Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima, 727-0023, Japan.
Division of Bioscience and Biotechnology Department of Agricultural and Life Sciences, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano, 399-4598, Japan.
Cell Mol Biol Lett. 2021 Apr 30;26(1):16. doi: 10.1186/s11658-021-00260-1.
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.
在哺乳动物中，有两种 Grb2 相关的 ERK/MAPK 调节因子（GAREM）亚型，这是一种位于细胞生长因子受体下游的衔接蛋白。GAREM1 广泛表达，而 GAREM2 主要在大脑中表达。然而，在生长因子刺激的细胞中，每种 GAREM 亚型的易位的确切机制仍不清楚。
在这项研究中，使用针对每种 GAREM 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，以分析每种 GAREM 亚型在细胞生长刺激依赖性下的亚细胞定位。我们还对其亚细胞定位与 GAREM2 的酪氨酸磷酸化之间的相关性进行了生化分析。
我们发现，内源性和外源性表达的 GAREM2 特异性地在 NGF 刺激的 PC-12 细胞和 EGF 刺激的 COS-7 细胞中聚集并形成颗粒。基于观察到的嵌合 GAREM1 和 GAREM2 蛋白的亚细胞定位，仅在 GAREM2 中存在的富含甘氨酸的区域是观察到的颗粒形成所必需的。该区域还调节 EGF 刺激依赖性的 GAREM2 酪氨酸磷酸化的程度。
我们的结果表明，GAREM2 对 EGF 刺激的聚集依赖于富含甘氨酸的区域，这表明 GAREM2 的聚集可能与神经退行性疾病有关。
