Purification and characterization of prostacyclin receptor in mastocytoma cells

肥大细胞瘤细胞中前列环素受体的纯化和表征

• 批准号: 03671048
• 负责人: NEGISHI Manabu
• 金额: $ 1.28万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03671048/
• 关键词: Prostacyclin; Adenylate cyclase; Cloning; Receptor; Prostaglandin E2; Mast cell; Photoaffinity; cAMP; トロンボキサンA_2; ホモロジースクリーニング; PCR法; フォトアフィニティ-; 癌化肥満細胞

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.

小鼠肥大细胞瘤P-815细胞的前列环蛋白（PGI_2）受体的特征是通过稳定的PGI_2模拟[15-^3H_1] -19-（3-氮杂苯基）-20- norisocarbacyclin（[^3H] APNIC）使用的Podobseor概率的受体来表征。 [^3H]与肥大细胞瘤膜结合，具有高亲和力，可饱和。 SCATCHARD图分析表明，单个结合位点，KD为4.7 nm，BMAX为0.58 pmol/mg蛋白。 [^3H] apnic的结合因apnic和iLoprost（另一种稳定的PGI_2激动剂，而在PGE_2的低点程度上）依赖于剂量。放射线的结合显示对鸟嘌呤核苷酸鸟嘌呤5'-摩西亚 - （3- thiotriphosphate）（GTPGAMMAS）的敏感性。 [^3H] APNIC-PRELAIL标签的膜的光解会导致放射性映射掺入约43 kDa的蛋白质中。 PGI_2激动剂和其他对PGI_2受体的特异性抑制了光标记，并由GTPGAMMAS调节。在猪血小板的膜中，[^3H] apnic也标记了大约45 kDa的蛋白质，该蛋白质在PGI_2受体中已知丰富的膜。这些结果表明，[^3H] APNIC专门标记可能代表PGI_2受体的蛋白质，并且该辐射探针将是进一步表征和纯化PGI_2受体的有用试剂。

项目成果

Manabu negishi: "Differential regulation of thrombinー of ATPーinduced mobilization of intracellular Ca^<2+> bu prostacyclin recptor in mouse mastocytome" Biochemical and Biophysical Research Communication. 176. 102-107 (1991)

Manabu negishi：“小鼠肥大细胞中ATP诱导的细胞内Ca ^ 2+ bu前列环素受体的凝血酶的差异调节”生物化学和生物物理研究通讯。176。102-107（1991）。

Satoru Takahashi: "Cytosol promotes the guanine nucleotide-induced release of the α subunit of Gi_2 from the membrane of mouse mastocytoma P-815 cells" The Journal of Biological Chemistry. 266. 5367-5370 (1991)

Satoru Takahashi：“胞质溶胶促进鸟嘌呤核苷酸诱导的小鼠肥大细胞瘤 P-815 细胞膜上 Gi_2 α 亚基的释放”《生物化学杂志》266. 5367-5370 (1991)。

Yukihiko Sugimoto: "Cloning and expression of a cDNA for mouse prostaglandin E receptor EP_3 subtype" The Journal of Biological Chemistry. 267. (1992)

Yukihiko Sugimoto：“小鼠前列腺素 E 受体 EP_3 亚型 cDNA 的克隆和表达”《生物化学杂志》。

S. Takahashi: "Cytosol promotes the guanine nucleotide-induced release of the alpha subunit of Gi2 from the membrane of mouse mastocytoma P-815 cells" J.Biol.Chem.266. 5367-5370 (1991)

S. Takahashi：“胞质溶胶促进鸟嘌呤核苷酸诱导的小鼠肥大细胞瘤 P-815 细胞膜上 Gi2 α 亚基的释放”J.Biol.Chem.266。

Mamabu Negishi: "Translocation of α subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells" The Journal of Biological Chemistry. 267. 2364-2369 (1992)

Mamabu Negishi：“通过刺激小鼠肥大细胞瘤细胞中的前列环素受体来实现刺激性鸟嘌呤核苷酸结合蛋白的 α 亚基的易位”《生物化学杂志》267. 2364-2369 (1992)。