Hayes J S, Lawler O A, Walsh M T, Kinsella B T
Department of Biochemistry, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.
J Biol Chem. 1999 Aug 20;274(34):23707-18. doi: 10.1074/jbc.274.34.23707.
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+) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of Ca(2+) 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+) 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.
前列环素受体（IP）是一种G蛋白偶联受体，介导前列腺素前列环素及其类似物的作用。来自多个物种的IP各自含有完全保守的假定异戊二烯化CAAX基序，每个基序的序列为CSLC。在[（3）H]甲羟戊酸内酯存在下，对稳定过表达血凝素表位标签化IP的人胚肾（HEK）293细胞进行代谢标记，确定小鼠IP被异戊二烯化。涉及体外试验的研究证实，人和小鼠IP的重组形式被碳15法尼基异戊二烯类修饰。通过将CAAX基序内的半胱氨酸（Cys）414定点突变为丝氨酸（Ser）414来破坏异戊二烯化，消除了IP（SSLC）在体外和转染细胞中的异戊二烯化。对野生型（IP）和突变型（IP（SSLC））受体的Scatchard分析证实，每种受体对[（3）H]伊洛前列素均表现出高亲和力和低亲和力结合位点，不受受体异戊二烯化的影响。相对于未转染的细胞，稳定过表达IP的细胞系产生显著的激动剂（伊洛前列素和西卡前列素）介导的环磷酸腺苷（cAMP）增加，而IP（SSLC）细胞产生的cAMP与未转染的对照HEK 293细胞无显著差异。此外，Gs的α（α）亚基共表达使IP细胞而非IP（SSLC）细胞的cAMP显著增加。与未转染的HEK 293细胞相比，IP对伊洛前列素或西卡前列素的反应也表现出显著的、剂量依赖性的细胞内钙离子浓度（[Ca（2+）]i）增加，而IP（SSLC）对[Ca（2+）]i的动员显著受损。用Gαq或Gα11共转染细胞导致IP细胞而非IP（SSLC）细胞或对照HEK 293细胞的激动剂介导的[Ca（2+）]i动员显著增加。此外，与未异戊二烯化的β2肾上腺素能受体或未处理的细胞相比，洛伐他汀处理抑制异戊二烯化显著降低了IP激动剂介导的cAMP生成。因此，IP的异戊二烯化不影响配体结合，但对于有效偶联效应器腺苷酸环化酶和磷脂酶C是必需的。
