Abstract

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 (N⁷ and N⁷⁸). However, the role of glycosylation is unknown. Mutant receptors (N⁷-Q⁷,N⁷⁸-Q⁷⁸and N⁷,N⁷⁸-Q⁷,Q⁷⁸) were generated by replacing N⁷ and/or N⁷⁸ with Q's. Receptor glycosylation was similar in the wild-type and N⁷-Q⁷ and was inhibited with tunicamycin. N⁷⁸-Q⁷⁸ and N⁷,N⁷⁸-Q⁷,Q⁷⁸demonstrated 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 N⁷-Q⁷ 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 forB_max (0.35 ± 0.03 fmol/mg of protein versus 3.34 ± 0.52 fmol/mg of protein, n = 4). Binding to N⁷⁸-Q⁷⁸(B_max = 0.27 ± 0.03 fmol/mg of protein, n = 3;K_d = 149.1 ± 11.1,n = 3) and N⁷,N⁷⁸-Q⁷,Q⁷⁸ (no specific binding) was further impaired. Agonist-induced adenylyl cyclase activation was reduced in N⁷-Q⁷ cells, whereas N⁷⁸-Q⁷⁸ cells responded only to high concentrations of iloprost and N⁷,N⁷⁸-Q⁷,Q⁷⁸ were unresponsive. Inositol phosphate generation was evident only with the wild-type. Only the wild-type and N⁷-Q⁷receptors underwent agonist-induced sequestration. Our findings demonstrate greater glycosylation at N⁷⁸ compared with N⁷. The extent of N-linked glycosylation of hIP may be important for membrane localization, ligand binding, and signal transduction.