Effects of extracellular ATP on Ca^<2+> movement, and phosphoinositide metabolism, leading to a functional change of EGF receptor

细胞外ATP对Ca^2运动和磷酸肌醇代谢的影响，导致EGF受体功能改变

• 批准号: 62570840
• 负责人: HOSOI Kazuo
• 金额: $ 1.54万
• 依托单位: Meikai 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: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570840/
• 关键词: P_2 Purinoceptors; EGF Receptors; Protein phosphosphorylation; Inositol metabolism; Intracellular calcium; Calcium influx; 細胞外ATP; プリン受容体; 細胞内Ca^<2+>; Ca^<2+>輸送; ホスファチジールイノシトール燐酸代謝; 燐酸化; ホスファチジールイノシトールりん酸代謝; りん酸化

Using a human epidemoidal carcinoma cell line, A-431, we have investigated ligand specificity of P_2 purinoceptor by measuring the ability to mobilize and elevate intracellular calcium, [Ca^<2+>]i and to influx ^<45>Ca^<2+>. We also investigated the effects of extracellular ATP on inositolphosphate metabolism, calcium mobilization, ^<45>Ca^<2+> influx, phosphorylation of EGF receptor protein, analysis of EGF receptor by Scatchard plotting. The substrate specificity of ecto-ATPase was also studied. The following results were obtained:(1)[Ca^<2+>]i elevation was very rapid and maximum [Ca^<2+>]i concentration was obtained within 30 s after the stimulation with ATP. The onset of ^<45>Ca^<2+> influx was triggered thereafter with the maximum velocity being achieved in approximately 2 min.(2)The [Ca^<2+>]i elevation and ^<45>Ca^<2+> influx were stimulated by not only ATP but also GTP, UTP, ADP, and UDP. Other nucleotides or nucleosides were ineffective. Six ATP/ADP analogues, ATP-gamma S, AM … More P- PNP, AMPPCP, AMPCPP, ADP-beta S, AMPCP, were found that these compounds do not inhibit the activity described above. Two of them, ATP-gamma S and AMPPNP rather stimulated the mobilization of Ca^<2+> and ^<45>Ca^<2+> influx.(3)From the ligand specificity, the receptors responsible in provoking such cellular activity were suggested to be P_2-type purinoceptors. Of a couple of azido-compounds examined, an azido-AMPPNP derivative was found to be able to photoaffinity label the present receptors.(4)The calcium ion stored in the cells were essential to produce IP_3 and hence elevate [Ca^<2+>]i and influx ^<45>Ca^<2+> when cells were stimulated with ATP.(5)The specificity of ecto-ATPase associated with A431 cells were quite different from the ligand specifity of P_2 prinoceptor, indicating that the two proteins were involved in different systems.(6)IP_3 formation and [Ca^<2+>]i elevation by ATP suggest the increase of DG, which is expected to stimulate protein kinase C. As a matter of fact, we observed the enhanced phosphorylation of serine and threonine residues of EGF receptor protein, and also the changes of affinity (affinity decreased) of the receptor for EGF.(7)The incorporation of [^3H]-inositol into cells and/or membrane protein were stimulated with ATP. The analysis of PCA insoluble membrane phospholipids showed that the main compound formed by stimulation with ATP was phosphatidylinositol. The synthesis of phosphatidylinositol required the presence of extracellular Ca^<2+>. Less

我们用人卵巢癌细胞系A-431，通过测定P_2嘌呤受体动员和升高细胞内[Ca^（2+）]i和内流[Ca ^（2+）]i的能力，研究了P_2嘌呤受体的配体特异性<45>。同时观察了细胞外ATP对磷酸肌醇代谢、钙动员、~（12）<45>Ca^&lt;2+&gt;内流、EGF受体蛋白磷酸化的影响，以及用Scatchard作图分析EGF受体。对胞外ATP酶的底物特异性进行了研究。结果表明：（1）ATP刺激后，[Ca ^&lt;2 +&gt;]i迅速升高，在30 s内达到最大值;此后<45>，在大约2分钟内达到最大速度时，触发了^ Ca^&lt;2+&gt;内流的开始。（2）<45>ATP、GTP、UTP、ADP和UDP均能引起[Ca^2 +]i升高和^ Ca^2+内流。其他核苷酸或核苷无效。六种ATP/ADP类似物，ATP-γ S，AM ...更多信息 发现P-PNP、AMPPCP、AMPCPP、ADP-β S、AMPCP不抑制上述活性。其中ATP-γ S和AMPPNP对Ca^&lt;2+&gt;动员和^<45>Ca^&lt;2+&gt;内流有较强的促进作用。（3）从配体的特异性来看，引起这种细胞活性的受体可能是P_2型嘌呤受体。一对夫妇的叠氮化合物检查，叠氮-AMPPNP衍生物被发现能够光亲和标记本受体。（4）细胞内贮存的Ca ~（2+）是产生IP_3所必需的，从而使ATP刺激细胞时[Ca^~（2+）]i升高，<45>内流增加。（5）与A431细胞相关的胞外ATP酶的特异性与P_2受体的配体特异性有很大差异，表明这两种蛋白参与不同的系统。(6)IP 3的形成和ATP引起的[Ca^&lt;2+&gt;]i的升高提示DG的增加，这可能是由于DG刺激蛋白激酶C所致。事实上，我们观察到EGF受体蛋白的丝氨酸和苏氨酸残基的磷酸化增强，以及受体对EGF的亲和力的变化（亲和力降低）。（7）ATP刺激[^3H]-肌醇掺入细胞和/或膜蛋白。PCA不溶性膜磷脂的分析表明，由ATP刺激形成的主要化合物是磷脂酰肌醇。磷脂酰肌醇的合成需要细胞外Ca^&lt;2+&gt;的存在.少

项目成果

Hosoi, K. et al: "A simple and rapid purification procedure for the tetra-methylrhodamine labeled antibodies." The Japanese Journal of Physiology 39(2), 317-324, 1989.

Hosoi, K. 等人：“四甲基罗丹明标记抗体的简单快速纯化程序。”

Hosoi, K. et al: "Production of a human-mouse hybrid cell line expressing both HLA and H-2 antigens." The Japanese Journal of Physiology.

Hosoi, K. 等人：“表达 HLA 和 H-2 抗原的人鼠杂交细胞系的生产。”

Hosoi, K. et al: Labeling and analysis of cell membrane, in "Laboratory Manual:Basic technique in study utilizing cells, antibodies, and genes", edited by Horio, T.Nankodo (Tokyo), (1990)

Hosoi, K. 等人：细胞膜的标记和分析，载于《实验室手册：利用细胞、抗体和基因进行研究的基本技术》，由 Horio, T.Nankodo（东京）编辑，（1990 年）

小玉昭子 他: "ROS17/2.8細胞による^<45>Ca^<2+>取り込みとアルカリフォスファタ-ゼ活性に与える各種骨代謝因子の作用" 明海大学歯学雑誌. 18(1). 82-87 (1989)

Akiko Kodama 等人：“各种骨代谢因素对 ROS17/2.8 细胞的 ^ 45 > Ca ^ 2+ 吸收和碱性磷酸酶活性的影响”Meikai University Journal of Dentistry 18(1)。 1989）

細井和雄,藤下まり子: 医学のあゆみ. (1989)

细井一夫、藤下真理子：医学史（1989）。