RT Journal Article
SR Electronic
T1 Glycosylation of the Human Prostacyclin Receptor: Role in Ligand Binding and Signal Transduction
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 480
OP 487
VO 60
IS 3
A1 Zhibing Zhang
A1 Sandra C. Austin
A1 Emer M. Smyth
YR 2001
UL http://molpharm.aspetjournals.org/content/60/3/480.abstract
AB 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(Kd = 21.7 ± 1.7 nM,n = 4) to that of the wild-type receptor (Kd = 24.3 ± 3.6 nM,n = 4), despite a reduced value forBmax (0.35 ± 0.03 fmol/mg of protein versus 3.34 ± 0.52 fmol/mg of protein, n = 4). Binding to N78-Q78(Bmax = 0.27 ± 0.03 fmol/mg of protein, n = 3;Kd = 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. The American Society for Pharmacology and Experimental Therapeutics