N 3 ). [ 125 I]l-SAP-N 3 irreversibly incorporated into the purified receptor yielding a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and indicated a molecular weight for the receptor of 50–51 kDa. The incorporation of the ligand could be inhibited by a variety of TXA 2 /PGH 2 analogues. In addition, photoaffinity labelling was inhibited in a stereoselective manner as demonstrated by the pair of enantiomers (d)- and (1)-S145. Digestion of photoaffinity labelled receptor with N-glycosidase F demonstrated the presence of at least two N-linked glycosylation sites. The time course of the digestion shows that one site is rapidly deglycosylated giving a molecular weight of the receptor of approximately 42 kDa. This is followed by a slower second deglycosylation resulting in a protein of 32 kDa molecular weight. No radioactivity in the photoaffinity labelled receptor was lost during (he deglycosylations. These results demonstrate that the TXA 2 /PGH 2 receptor is heavily N-glycosylated.