ADP-RIBOSYLATION CYCLES

ADP-核糖基化循环

• 批准号: 2576753
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2576753
• 关键词: ADP ribosylation; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; immunoprecipitation; integrins; laminin; molecular site; myoblasts; northern blottings; posttranslational modifications; protein sequence; protein structure function; receptor binding; tissue /cell culture; western blottings

Integrin alpha7 is a major substrate for a cell-surface ADP- ribosyltransferase in intact, differentiated mouse myoblasts (myotubes). To address the question of the role of post-translational modification of integrin alpha7, we studied interactions of the dimer of integrin alpha7 and beta1 with the extracellular matrix protein laminin in solution and in intact cells. It was observed that integrin alpha7beta1 bound to EHS laminin (laminin-1, composed of alpha1, beta1 and gamma1 chains), but did not bind to endogenous laminin expressed in C2C12 myotubes. Northern blot analysis demonstrated that C2C12 myotubes synthesized mRNA's for all laminin-1 subunits. C2C12 laminin was however, immunologically distinct from EHS laminin; it was not recognized by 5D3 anti-laminin-1 monoclonal antibody, whereas 5A2 and LT3 antibodies reacted equally well with C2C12 and EHS laminins. Following deglycosylation of EHS laminin, separation of the subunits by SDS-PAGE, Western blotting, and partial amino acid sequencing of the protein bands, the epitope recognized by 5D3 antibody was localized to the gamma1 laminin chain. It is postulated that integrin alpha7beta1 binding site is contained within the same laminin subunit. Incubation of intact C2C12 myotubes with EHS laminin or plating of C2C12 cells on EHS laminin-coated dishes resulted in the binding of EHS laminin to the cell surface and subsequent co-immunoprecipitation of laminin and integrin alpha7. The dose response analysis and the divalent cation dependence of the binding suggested that the interaction of EHS laminin and integrin alpha7beta1 occurred after lysis of cells rather than at the cell surface. EHS laminin and integrin alpha7beta1 were chemically cross-linked after they formed a complex in solution. The two proteins, however, did not undergo cross-linking at the cell surface, suggesting that in intact, resting myotubes integrin alpha7beta1 interacted poorly with EHS laminin, which may reflect a limited accesibility of integrin alpha7beta1 for laminin in the membrane to or an inactive state of the integrin.

整合素α7是细胞表面ADP-的主要底物 完整、分化的小鼠成肌细胞（肌管）中的核糖基转移酶。 解决翻译后修饰的作用问题 整合素α7，我们研究了整合素二聚体的相互作用 α7 和 β1 与细胞外基质蛋白层粘连蛋白 溶液和完整细胞中。 据观察，整合素α7β1 与 EHS 层粘连蛋白（laminin-1，由 alpha1、beta1 和 gamma1 组成）结合 链），但不与 C2C12 中表达的内源层粘连蛋白结合 肌管。 Northern 印迹分析表明 C2C12 肌管 合成了所有层粘连蛋白-1 亚基的 mRNA。 C2C12 层粘连蛋白是 然而，在免疫学上与 EHS 层粘连蛋白不同；它没有被识别 5D3 抗层粘连蛋白-1 单克隆抗体，而 5A2 和 LT3 抗体 与 C2C12 和 EHS 层粘连蛋白的反应同样良好。 下列的 EHS 层粘连蛋白的去糖基化，通过 SDS-PAGE 分离亚基， 蛋白质印迹和蛋白质条带的部分氨基酸测序， 5D3抗体识别的表位定位于gamma1 层粘连蛋白链。 推测整合素α7β1结合位点 包含在同一层粘连蛋白亚基内。 完整 C2C12 的孵育 带有 EHS 层粘连蛋白的肌管或将 C2C12 细胞铺在 EHS 层粘连蛋白上 培养皿导致 EHS 层粘连蛋白与细胞表面结合， 随后进行层粘连蛋白和整合素 α7 的免疫共沉淀。 这 剂量反应分析和结合的二价阳离子依赖性 表明EHS层粘连蛋白和整合素α7β1的相互作用 发生在细胞裂解后而不是在细胞表面。 环境健康安全 层粘连蛋白和整合素α7β1发生化学交联后 在溶液中形成络合物。 然而，这两种蛋白质并没有发生变化。 在细胞表面交联，表明在完整、静止状态下 肌管整合素 α7β1 与 EHS 层粘连蛋白相互作用较差， 可能反映出整合素α7β1对于层粘连蛋白的可及性有限 在膜中整合素处于非活性状态。