Purification and characterization of prostacyclin receptor in mastocytoma cells

用稳定的PGI_2类似物[15-^3H_1]-19-（3-氮杂苯基）-20-去甲异霉素（[^3H]APNIC）作为潜在的光亲和探针，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以高亲和力和饱和方式与肥大细胞瘤膜结合。Scatchard图分析显示，该蛋白的Kd为4.7 nM， Bmax为0.58 pmol/mg。APNIC和另一种稳定的PGI_2激动剂伊洛prost对[^3H]APNIC的结合有剂量依赖性抑制，PGE_2对[^3H]APNIC的作用较小。放射性配体的结合显示对鸟嘌呤核苷酸鸟苷5'- omicron -（3-硫代三磷酸）（GTPgammaS）敏感。[^3H] apnic预标记膜的光解导致放射性标记物结合到大约43 kDa的蛋白质中。PGI_2受体激动剂和其他前列腺素对PGI_2受体具有特异性，可抑制光标记，并受GTPgammaS调节。在猪血小板的膜上，一个大约45 kDa的蛋白也被[^3H]APNIC标记出来，这是已知的富含PGI_2受体的膜。这些结果表明[^3H]APNIC特异性标记了可能代表PGI_2受体的蛋白质，该放射性探针将成为进一步表征和纯化PGI_2受体的有用试剂。

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.