Arf GTP-binding proteins and membrane traffic

Arf GTP 结合蛋白和膜运输

• 批准号: 7968968
• 负责人: Julie G Donaldson
• 金额: $ 117.67万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7968968
• 关键词: Actins; Adaptor Signaling Protein; Adrenergic Agents; Back; Breast Cancer Cell; CD44 gene; Cancer cell line; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Clathrin; Clathrin Adaptors; Cytoskeleton; Endocytosis; Endocytosis Pathway; Endosomes; Exocytosis; Exposure to; Extracellular Protein; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glucose Transporter; Goals; Hela Cells; Human Cell Line; ICAM1 gene; Insulin; Laboratories; Ligand Binding; Ligands; Lipids; MCF7 cell; Major Histocompatibility Complex; Mass Spectrum Analysis; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Motor; Muscarinic M3 Receptor; Myosin ATPase; Organelles; Pathway interactions; Process; Proteins; Receptor Signaling; Recycling; Research; Role; Route; Secretory Vesicles; Signal Transduction; Sorting - Cell Movement; Structure; System; Transferrin; Transferrin Receptor; Travel; Tubular formation; Vesicle; adrenergic; base; interest; protein function; receptor; trafficking

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. In an attempt to discover the machinery and additional cargo proteins associated with this pathway, we have isolated clathrin-independent endosomes and used mass spectrometry to identify new candidate proteins. We have validated 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1 (Eyster et al 2009). We have confirmed that these proteins enter cells by clathrin-independent endocytosis in many human cell lines and in addition, in HeLa cells, we see a difference in intracellular itinerary followed by these proteins. CD55 and Glut1 follow a similar itinerary as MHCI, merging with endosomes containing the transferrin receptor before routing to the tubular recycling endosomes. By contrast, CD44, 98 and 147 after endocytosis do not enter transferrin-containing endosomes but are routed directly to the recycling endosome (Eyster et al 2009). This divergence in itinerary suggests that clathrin-independent cargo proteins are sorted within the cell and we are currently studying whether signals contained on these cargo proteins (extracellular, transmembrane or cytoplasmic) are directing their trafficking. In another project in the lab, we investigated whether this clathrin-independent endocytic pathway might be utilized for constitutive trafficking of signaling receptors. G protein-coupled receptors (GPCRs) are a large family of seven transmembrane-spanning proteins that respond to many cellular signals or ligands. In most cases, following ligand binding the GPCRs are rapidly internalized into cells by clathrin-dependent endocytosis. We found that both the b2 adrenergic and M3 muscarinic receptors entered cells via clathrin-independent endocytosis in the absence of ligand and traveled along the same recycling pathway as MHCI (Scarselli and Donaldson, 2009). Furthermore, the endogenous G proteins that are activated by these receptors, alpha s and alpha q, were localized also to these membrane systems. In the presence of ligand, now the GPCR entered cells via clathrin-dependent endocytosis, but the G proteins remained at the cell surface and associated with clathrin-independent endosomes. Interestingly, deletion of the third intracellular loop of the M3 receptor results in a receptor capable of full signaling but unable to enter cells via clathrin-mediated endocytosis. This truncated M3 receptor was still rapidly internalized upon exposure to ligand, but now entered cells via clathrin-independent endocytosis (Scarselli and Donaldson, 2009). This demonstrates that GPCRs represent cell surface proteins that can enter different types of endocytic pathways depending on whether the receptor is stimulated with ligand or when ligand is absent where it might undergo constitutive endocytic trafficking. In addition to regulating recycling of endosomal membrane back to the cell surface, Arf6 has also been implicated in some forms of regulated secretion. Both of these processes generally require the actin cytoskeleton for exocytosis of the membrane back to the plasma membrane. In a collaborative study, it was found that myosin Vc is associated with tubular endosomes in breast cancer cell line MCF7 and also with secretory granules (Jacobs et al 2009). Although it is not clear whether myosin Vc is associated with the recycling endosomes in HeLa cells, we are investigating roles for other motor proteins, including microtubular-based motors, in this process

该实验室有兴趣了解与Arf 6相关的不依赖网格蛋白的内吞途径。 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CD59。 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）的内体并与之融合。 从那里，MHCI和CD59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Rab11和22在内的几种其他调节剂的活性。 为了发现与该途径相关的机制和额外的货物蛋白，我们分离了不依赖网格蛋白的内体，并使用质谱法鉴定新的候选蛋白。 我们已经验证了7种新的货物蛋白，它们进入细胞并沿着沿着这种网格蛋白非依赖性途径运输：CD 44、CD 55、CD 98、CD 147、Lat1、ICAM 1和非胰岛素刺激的葡萄糖转运蛋白Glut 1（Eyster et al 2009）。 我们已经证实，这些蛋白质进入细胞的网格蛋白独立的内吞作用在许多人类细胞系，此外，在HeLa细胞中，我们看到细胞内行程的差异，其次是这些蛋白质。 CD55和Glut1遵循与MHCI类似的路线，在路由到管状再循环内体之前与含有转铁蛋白受体的内体合并。 相比之下，内吞作用后的CD 44、98和147不进入含转铁蛋白的内体，而是直接进入再循环内体（Eyster et al 2009）。 这种路线上的差异表明，不依赖网格蛋白的货物蛋白在细胞内被排序，我们目前正在研究这些货物蛋白（细胞外、跨膜或细胞质）上包含的信号是否正在指导它们的运输。 在实验室的另一个项目中，我们研究了这种不依赖网格蛋白的内吞途径是否可用于信号受体的组成性运输。 G蛋白偶联受体（GPCR）是一个由七种跨膜蛋白组成的大家族，它们对许多细胞信号或配体起反应。 在大多数情况下，配体结合后，GPCR通过网格蛋白依赖性内吞作用迅速内化到细胞中。 我们发现b2肾上腺素能受体和M3毒蕈碱受体在不存在配体的情况下通过网格蛋白非依赖性内吞作用进入细胞，并沿着与MHCI相同的再循环途径沿着行进（Scarselli和唐纳森，2009）。 此外，由这些受体α s和α q激活的内源性G蛋白也定位于这些膜系统。 在配体存在的情况下，GPCR通过网格蛋白依赖的内吞作用进入细胞，但G蛋白仍保留在细胞表面并与网格蛋白非依赖的内体结合。 有趣的是，M3受体的第三胞内环的缺失导致能够完全信号传导但不能通过网格蛋白介导的内吞作用进入细胞的受体。 这种截短的M3受体在暴露于配体后仍迅速内化，但现在通过网格蛋白非依赖性内吞作用进入细胞（Scarselli和唐纳森，2009）。 这表明GPCR代表细胞表面蛋白，其可以进入不同类型的内吞途径，这取决于受体是否被配体刺激或当配体不存在时，其可能经历组成性内吞运输。 除了调节内体膜再循环回到细胞表面之外，Arf 6还涉及某些形式的调节分泌。这两个过程通常需要肌动蛋白细胞骨架的胞吐作用的膜回到质膜。 在一项合作研究中，发现肌球蛋白Vc与乳腺癌细胞系MCF7中的管状内体以及分泌颗粒相关（Jacobs et al 2009）。 虽然还不清楚肌球蛋白Vc是否与HeLa细胞内体的再循环有关，但我们正在研究其他马达蛋白，包括微管马达，在这一过程中的作用