The prostacyclin receptor is isoprenylated - Isoprenylation is required for efficient receptor-effector coupling

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics, IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC, Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitopetagge d IP in the presence of [H-3]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombin ant forms of the human and mouse IP are modified by carbon 15 farnesyl isop renoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys( 414) to Ser(414) within the CAAX motif, abolished isoprenylation of IPSSLC both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IPSSLC) receptor confirmed that each receptor exhibited h igh and low affinity binding sites for [H-3]iloprost, which were not influe nced by receptor isoprenylation. Whereas stable cell lines overexpressing I P generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IPSSLC cells was not significantly different from the control, nontransfected HEK 293 ce lls. Moreover, co-expression of the alpha (alpha) subunit of Gs generated s ignificant augmentations in cAMP by IP but not by IPSSLC cells, Whereas IP also demonstrated significant, dose-dependent increases in [Ca2+](i) in res ponse to iloprost or cicaprost compared with the nontransfected HEK 293 cel ls, mobilization of [Ca2+](i) by IPSSLC was significantly impaired. Co-tran sfection of cells with either G alpha(q) or G alpha(11) resulted in signifi cant augmentation of agonist-mediated [Ca2+](i) mobilization by IP cells bu t not by IPSSLC cells or by the control, HEK 293 cells. In addition, inhibi tion of isoprenylation by lovastatin treatment significantly reduced agonis t-mediated cAMP generation by IP in comparison to the nonisoprenylated beta (2) adrenergic receptor or nontreated cells. Hence, isoprenylation of TP do es not influence ligand binding but is required for efficient coupling to t he effecters adenylyl cyclase and phospholipase C.