Zolkiewska A, Moss J
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434, USA.
J Biol Chem. 1995 Apr 21;270(16):9227-33. doi: 10.1074/jbc.270.16.9227.
Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol-anchored, arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta 1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.
整合素α7是骨骼肌细胞中细胞表面糖基磷脂酰肌醇锚定的精氨酸特异性ADP核糖基转移酶的主要底物。由于负责切割ADP-核糖基精氨酸键的酶ADP-核糖基精氨酸水解酶是一种胞质酶，并且是假定的ADP核糖基化循环中转移酶的一个组成部分，因此对ADP-核糖基化整合素α7的加工过程进行了研究。将分化的小鼠C2C12成肌细胞与[腺苷酸-32P]NAD一起孵育，并在还原条件下通过SDS-聚丙烯酰胺凝胶电泳进行分析，分离出两种[32P]ADP-核糖基化形式的整合素α7。通过脉冲追踪以及在层粘连蛋白亲和柱上纯化放射性标记的蛋白质，结果表明105 kDa的ADP-核糖基化形式源自单ADP-核糖基化的102 kDa形式，代表在多个位点修饰的整合素α7。在较高NAD浓度下利用的额外修饰位点位于整合素α7的63 kDa N端片段中。两种[32P]ADP-核糖基化整合素均与细胞骨架松散结合，与层粘连蛋白亲和柱结合，并能用整合素β1抗体进行免疫沉淀。在修饰的任一部位，[32P]ADP-核糖基化整合素α7上的32P标记都能迅速去除，这一过程受到游离ADP-核糖或5'-核苷酸磷酸二酯酶的替代底物对硝基苯胸苷-5'-单磷酸的抑制。尽管表面整合素α7的量保持恒定，但加工后的整合素α7无法进行后续的ADP核糖基化。在加工过程中，用[14C]NAD进行放射性标记的整合素α7（其烟酰胺近端核糖中含有14C）未观察到标记损失，这与细胞表面5'-核苷酸磷酸二酯酶对ADP-核糖部分的降解一致。因此，与细胞内ADP核糖基化不同，细胞表面ADP核糖基化不易被ADP-核糖基精氨酸水解酶逆转，并且似乎在假定的ADP核糖基化循环之外起作用。
