Smyth E M, Nestor P V, FitzGerald G A
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1996 Dec 27;271(52):33698-704. doi: 10.1074/jbc.271.52.33698.
An epitope-tagged human prostacyclin receptor (HAhIP) was constructed and stably transfected into human embryonic kidney 293 cells. The receptor exhibited high (Kd = 0.4 +/- 0.08 nM, Bmax = 0.7 +/- 0.2 pmol/mg protein; n = 4) and low (Kd = 75 +/- 27.4 nM, Bmax = 7.1 +/- 3.6 pmol/mg protein; n = 4) affinity for iloprost and coupled to both cAMP (EC50 = 0.1 +/- 0.03 nM) and inositol phosphate (EC50 = 43.1 +/- 10 nM) production. The receptor resolved on SDS-polyacrylamide gel electrophoresis as a broad complex with a molecular mass of 44-62 kDa and is glycosylated and phosphorylated. Stimulation of transfected cells with iloprost induced a rapid time- and concentration-dependent phosphorylation of HAhIP. Pretreatment of cells with a protein kinase C (PKC) inhibitor (GF109203X; 5 microM) abolished basal phosphorylation and dramatically reduced iloprost-induced HAhIP phosphorylation. A protein kinase A (PKA) inhibitor (H89) was largely ineffective under the same conditions. HAhIP phosphorylation was stimulated by receptor-dependent (thrombin, 2 units/ml) or receptor-independent (phorbol 12-myristate 13-acetate, 5 microM) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor-independent (forskolin (5 microM) or dibutyryl cAMP (1 microM)) activation of PKA did not induce HAhIP phosphorylation. These results indicate that the human prostacyclin receptor may be regulated by agonist-dependent phosphorylation. This appears to be mediated, in part, by activation of PKC but not by PKA.
构建了一个带有表位标签的人前列环素受体（HAhIP），并将其稳定转染到人胚肾293细胞中。该受体对伊洛前列素表现出高亲和力（Kd = 0.4 +/- 0.08 nM，Bmax = 0.7 +/- 0.2 pmol/mg蛋白；n = 4）和低亲和力（Kd = 75 +/- 27.4 nM，Bmax = 7.1 +/- 3.6 pmol/mg蛋白；n = 4），并与cAMP（EC50 = 0.1 +/- 0.03 nM）和肌醇磷酸（EC50 = 43.1 +/- 10 nM）的产生偶联。该受体在SDS-聚丙烯酰胺凝胶电泳上呈现为分子量为44-62 kDa的宽复合物，且进行了糖基化和磷酸化修饰。用伊洛前列素刺激转染细胞可诱导HAhIP快速的时间和浓度依赖性磷酸化。用蛋白激酶C（PKC）抑制剂（GF109203X；5 microM）预处理细胞可消除基础磷酸化，并显著降低伊洛前列素诱导的HAhIP磷酸化。在相同条件下，蛋白激酶A（PKA）抑制剂（H89）基本无效。HAhIP磷酸化可通过受体依赖性（凝血酶，2单位/ml）或受体非依赖性（佛波醇12-肉豆蔻酸酯13-乙酸酯，5 microM）的PKC激活来刺激；用GF109203X预处理细胞均可消除这两种激活。相反，受体非依赖性（福斯可林（5 microM）或二丁酰环磷腺苷（1 microM））的PKA激活并未诱导HAhIP磷酸化。这些结果表明，人前列环素受体可能受激动剂依赖性磷酸化调节。这似乎部分是由PKC激活介导的，而非PKA。
