Conklin Matthew W, Ada-Nguema Aude, Parsons Maddy, Riching Kristin M, Keely Patricia J
Dept of Pharmacology, Laboratory for Molecular Biology and University of Wisconsin Carbone Cancer Center, University of Wisconsin, 1525 Linden Dr, Madison, WI 53706, USA.
BMC Cell Biol. 2010 Feb 18;11:14. doi: 10.1186/1471-2121-11-14.
Integrin-mediated cell adhesion and spreading is dramatically enhanced by activation of the small GTPase, R-Ras. Moreover, R-Ras localizes to the leading edge of migrating cells, and regulates membrane protrusion. The exact mechanisms by which R-Ras regulates integrin function are not fully known. Nor is much known about the spatiotemporal relationship between these two molecules, an understanding of which may provide insight into R-Ras regulation of integrins.
GFP-R-Ras localized to the plasma membrane, most specifically in membrane ruffles, in Cos-7 cells. GFP-R-Ras was endocytosed from these ruffles, and trafficked via multiple pathways, one of which involved large, acidic vesicles that were positive for Rab11. Cells transfected with a dominant negative form of GFP-R-Ras did not form ruffles, had decreased cell spreading, and contained numerous, non-trafficking small vesicles. Conversely, cells transfected with the constitutively active form of GFP-R-Ras contained a greater number of ruffles and large vesicles compared to wild-type transfected cells. Ruffle formation was inhibited by knock-down of endogenous R-Ras with siRNA, suggesting that activated R-Ras is not just a component of, but also an architect of ruffle formation. Importantly, beta1-integrin co-localized with endogenous R-Ras in ruffles and endocytosed vesicles. Expression of dominant negative R-Ras or knock down of R-Ras by siRNA prevented integrin accumulation into ruffles, impaired endocytosis of beta1-integrin, and decreased beta1-integrin-mediated adhesion. Knock-down of R-Ras also perturbed the dynamics of another membrane-localized protein, GFP-VSVG, suggesting a more global role for R-Ras on membrane dynamics. However, while R-Ras co-internalized with integrins, it did not traffic with VSVG, which instead moved laterally out of ruffles within the plane of the membrane, suggesting multiple levels of regulation of and by R-Ras.
Our results suggest that integrin function involves integrin trafficking via a cycle of membrane protrusion, ruffling, and endocytosis regulated by R-Ras, providing a novel mechanism by which integrins are linked to R-Ras through control of membrane dynamics.
小GTP酶R-Ras的激活可显著增强整合素介导的细胞黏附与铺展。此外，R-Ras定位于迁移细胞的前沿，并调节膜突出。R-Ras调节整合素功能的确切机制尚不完全清楚。这两种分子之间的时空关系也知之甚少，而了解这一点可能有助于深入了解R-Ras对整合素的调节。
在Cos-7细胞中，绿色荧光蛋白标记的R-Ras（GFP-R-Ras）定位于质膜，最显著地定位于膜皱褶中。GFP-R-Ras从这些皱褶处被内吞，并通过多种途径运输，其中一条途径涉及对Rab11呈阳性的大型酸性囊泡。用GFP-R-Ras的显性负性形式转染的细胞不形成皱褶，细胞铺展减少，并且含有许多不进行运输的小囊泡。相反，与野生型转染细胞相比，用GFP-R-Ras的组成型活性形式转染的细胞含有更多的皱褶和大型囊泡。用小干扰RNA（siRNA）敲低内源性R-Ras可抑制皱褶形成，这表明活化的R-Ras不仅是皱褶形成的一个组成部分，而且是皱褶形成的构建者。重要的是，β1整合素与内源性R-Ras在皱褶和内吞囊泡中共定位。显性负性R-Ras的表达或用siRNA敲低R-Ras可阻止整合素在皱褶中积累，损害β1整合素的内吞作用，并降低β1整合素介导的黏附。敲低R-Ras还扰乱了另一种膜定位蛋白绿色荧光蛋白标记的水泡性口炎病毒糖蛋白（GFP-VSVG）的动力学，这表明R-Ras在膜动力学方面具有更广泛的作用。然而，虽然R-Ras与整合素共同内化，但它不与VSVG一起运输，VSVG而是在膜平面内从皱褶横向移出，这表明R-Ras存在多层次的调节以及对R-Ras的调节。
我们的结果表明，整合素功能涉及通过由R-Ras调节的膜突出、皱褶形成和内吞循环进行的整合素运输，这提供了一种通过控制膜动力学将整合素与R-Ras联系起来的新机制。
