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

Thromboxane A(2) receptor (TXA(2)R) was expressed in insect Sf21 cells and demonstrated to interact with 8-iso-PGF(2) alpha and 9 alpha 11 beta-PGF(2) alpha with a potency similar to that of TXA, agonist U46619. TXA(2)R was shown to be a glycoprotein. The role of N-glycosylation of TXA(2)R in ligand binding was investigated in the insect cells overexpressed with recombinant TXA(2)R, 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-18) abolished the Ligand binding of TXA(2)R, suggesting that N-glycosylation is crucial for binding function. Mutation of either Asn-4 or Asn-18 to a leucine did not have much effect on maximaI binding. However, the mutant receptors possess lower binding affinity toward TXA(2)R antagonist [H-3]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. (C) 1998 Academic Press.