Lupi R, Corda D, Di Girolamo M
Department of Cell Biology and Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy.
J Biol Chem. 2000 Mar 31;275(13):9418-24. doi: 10.1074/jbc.275.13.9418.
Mono-ADP-ribosylation is a post-translational modification of cellular proteins that has been implicated in the regulation of signal transduction, muscle cell differentiation, protein trafficking, and secretion. In several cell systems we have observed that the major substrate of endogenous mono-ADP-ribosylation is a 36-kDa protein. This ADP-ribosylated protein was both recognized in Western blotting experiments and selectively immunoprecipitated by a G protein beta subunit-specific polyclonal antibody, indicating that this protein is the G protein beta subunit. The ADP-ribosylation of the beta subunit was due to a plasma membrane-associated enzyme, was sensitive to treatment with hydroxylamine, and was inhibited by meta-iodobenzylguanidine, indicating that the involved enzyme is an arginine-specific mono-ADP-ribosyltransferase. By mutational analysis, the target arginine was located in position 129. The ADP-ribosylated beta subunit was also deribosylated by a cytosolic hydrolase. This ADP-ribosylation/deribosylation cycle might be an in vivo modulator of the interaction of betagamma with specific effectors. Indeed, we found that the ADP-ribosylated betagamma subunit is unable to inhibit calmodulin-stimulated type 1 adenylyl cyclase in cell membranes and that the endogenous ADP-ribosylation of the beta subunit occurs in intact Chinese hamster ovary cells, where the NAD(+) pool was labeled with [(3)H]adenine. These results show that the ADP-ribosylation of the betagamma subunit could represent a novel cellular mechanism in the regulation of G protein-mediated signal transduction.
单 ADP - 核糖基化是细胞蛋白质的一种翻译后修饰，与信号转导、肌肉细胞分化、蛋白质运输和分泌的调节有关。在多个细胞系统中，我们观察到内源性单 ADP - 核糖基化的主要底物是一种 36 kDa 的蛋白质。这种 ADP - 核糖基化蛋白在蛋白质印迹实验中被识别，并被 G 蛋白β亚基特异性多克隆抗体选择性免疫沉淀，表明该蛋白是 G 蛋白β亚基。β亚基的 ADP - 核糖基化是由一种与质膜相关的酶引起的，对羟胺处理敏感，并被间碘苄胍抑制，表明所涉及的酶是一种精氨酸特异性单 ADP - 核糖基转移酶。通过突变分析，目标精氨酸位于第 129 位。ADP - 核糖基化的β亚基也被一种胞质水解酶去核糖基化。这种 ADP - 核糖基化/去核糖基化循环可能是体内调节βγ与特定效应器相互作用的一种调节剂。事实上，我们发现 ADP - 核糖基化的βγ亚基无法抑制细胞膜中钙调蛋白刺激的 1 型腺苷酸环化酶，并且β亚基的内源性 ADP - 核糖基化发生在完整的中国仓鼠卵巢细胞中，其中 NAD(+)池用 [(3)H]腺嘌呤标记。这些结果表明，βγ亚基的 ADP - 核糖基化可能代表了一种调节 G 蛋白介导的信号转导的新细胞机制。
