Kuwayama H, Van Haastert P J
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
J Biol Chem. 1996 Sep 27;271(39):23718-24. doi: 10.1074/jbc.271.39.23718.
Chemoattractants transiently activate guanylyl cyclase in Dictyostelium discoideum cells. Mutant analysis demonstrates that the produced cGMP plays an essential role in chemotactic signal transduction, controlling the actomyosin-dependent motive force. Guanylyl cyclase activity is associated with the particulate fraction of a cell homogenate. The addition of the cytosol stimulates guanylyl cyclase activity, whereas the cytosol plus ATP/Mg2+ inhibits enzyme activity. We have analyzed the regulation of guanylyl cyclase in chemotactic mutants and present evidence that a cGMP-binding protein mediates both stimulation and ATP-dependent inhibition of guanylyl cyclase. Upon chromatography of cytosolic proteins, cGMP binding activity co-elutes with both guanylyl cyclase-stimulating and ATP-dependent-inhibiting activities. In addition, ATP-dependent inhibition of guanylyl cyclase activity is enhanced by the cGMP analogue 8-Br-cGMP, suggesting that a cGMP-binding protein regulates guanylyl cyclase activity. Mutant KI-4 has an aberrant cGMP binding activity with very low Kd and shows a very small chemoattractant-mediated cGMP response; the cytosol from this mutant does not stimulate guanylyl cyclase. In contrast to KI-4, the aberrant cGMP binding activity of mutant KI-7 has a very high Kd and chemoattractants induce a prolonged cGMP response. The cytosol of this mutant stimulates guanylyl cyclase activity, but ATP does not inhibit the enzyme. Thus, two previously isolated chemotactic mutants are defective in the activation and inhibition of guanylyl cyclase, respectively. The positive and negative regulation of guanylyl cyclase by its product cGMP may well explain how cells process the temporospatial information of chemotactic signals, which is necessary for sensing the direction of the chemoattractant.
趋化因子可瞬时激活盘基网柄菌细胞中的鸟苷酸环化酶。突变分析表明，产生的环鸟苷酸（cGMP）在趋化信号转导中起关键作用，控制着肌动球蛋白依赖性驱动力。鸟苷酸环化酶活性与细胞匀浆的颗粒部分相关。加入胞质溶胶可刺激鸟苷酸环化酶活性，而胞质溶胶加ATP/Mg2+则抑制酶活性。我们分析了趋化突变体中鸟苷酸环化酶的调节，并提供证据表明一种cGMP结合蛋白介导了鸟苷酸环化酶的刺激和ATP依赖性抑制。对胞质蛋白进行层析时，cGMP结合活性与鸟苷酸环化酶刺激活性和ATP依赖性抑制活性共同洗脱。此外，cGMP类似物8-溴-cGMP增强了ATP对鸟苷酸环化酶活性的抑制作用，表明一种cGMP结合蛋白调节鸟苷酸环化酶活性。突变体KI-4具有异常的cGMP结合活性，解离常数（Kd）极低，且趋化因子介导的cGMP反应非常小；该突变体的胞质溶胶不刺激鸟苷酸环化酶。与KI-4相反，突变体KI-7的异常cGMP结合活性的Kd非常高，趋化因子可诱导延长的cGMP反应。该突变体的胞质溶胶刺激鸟苷酸环化酶活性，但ATP不抑制该酶。因此，两个先前分离的趋化突变体分别在鸟苷酸环化酶的激活和抑制方面存在缺陷。鸟苷酸环化酶受其产物cGMP的正负调节，这很可能解释了细胞如何处理趋化信号的时空信息，而这对于感知趋化因子的方向是必要的。
