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 epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.
Hayes
J S
JS
Department of Biochemistry, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.
Lawler
O A
OA
Walsh
M T
MT
Kinsella
B T
BT
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D016285:Iloprost / Q000378:metabolism; D008148:Lovastatin / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D017368:Protein Prenylation; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000378:metabolism; D012717:Sesquiterpenes; D013329:Structure-Activity Relationship; D010738:Type C Phospholipases / Q000378:metabolism
D011106:Polyisoprenyl Phosphates; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D008148:Lovastatin; D000242:Cyclic AMP; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases; D016285:Iloprost; D002118:Calcium