Subcellular organization of Adhesion G-Protein Coupled Receptor (aGPCR) signalling.

我的研究项目的长期目标是了解G蛋白偶联受体的功能。G蛋白偶联受体(GPCRs)感知和传递来自众多效应分子的信号，在协调细胞对环境快速反应的能力方面发挥着重要作用。具体地说，我的研究重点是了解非规范激活的G蛋白偶联受体(GPCRs)的功能。这项提案将重点放在粘附性G蛋白偶联受体G1(ADGRG1；也称为GPR56)上。ADGRG1是自催化激活的GPCRs黏附家族的成员。*ADGRG1在被称为GPCR蛋白分解位点(GPS)的位置经历了一次自动催化事件，导致细胞表面表达由两条链、一个大的胞外N端片段(ADGRG1-NT)和一个膜结合的C端片段(ADGRG1-CT)组成的异二聚体，这两个片段保持结合和非共价连接。ADGRG-NT被认为是通过与细胞外基质成分的结合和剪切力从ADGRG-CT中去除的，导致ADGRG1-CT去抑制、释放和结合拴系配体和受体信号，通过G-蛋白质和-arrestin。*ADGRG1独特的蛋白分解激活模式导致不可逆转的激活，这些受体通常被描述为“一次完成”GPCRs。这与可溶性激素的可逆结合形成对比，后者能很好地激活GPCRs，如2-肾上腺素能受体(2-AR)或血管紧张素II受体1型(AT1)，这些受体在配体内化和从受体解离后经常循环回到细胞表面。因此，粘附性GPCRs可能参与了与可溶性激素结合GPCRs不同的细胞内分选机制和分区信号传递。*总体假设：由于不可逆的自动催化激活，ADGRG1参与了不同于其他激素激活的GPCR(如2-AR和AT1)的细胞内囊泡运输和信号通路。*为了了解ADGRG1信号和运输，在短期内，我们将：*目标1.定义ADGRG1 C末端的尾部氨基酸序列，调节与--arrestins的相互作用。*目标2.定义ADGRG1内化的囊泡转运途径和从头合成的受体运输到细胞膜的囊泡转运途径。*目的3.定义ADGRG1的细胞表面和内体信号转导系统。*意义：这些研究将解决有关非规范激活的gpr的信号和囊泡运输机制的基本问题。鉴于这类受体的重要生理作用和独特的激活机制，这些研究很可能揭示调控GPCRs信号和内吞命运的途径和机制的多样性。

The long-term objectives of my research program are to understand G-protein coupled receptor function. G-protein-coupled receptors (GPCRs) sense and traduce signals from numerous effector molecules and play an essential role in coordinating the ability of cells to rapidly respond to its environment. Specifically, my research is focused on understanding function of non-canonically activated G-protein coupled receptors (GPCRs). This proposal will focus on the Adhesion G-protein-coupled Receptor-G1 (ADGRG1; also known as GPR56). ADGRG1 is a member of the autocatalytically activated Adhesion Family of GPCRs.******ADGRG1 undergoes an auto-catalytic event at a site called the GPCR proteolysis site (GPS), that results in the cell surface expression of a heterodimer consisting of 2 chains, a large extracellular N-terminal fragment (ADGRG1-NT) and a membrane-bound C-terminal fragment (ADGRG1-CT) which remain associated and non-covalently linked. It is believed that the ADGRG-NT is removed from ADGRG-CT through binding to extracellular matrix components and shear force resulting in ADGRG1-CT disinhibition, release and engagement of a tethered ligand and receptor signalling through G--proteins and --arrestin.******The unique proteolytic mode of activation for ADGRG1 results in irreversible activation and these receptors are often described as “one and done” GPCRs. This is in contrast to the reversible binding of a soluble hormone that activates most well studies GPCRs such as the 2--adrenergic receptor (2-AR) or the angiotensin II receptor type 1 (AT1) that are frequently recycled back to the cell surface following internalization and dissociation of the ligand from the receptor. It follows that adhesion GPCRs might engage intracellular sorting mechanisms and compartmentalized signalling that are distinct from those for soluble hormone binding GPCRs.******Overarching hypothesis: ADGRG1 engages intracellular vesicular trafficking and signalling pathways that are distinct from other hormone activated GPCRs such as 2-AR and AT1 as a result of irreversible auto-catalytic activation.******In order to understand ADGRG1 signalling and trafficking, in the short-term, we will:******Aim 1. Define ADGRG1 C-terminal tail amino acid sequences regulating interaction with the --arrestins.***Aim 2. Define the vesicular trafficking route for internalization of ADGRG1 and the vesicular trafficking route for de-novo synthesized receptor trafficking to the cell membrane.***Aim 3. Define the cell surface and endosomal signalling repertoire for ADGRG1. ******Significance: These studies will address fundamental questions regarding the signaling and vesicular transport mechanisms of a non-canonically activated GPCR. Given the important physiological roles and unique mechanisms of activation of this class of receptors, it is likely that these studies will reveal the diversity of pathways and mechanisms regulating the signaling and endocytic fate of GPCRs.