In mammals, there are two subtypes of Grb2-associated regulator of Erk/MAPK (GAREM), an adaptor protein that functions downstream of the cell growth factor receptor. GAREM1 is ubiquitously expressed, whereas GAREM2 is mainly expressed in the brain. However, the precise mechanism of the translocation of each GAREM subtype in growth factor-stimulated cells is still unclear. In this study, immunofluorescence staining with specific antibodies against each GAREM subtype and time-lapse analysis using GFP fusion proteins were used to analyze the subcellular localization of each GAREM subtype in a cell growth stimulus-dependent manner. We also biochemically analyzed the correlation between its subcellular localization and tyrosine phosphorylation of GAREM2. We found that endogenously and exogenously expressed GAREM2 specifically aggregated and formed granules in NGF-stimulated PC-12 cells and in EGF-stimulated COS-7 cells. Based on the observed subcellular localizations of chimeric GAREM1 and GAREM2 proteins, a glycine-rich region, which is present only in GAREM2, is required for the observed granule formation. This region also regulates the degree of EGF-stimulation-dependent tyrosine phosphorylation of GAREM2. Our results, showing that aggregation of GAREM2 in response to EGF stimulation is dependent on a glycine-rich region, suggest that GAREM2 aggregation may be involved in neurodegenerative diseases.

中文翻译：

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GAREM2 中富含甘氨酸的结构域对其独特的亚细胞定位对 EGF 刺激的影响

在哺乳动物中，Erk/MAPK (GAREM) 的 Grb2 相关调节剂有两种亚型，这是一种在细胞生长因子受体下游起作用的衔接蛋白。GAREM1 普遍表达，而 GAREM2 主要在大脑中表达。然而，在生长因子刺激的细胞中每个 GAREM 亚型易位的确切机制仍不清楚。在这项研究中，使用针对每种 GAEM 亚型的特异性抗体进行免疫荧光染色，并使用 GFP 融合蛋白进行延时分析，以细胞生长刺激依赖性方式分析每种 GAREM 亚型的亚细胞定位。我们还生化分析了其亚细胞定位与 GAREM2 酪氨酸磷酸化之间的相关性。我们发现内源性和外源性表达的 GAREM2 在 NGF 刺激的 PC-12 细胞和 EGF 刺激的 COS-7 细胞中特异性聚集并形成颗粒。根据观察到的嵌合 GAREM1 和 GAREM2 蛋白的亚细胞定位，观察到的颗粒形成需要一个仅存在于 GAREM2 中的富含甘氨酸的区域。该区域还调节 GAREM2 的 EGF 刺激依赖性酪氨酸磷酸化的程度。我们的结果表明，GAREM2 响应 EGF 刺激的聚集依赖于富含甘氨酸的区域，表明 GAREM2 聚集可能与神经退行性疾病有关。仅存在于 GAREM2 中，是观察到的颗粒形成所必需的。该区域还调节 GAREM2 的 EGF 刺激依赖性酪氨酸磷酸化的程度。我们的结果表明，GAREM2 响应 EGF 刺激的聚集依赖于富含甘氨酸的区域，表明 GAREM2 聚集可能与神经退行性疾病有关。仅存在于 GAREM2 中，是观察到的颗粒形成所必需的。该区域还调节 GAREM2 的 EGF 刺激依赖性酪氨酸磷酸化的程度。我们的结果表明，GAREM2 响应 EGF 刺激的聚集依赖于富含甘氨酸的区域，表明 GAREM2 聚集可能与神经退行性疾病有关。