Arf GTP-binding proteins and membrane traffic

该实验室有兴趣了解与Arf 6相关的网格蛋白独立内吞途径。 我们已经表明，这一途径是负责内化质膜（PM）蛋白，缺乏允许识别的网格蛋白和衔接蛋白机器的序列。 通过这种内吞作用进入细胞的蛋白质包括主要组织相容性复合物I类蛋白（MHCI）和脂质锚定蛋白CD 59。 在胞吞作用之后，囊泡中的MHCI和⑶ 59被递送至含有来自网格蛋白依赖性胞吞作用途径的货物蛋白（如转铁蛋白受体）的内体并与其融合。 从那里，MHCI和CD 59可以继续进入晚期内体区室，在那里它们被降解，或者它们可以通过独特的管状再循环内体再循环回到PM。 在HeLa细胞中，这些再循环内体仅包含通过网格蛋白非依赖性内吞作用进入的货物，并且它们返回PM取决于Arf 6和包括Rab 11和22在内的几种其他调节剂的活性。 我们以前已经表明，除了Arf 6，Rac，Rho GTP结合蛋白，改变PM肌动蛋白细胞骨架，与网格蛋白独立的内体膜，并在其激活导致PM皱褶和巨胞饮，刺激形式的网格蛋白独立的内吞作用。 最近，我们研究了其他信号分子（除了Arf 6和Rac）是否可以与这个膜系统。 我们发现H-Ras与这些膜相关并与MHCI共定位，但随后在EGF处理后，H-Ras被激活，导致PM皱褶和诱导巨胞饮作用（Porat-Shliom等2008）。 由于已知H-Ras激活导致磷脂酰肌醇3-激酶的激活，因此我们研究了巨胞饮过程中磷酸肌醇组合物的变化。 在表达H-Ras组成型活性突变体的细胞中，传入的巨胞饮体最初含有磷脂酰肌醇4，5-二磷酸（PIP 2）和磷脂酰肌醇3，4，5-三磷酸（PIP 3）。 在大胞饮体成熟过程中，PIP 2首先丢失，随后是Rab 5的募集，然后是PIP 3的丢失。 磷酸肌醇含量的这些连续变化也见于Arf 6活化形成的巨胞饮体。 然而，响应于Arf 6或Ras的激活而形成的大胞饮体确实募集不同的效应物，例如Ras募集Erk和Akt。 在另一个项目中，我们分离了网格蛋白独立的内体，试图鉴定与该途径相关的机械和额外的货物蛋白。 质谱用于鉴定新的候选蛋白质。 我们已经验证了7个新的货物蛋白进入细胞和交通沿着这个网格蛋白的独立途径。 新的蛋白质是：CD 44，CD 55，CD 98，CD 147，Lat 1，ICAM 1和非胰岛素刺激的葡萄糖转运蛋白Glut 1。 我们目前正在详细研究它们在细胞内的内吞和运输模式。

The laboratory is interested in understanding the clathrin-independent endocytosis pathway that is associated with Arf6. We have shown that this pathway is responsible for internalizing plasma membrane (PM) proteins that lack sequences that allow recognition by the clathrin and adaptor protein machinery. Among the proteins that enter cells via this mode of endocytosis are the major histocompatibility complex Class I protein (MHCI) and the lipid anchored protein CD59. After endocytosis, MHCI and CD59 in vesicles are delivered to and fuse with endosomes containing cargo proteins from the clathrin-dependent endocytosis pathway such as transferrin receptor. From there, MHCI and CD59 can proceed on to late endosomal compartments where they are degraded or they can be recycled back out to the PM via unique tubular recycling endosomes. In HeLa cells these recycling endosomes contain only cargo that had entered via clathrin-independent endocytosis and their return to the PM is dependent upon the activity of Arf6 and several other regulators including Rab11 and 22. We had previously shown that in addition to Arf6, Rac, a Rho GTP-binding protein that alters PM actin cytoskeleton, was associated with the clathrin-independent endosomal membranes and upon its activation resulted in PM ruffling and macropinocytosis, a stimulated form of clathrin-independent endocytosis. Recently we examined whether other signaling molecules (in addition to Arf6 and Rac) could be associated with this membrane system. We found that H-Ras is associated with these membranes and colocalizes with MHCI but then upon EGF treatment H-Ras is activated, resulting in PM ruffling and induction of macropinocytosis (Porat-Shliom et al 2008). Since H-Ras activation is known to result in the activation of a phosphotidylinositol 3-kinase, we examined the changes in phosphoinositide composition during the process of macropinocytosis. In cells expressing constitutively active mutant of H-Ras incoming macropinosomes initially contained phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). During maturation of the macropinosome, PIP2 was lost first, followed by the recruitment of Rab5 and then loss of PIP3. These sequential changes in phosphoinositide content were also seen in macropinosomes formed by activation of Arf6. The macropinosomes that form in response to activation of Arf6 or Ras do however recruit distinct effectors, for example Ras recruits Erk and Akt. In another project, we have isolated clathrin-independent endosomes in an attempt to identify machinery and additional cargo proteins associated with this pathway. Mass spectroscopy was used to identify new candidate proteins. We have validated 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway. The new proteins are: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1. We are currently examining in detail their pattern of endocytosis and trafficking within cells.