Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 7967359
• 负责人: James Hurley
• 金额: $ 28.82万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/7967359
• 关键词: Adaptor Signaling Protein; Affinity; Binding; Binding Sites; Biological Assay; C-terminal; Cells; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Endocytosis; Eukaryotic Cell; Family; Goals; Human; In Vitro; Integral Membrane Protein; Laboratories; Link; Measures; Membrane; N-terminal; Names; Pathway interactions; Play; Proteins; Role; Shapes; Signal Transduction; Sorting - Cell Movement; Structure; Vesicle; Yeasts; golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein; in vivo; member; molecular recognition; protein complex; receptor; scaffold

Molecular Recognition by Clathrin Adaptors The clathrin coat plays a ubiquitous and fundamental role in endocytosis and in endosomal sorting within the eukaryotic cell. Clathrin forms a cage that surrounds cargo-bearing vesicles, but clathrin itself does not directly bind to cargo. Cargo is sorted into clathrin-coated vesicles by adaptor proteins that physically bridge cargo and clathrin. The best-known general purpose adaptors are the heterotetrameric adaptor protein complexes (AP complexes) and the multimodular GGA adaptor proteins. The overall goals of this project are 1) to identify the binding sites for cargo on adaptor proteins and measure their affinities quantitatively; 2) to determine the crystal structures of complexes between adaptors and soluble cargo fragments; and 3) to relate structure and function using mutational analysis. The subunits of the AP complexes and the GGAs define conserved structural domains that are also found in other endocytic proteins. In the past year, we collaborated with Beverly Wendland (JHU) to define the structures of the domains of the endocytic protein Syp1. Syp1 is proposed to be a founding member of the newly-named muniscin family of endocytic adaptors that is conserved from yeast to humans. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding mu homology domains (muHD). In vitro and in vivo assays in the Wendland laboratory confirmed membrane tubulation activity for muniscin EFC/F-BAR domains. The mHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. A transmembrane protein previously implicated in polarized Rho1 signaling was identified as a cargo of the yeast adaptor protein. We propose that muniscins directly link cargo selection with vesicle formation, thereby regulating receptors that stimulate cell polarization pathways.

网格蛋白衔接子的分子识别 网格蛋白外壳在真核细胞内的内吞作用和内体分选中起着普遍存在的和基本的作用。网格蛋白形成一个笼，围绕货物轴承囊泡，但网格蛋白本身不直接结合货物。货物通过衔接蛋白被分选到网格蛋白包被的囊泡中，衔接蛋白物理桥接货物和网格蛋白。最著名的通用衔接子是异源四聚体衔接子蛋白复合物（AP复合物）和多模块GGA衔接子蛋白。该项目的总体目标是：1）鉴定衔接蛋白上货物的结合位点并定量测量其亲和力; 2）确定衔接蛋白和可溶性货物片段之间复合物的晶体结构; 3）使用突变分析将结构和功能联系起来。 AP复合物和GGA的亚基定义了在其他内吞蛋白中也发现的保守结构域。在过去的一年中，我们与Beverly Wendland（JHU）合作，定义了内吞蛋白Syp 1结构域的结构。Syp 1被认为是新命名的muniscin家族的内吞衔接子的创始成员，该家族在酵母和人类之间是保守的。解析酵母免疫球蛋白结构域的结构证实了与新月形膜微管EFC/F-BAR结构域同源的N-末端结构域和与货物结合μ同源结构域（muHD）同源的C-末端结构域的独特组合。Wendland实验室的体外和体内测定证实了muniscin EFC/F-BAR结构域的膜滋养活性。mHD结构域与内吞适配器/支架Ede 1/eps 15具有保守的相互作用，其影响免疫球蛋白定位。一个跨膜蛋白先前牵连在极化Rho 1信号被确定为货物的酵母衔接蛋白。我们提出，免疫球蛋白直接连接货物选择与囊泡形成，从而调节受体刺激细胞极化途径。