Klueppelberg U G, Gates L K, Gorelick F S, Miller L J
Gastroenterology Basic Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
J Biol Chem. 1991 Feb 5;266(4):2403-8.
The present study was undertaken to determine if the cholecystokinin (CCK) receptor may be phosphorylated, and to gain insight into its regulation. For this, the ATP pool of rat pancreatic acini was prelabeled with 32P, and the cells were stimulated with various secretagogues. CCK receptors from treated cells were enriched by sequential fractionation to produce plasmalemma, and subsequent solubilization and lectin-affinity chromatography. This protocol detected a phosphorylated Mr = 85,000-95,000 plasma membrane glycoprotein with features similar to the CCK receptor. Phosphorylation of this protein occurred rapidly (less than 2 min) and in a concentration-dependent manner in response to CCK, and was inhibited by the CCK receptor antagonist L-364,718. Further evidence that this represented the CCK receptor included comigration of phosphorylated and CCK radioligand affinity-labeled proteins on sodium dodecyl sulfate-polyacrylamide gels, both in native forms and after endoglycosidase F deglycosylation, and the specific adsorption of the phosphoprotein to a CCK analogue affinity resin. Phosphorylation occurred predominantly on serine residues of the receptor protein. Phosphorylation of this protein was also enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in response to direct activation of this enzyme by 12-O-tetradecanoylphorbol 13-acetate. Thus, the pancreatic CCK receptor is phosphorylated in a regulated manner, in response to both homologous and heterologous secretagogues, and to protein kinase C activation.
本研究旨在确定胆囊收缩素（CCK）受体是否会被磷酸化，并深入了解其调控机制。为此，先用³²P对大鼠胰腺腺泡的ATP池进行预标记，然后用各种促分泌剂刺激细胞。通过连续分级分离处理细胞的CCK受体，以制备质膜，随后进行溶解和凝集素亲和层析。该方案检测到一种磷酸化的分子量为85,000 - 95,000的质膜糖蛋白，其特征与CCK受体相似。该蛋白的磷酸化迅速发生（不到2分钟），且对CCK呈浓度依赖性，并且被CCK受体拮抗剂L - 364,718抑制。进一步证明这代表CCK受体的证据包括，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上，磷酸化蛋白和CCK放射性配体亲和标记蛋白在天然形式以及经内切糖苷酶F去糖基化后均共迁移，以及该磷蛋白对CCK类似物亲和树脂的特异性吸附。磷酸化主要发生在受体蛋白的丝氨酸残基上。该蛋白的磷酸化在响应其他促分泌剂时也会增强，这些促分泌剂与CCK一样，刺激导致蛋白激酶C激活的级联反应，并且在响应12 - O - 十四酰佛波醇13 - 乙酸酯对该酶的直接激活时也会增强。因此，胰腺CCK受体以一种受调控的方式被磷酸化，以响应同源和异源促分泌剂以及蛋白激酶C的激活。
