Purification and characterization of prostacyclin receptor in mastocytoma cells

用稳定的前列环素（PGI_2）类似物[15-^3H_1]-19-（3-叠氮苯基）-20-去甲异碳环素（[^3H]APNIC）作为光亲和探针，对小鼠肥大细胞瘤P-815细胞的PGI_2受体进行了光亲和标记。[^3H]APNIC与肥大细胞瘤膜以高亲和力和可饱和的方式结合。Scatchard图分析表明，一个单一的结合位点，Kd为4.7 nM，Bmax为0.58 pmol/mg蛋白。[^3H]APNIC的结合被APNIC和另一种稳定的PGI_2激动剂伊洛前列素剂量依赖性地抑制，而被PGE_2抑制的程度要小得多。放射性配体的结合显示出对鸟嘌呤核苷酸鸟苷5 '-OMICRON-（3-硫代三磷酸）（GTP γ S）的敏感性。[^3H] APNIC预标记的膜的光解导致放射性标记掺入到一个约43 kDa的蛋白质中。光标记被PGI_2受体激动剂和其它对PGI_2受体具有特异性的野牡丹素抑制，并被GTP γ S调节。在猪血小板膜中，[^3H]APNIC还标记了一种分子量约为45 kDa的蛋白质，这种膜富含PGI_2受体。这些结果表明，[^3H]APNIC特异性标记了一种可能代表PGI_2受体的蛋白质，这种放射性探针将为进一步鉴定和纯化PGI_2受体提供有用的试剂。

The prostacyclin (PGI_2) receptor of mouse mastocytoma P-815 cells was characterized by photoaffinity labeling with the stable PGI_2 analogue [15-^3H_1]-19-(3-azidophenyl)-20-norisocarbacyclin ([^3H]APNIC) used as a potential photoaffinity probe for the receptor. [^3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [^3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI_2 agonist, and to a much lessor extent by PGE_2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosins 5'-OMICRON-(3-thiotriphosphate) (GTPgammaS). Photolysis of [^3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI_2 agonists and other prostaglandins with specificity for the PGI_2 receptor and was modulated by GTPgammaS. A protein of approximately 45 kDa was also labeled by [^3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI_2 receptors. These results demonstrated that [^3H]APNIC specifically labels a protein that may represent the PGI_2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI_2 receptor.