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

Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, acts 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 receptors (N(7)-Q(7),N(78)-Q(78) and N(7),N(78)-Q(7),Q(78)) were generated by replacing 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 localization was reduced for each mutant concomitant with impaired glycosylation. Partial localization to the plasma membrane allowed direct examination of the effect of glycosylation on IP function. High-affinity binding to N(7)-Q(7) was 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). Binding 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 in N(7)-Q(7) cells, whereas N(78)-Q(78) cells responded only to high concentrations of iloprost and N(7),N(78)-Q(7),Q(78) were unresponsive. Inositol phosphate generation was evident only with the wild-type. Only the wild-type and N(7)-Q(7) receptors underwent agonist-induced sequestration. Our findings demonstrate greater glycosylation at N(78) compared with N(7). The extent of N-linked glycosylation of hIP may be important for membrane localization, ligand binding, and signal transduction.