The Role of N-Glycosylation of Human Thromboxane A2Receptor in Ligand Binding

Thromboxane A2receptor (TXA2R) was expressed in insectSf21 cells and demonstrated to interact with 8-iso-PGF2α and 9α,11β-PGF2α with a potency similar to that of TXA2agonist U46619. TXA2R was shown to be a glycoprotein. The role of N-glycosylation of TXA2R in ligand binding was investigated in the insect cells overexpressed with recombinant TXA2R. Deletion of the carbohydrate moiety by adding tunicamycin during infection ofSf21 cells or mutation of both potential N-glycosylation sites (Asn-4 and Asn-16) abolished the ligand binding of TXA2R, suggesting that N-glycosylation is crucial for binding function. Mutation of either Asn-4 or Asn-16 to a leucine did not have much effect on maximal binding. However, the mutant receptors possess lower binding affinity toward TXA2R antagonist [3H]SQ29548. Furthermore, the binding specificity of the mutant receptors was shown to be altered. Our data suggest that both Asn-4 and Asn-16 are glycosylated and glycosylation on either site is sufficient for ligand recognition. However, glycosylation on both sites is required to maintain binding affinity and specificity.