The role of N-glycosylation of human thromboxane A2 receptor in ligand binding.

Thromboxane A2 receptor (TXA2R) was expressed in insect Sf21 cells and demonstrated to interact with 8-iso-PGF2 alpha and 9 alpha, 11 beta-PGF2 alpha with a potency similar to that of TXA2 agonist U46619. TXA2R was shown to be a glycoprotein. The role of N-glycosylation of TXA2R in ligand binding was investigated in the insect cells over-expressed with recombinant TXA2R. Deletion of the carbohydrate moiety by adding tunicamycin during infection of Sf21 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.