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

以人鳞状细胞癌细胞系A-431为研究对象，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嘌呤受体的配基特异性。我们还研究了细胞外ATP对肌醇磷酸代谢、钙动员、钙离子内流、EGF受体蛋白磷酸化、Scatchard图分析EGF受体的影响。还研究了胞外ATPase的底物专一性。结果表明：(1)ATP刺激后[Ca~(2+)]i升高非常迅速，在30 S内达到峰值。随后触发[Ca^lt；45；Ca^&gt；2+]内流的开始，最大速度在大约2分钟内达到。(2)[Ca^&lt；2+]i升高和^&lt；45；Ca^&gt；2+内流不仅受到ATP的刺激，而且还受到GTP、UTP、ADP和UDP的刺激。其他核苷酸或核苷无效。6个三磷酸腺苷/腺苷二磷酸类似物，三磷酸腺苷-伽马S，AM…更多的是P-PNP、AMPPCP、AMPCPP、腺苷二磷酸-β-S、AMPCP，这些化合物不抑制上述活性。其中S和AMPPNP较强地刺激了Ca~(2+)的动员和Ca~(2+)的内流。(3)从配体的特异性来看，激活细胞活性的受体可能是P_2型嘌呤受体。在被检测的几种叠氮化合物中，发现一种叠氮-AMPPNP衍生物能够对当前的受体进行光亲和标记。(4)细胞内储存的钙离子是产生IP_3所必需的，因此当细胞受到ATP刺激时，[Ca^&lt；45；Ca^&lt；2+&gt；]i和内流是必需的。(5)与A431细胞相关的ecto-ATPase的特异性与P_2原受体的配体特异性有很大的不同，表明这两种蛋白质参与了不同的系统。ATP引起的[~(2+)]i升高提示DG增加，这可能刺激蛋白激酶C。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EGF受体亲和力的变化。(7)ATP刺激细胞和/或膜蛋白掺入[~3H]-肌醇。PCA不溶性膜磷脂分析表明，ATP刺激后形成的主要化合物为磷脂酰肌醇。磷脂酰肌醇的合成需要细胞外钙离子的存在。较少

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