Klein P, Theibert A, Fontana D, Devreotes P N
J Biol Chem. 1985 Feb 10;260(3):1757-64.
We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.
我们最近通过用8-N3-[32P]cAMP对完整的盘基网柄菌细胞进行特异性光亲和标记，鉴定出一种细胞表面cAMP结合蛋白。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳放射自显影片中，主要的光标记蛋白呈现为双峰（Mr = 40,000 - 43,000）。在本研究中，该双峰显示出具有负责趋化作用和cAMP信号传导的cAMP受体的特征。双峰的特异性光亲和标记和8-N3-[32P]cAMP的结合都是可饱和的（KD = 0.3 microM），两者的水平在5小时达到峰值，并且都被cAMP和几种cAMP类似物以相同的效力顺序抑制，其K1值与测量的抑制[3H]cAMP结合的值相似。当cAMP结合活性被部分纯化（40倍）然后进行光亲和标记时，观察到相同的条带（Mr = 40,000 - 43,000）。双峰的上带和下带的相对强度以与cAMP合成中的自发振荡相同的频率交替。当振荡被抑制时，双峰的下带占主导。加入cAMP后，相对强度逐渐转移到上带。当去除cAMP时，下带形式逐渐恢复。我们提出受体的下带形式激活趋化作用和cAMP信号传导，而上带形式则不然。这种可逆的受体修饰可能是适应的机制，即生理反应不再被持续的cAMP刺激的过程。据报道，盘基网柄菌中的几个发育调控基因被cAMP脉冲诱导或抑制（适应性调控），其他基因被持续的cAMP抑制（非适应性调控）。如果两种以相同亲和力结合cAMP的受体形式独立影响基因表达，那么这里报道的受体修饰可能解释这些观察结果。
