Barry S T, Flinn H M, Humphries M J, Critchley D R, Ridley A J
Department of Biochemistry, University of Leicester, UK.
Cell Adhes Commun. 1997 Mar;4(6):387-98. doi: 10.3109/15419069709004456.
Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.
在无血清培养基中对瑞士3T3细胞进行过夜培养会导致粘着斑和相关的肌动蛋白应力纤维消失，尽管细胞仍能很好地铺展。小GTP结合蛋白Rho对于血清饥饿的瑞士3T3细胞中由溶血磷脂酸（LPA）等生长因子诱导的应力纤维和粘着斑的形成，以及LPA诱导的几种粘着斑蛋白的酪氨酸磷酸化是必需的。在没有添加生长因子的情况下，将细胞接种到细胞外基质蛋白上也会刺激蛋白酪氨酸磷酸化以及应力纤维和粘着斑的形成。这些反应在刮擦加载了Rho抑制剂C3转移酶的细胞中受到抑制。添加肽GRGDS也会触发粘着斑和应力纤维的形成，GRGDS被多种整合素识别，并且包含在多种细胞外基质蛋白的细胞结合域内。通过向细胞显微注射C3转移酶可阻断GRGDS肽的活性，这表明肽与整合素的结合会刺激粘着斑的Rho依赖性组装。这些实验表明Rho参与整合素下游的信号传导。
