title: The role of N-glycosylation of human thromboxane A2 receptor in ligand binding.
abstract: 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.
authors.affiliation: Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536-0082, USA.
authors.forename: N
authors.initials: N
authors.lastname: Chiang
authors.forename: H H
authors.initials: HH
authors.lastname: Tai
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D019086:Bridged Bicyclo Compounds, Heterocyclic; D019556:COS Cells; D002478:Cells, Cultured; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism; D028441:F2-Isoprostanes; D005231:Fatty Acids, Unsaturated; D006023:Glycoproteins / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D008024:Ligands; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017482:Receptors, Thromboxane / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D028441:F2-Isoprostanes; D005231:Fatty Acids, Unsaturated; D006023:Glycoproteins; D006834:Hydrazines; D008024:Ligands; D012333:RNA, Messenger; D017482:Receptors, Thromboxane; D011994:Recombinant Proteins; D014415:Tunicamycin; C075750:8-epi-prostaglandin F2alpha; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; C045749:SQ 29548; D015237:Dinoprost