Glycosylation of the human prostacyclin receptor: Role in ligand binding and signal transduction

Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, a cts through a cell-surface G protein-coupled receptor [prostacyclin (IP)]. The human (h) IP contains two consensus sites for N-linked glycosylation (N -7 and N-78). However, the role of glycosylation is unknown. Mutant recepto rs (N-7-Q(7),N-78-Q(78) and N-7,N-78-Q(7),Q(78)) were generated by replacin g N-7 and/or N-78 with Q's. Receptor glycosylation was similar in the wild- type and N-7-Q(7) and was inhibited with tunicamycin. N-78-Q(78) and N-7,N- 78-Q(7),Q(78) demonstrated little or no glycosylation. Membrane localizatio n was reduced for each mutant concomitant with impaired glycosylation. Part ial localization to the plasma membrane allowed direct examination of the e ffect of glycosylation on IP function. High-affinity binding to N-7-Q(7) wa s similar (K-d = 21.7 +/- 1.7 nM, n = 4) to that of the wild-type receptor (K-d = 24.3 +/- 3.6 nM, n = 4), despite a reduced value for B-max (0.35 +/- 0.03 fmol/mg of protein versus 3.34 +/- 0.52 fmol/mg of protein, n = 4). B inding to N-78-Q(78) (B-max = 0.27 +/- 0.03 fmol/mg of protein, n = 3; K-d = 149.1 +/- 11.1, n = 3) and N-7,N-78-Q(7),Q(78) (no specific binding) was further impaired. Agonist-induced adenylyl cyclase activation was reduced i n N-7-Q(7) cells, whereas N-78-Q(78) cells responded only to high concentra tions of iloprost and N-7,N-78-Q(7),Q(78) were unresponsive. Inositol phosp hate generation was evident only with the wild-type. Only the wild-type and N-7-Q(7) receptors underwent agonist-induced sequestration. Our findings d emonstrate greater glycosylation at N-78 compared with N-7. The extent of N -linked glycosylation of hIP may be important for membrane localization, li gand binding, and signal transduction.