Barletta E, Bremer E G, Culp L A
Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
Exp Cell Res. 1991 Mar;193(1):101-11. doi: 10.1016/0014-4827(91)90543-4.
Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.
F11神经杂交细胞（神经母细胞瘤×背根神经节神经元）的亚克隆在三种底物上分离出不同和/或重叠的神经突生成机制，这三种底物分别是具有多种受体活性的血浆纤连蛋白（pFN）、用于结合神经节苷脂GM1的霍乱毒素B亚基（CTB）以及用于结合硫酸乙酰肝素蛋白聚糖的血小板因子-4（PF4）。在本研究中，使用代表三种分化类型（仅在pFN上、仅在CTB上或在所有三种底物上形成神经突）的F11亚克隆，通过几种实验范式测试了三种底物的特定细胞表面受体活性在神经突生成过程中的调节作用。当在活跃期将放线菌酮加入培养基以抑制蛋白质合成时，所有三种底物上的所有亚克隆的神经突形成均显著增加，实际上消除了由细胞表面整合素、神经节苷脂GM1或硫酸乙酰肝素蛋白聚糖介导的分化对底物的选择性。因此，一种或多种不稳定蛋白（称为去整合素）必须调节介导神经突形成的基质受体（如整合素）的功能。为了验证放线菌酮诱导的神经突生成是否也受整合素与细胞表面GM1相互作用的调节，采用了两种方法。当将含（精氨酸-甘氨酸-天冬氨酸-丝氨酸）的肽A加入培养基时，它完全抑制了所有亚克隆在所有三种底物上的放线菌酮诱导的神经突生成，表明在这些机制中对细胞表面整合素功能有严格要求。相反，当CTB或抗GM1单克隆抗体也加入培养基时，放线菌酮诱导的神经突生成在pFN上进一步增强，并且对肽A抑制的敏感性消失。因此，在某些情况下，神经节苷脂GM1必须在细胞表面与整合素受体复合以调节其功能。这些结果还表明：（a）放线菌酮处理导致神经突生成中底物选择性的丧失；（b）神经突生长的这种负调节受整合素受体与不稳定调节蛋白（去整合素）以及与GM1的关联影响；（c）多价GM1结合蛋白对GM1的复合将神经突生成从依赖RGDS的整合素机制转变为不依赖RGDS的受体机制。
