Nandan D, Cates G A, Ball E H, Sanwal B D
Department of Biochemistry, Faculty of Medicine, University of Western Ontario, London, Canada.
Exp Cell Res. 1988 Nov;179(1):289-97. doi: 10.1016/0014-4827(88)90368-0.
We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.
我们之前已证明一种46 kDa的糖蛋白参与大鼠骨骼肌成肌细胞的分化。我们现在表明，这种糖蛋白与胶原蛋白和明胶的结合会被含精氨酸 - 甘氨酸 - 天冬氨酸（RGD）的肽破坏，但不会被精氨酸 - 甘氨酸 - 谷氨酸（RGE）破坏。前一种肽还能选择性地洗脱与明胶 - 琼脂糖结合的46 kDa糖蛋白。由于所有其他结合RGD序列的蛋白质都已在细胞表面被发现，我们试图通过免疫荧光染色和放射性碘标记来定位46 kDa的糖蛋白。令人惊讶的是，发现大部分蛋白质定位于内质网中。对微粒体部分进行蛋白酶处理表明该蛋白质在内质网内部。使用针对46 kDa蛋白质的多克隆抗体进行的免疫沉淀实验表明，成肌细胞中不存在密切相关的蛋白质，也证实该蛋白质不是细胞表面定位蛋白质的片段。这些发现表明RGD序列也用于细胞内的蛋白质识别。
