Theibert A, Klein P, Devreotes P N
J Biol Chem. 1984 Oct 25;259(20):12318-21.
The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.
最近观察到硫酸铵可稳定盘基网柄菌细胞表面的[3H]环磷酸腺苷（cAMP）结合（范·哈斯特尔特，P.，和基恩，E.（1983年）《生物化学杂志》258卷，9636 - 9642页），这促使我们尝试使用这种稳定技术，通过用8 - 叠氮基 - [32P]cAMP进行光亲和标记来鉴定表面cAMP受体。8 - 叠氮基 - [32P]cAMP特异性标记了一种多肽，该多肽在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上以紧密间隔的双峰形式迁移（相对分子质量 = 40,000至43,000）。发现超过60%的标记多肽与膜相关。通过发育调控、特异性和亚细胞定位的差异，将这种蛋白质与cAMP依赖性蛋白激酶的胞质调节亚基（相对分子质量 = 41,000）区分开来。通过蛋白质印迹分析在膜中未检测到激酶调节亚基。我们的初步观察表明，这种双峰的标记与cAMP结合活性密切相关，表明它是介导趋化性和cAMP信号传导的表面受体。
