Mechanisms Regulating GPCR Trafficking

描述（由申请人提供）：G蛋白偶联受体（gpcr）是哺乳动物基因组中最大的细胞表面信号受体家族，介导细胞对各种刺激的反应并控制大量的生理反应。失调的GPCR信号与多种人类病理状况有关，使这类受体成为临床使用的近一半药物的靶点。除了快速脱敏外，GPCR转运对于受体信号的时空控制至关重要。然而，通过内吞系统运输gpcr的机制仍然知之甚少。激活的gpcr内化通过网格蛋白和动力蛋白依赖的途径发生，需要抑制。然而，逮捕并不是所有gpcr内化的必要条件。包括蛋白酶激活受体-1 （PAR1）在内的几种gpcr通过网格蛋白和动力蛋白依赖的途径内化，不依赖于抑制因子，这表明不同的gpcr内化机制不同。一旦内化，gpcr被去磷酸化并以重敏状态循环回到细胞表面，能够再次发出信号或被分类到溶酶体降解途径，这是一个对受体信号终止至关重要的过程。其他gpcr留在内吞室中，信号独立于g蛋白。我们之前的研究表明，与大多数内化和再循环的gpcr不同，活化的PAR1被内化，直接分类到溶酶体并被降解；这一过程对于关闭激活的PAR1信号至关重要。PARs被降解的效率使这类受体成为研究GPCR溶酶体降解分子基础的良好模型系统。包括蛋白酶活化受体-2 （PAR2）在内的许多gpcr都被泛素修饰，泛素有助于溶酶体通过运输所需的内体分选复合物（ESCRT）途径进行运输。然而，一些gpcr可以独立于泛素化和ESCRT复合体的某些组分来排序溶酶体。值得注意的是，我们发现活化的PAR1从核内体转运到溶酶体，不依赖于泛素化和泛素结合的ESCRT成分。PAR1和其他gpcr独立于泛素化而向溶酶体排序的分子机制尚不清楚。这一建议的重点是描述调控泛素非依赖性溶酶体分选的gpcr的分子机制。该建议的具体目的是：1)确定通过泛素依赖型和非依赖型溶酶体途径进行分选的gpcr是否通过相同或不同的网格蛋白包覆的凹穴进行内化，并通过相同或不同的早期或晚期内体室进行分选；2)定义新型泛素非依赖型gpcr内体到溶酶体分选的分选信号和机制；3)描述了GPCRs对独立于泛素化和泛素结合ESCRT成分的多泡体（MVBs）的腔内囊泡（ILVs）的独特排序的分子途径。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) comprise the largest family of cell surface signaling receptors in the mammalian genome, mediate cellular responses to diverse stimuli and control a vast physiological responses. Dysregulated GPCR signaling has been implicated in multiple human pathological conditions, making this receptor class the target of nearly half the drugs used clinically. In addition to rapid desensitization, GPCR trafficking is crucial for the temporal and spatial control of receptor signaling. However, the mechanisms responsible for trafficking of GPCRs through the endocytic system remains poorly understood. Internalization of activated GPCRs occurs through a clathrin- and dynamin-dependent pathway that requires arrestins. However, arrestins are not essential for internalization of all GPCRs. Several GPCRs including protease- activated receptor-1 (PAR1) internalize through a clathrin- and dynamin-dependent pathway independent of arrestins, suggesting that distinct mechanisms regulate internalization of different GPCRs. Once internalized, GPCRs are dephosphorylated and recycled back to the cell surface in a resensitized state competent to signal again or sorted to a lysosomal degradation pathway, a process critical for termination of receptor signaling. Other GPCRs remain in endocytic compartments and signal independent of G-proteins. We previously showed that unlike most GPCRs, which internalize and recycle, activated PAR1 is internalized, sorted directly to lysosomes and degraded; a process critical for shutting-off activated PAR1 signaling. The efficiency with which PARs are degraded makes this receptor class an excellent model system to investigate the molecular basis of GPCR lysosomal degradation. Many GPCRs including protease-activated receptor-2 (PAR2) are modified with ubiquitin, which facilitates lysosomal trafficking through the endosomal-sorting complex required for transport (ESCRT) pathway. However, several GPCRs sort to lysosomes independent of ubiquitination and some components of the ESCRT complex. Remarkably, we found that activated PAR1 trafficks from endosomes to lysosomes independent of ubiquitination and ubiquitin-binding ESCRT components. The molecular mechanism by which PAR1 and other GPCRs sort to lysosomes independent of ubiquitination is not known. This proposal is focused on delineating the molecular mechanisms that regulate ubiquitin-independent lysosomal sorting of GPCRs. The specific aims of the proposal are to: 1) determine whether GPCRs that sort through ubiquitin- dependent versus -independent lysosomal pathways internalize through the same or distinct clathrin-coated pits, and sort through the same or distinct early or late endosomal compartments, 2) define the sorting signals and mechanism for the novel ubiquitin-independent endosome-to-lysosome sorting of GPCRs, and 3) delineate the molecular pathway by which GPCRs uniquely sort to intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) independent of ubiquitination and the ubiquitin-binding ESCRT components. PUBLIC HEALTH RELEVANCE: G protein-coupled receptors (GPCRs) signal in response to diverse stimuli, control vast physiological responses and are implicated in multiple human pathological diseases. In addition to rapid GPCR desensitization, GPCR trafficking is important for the spatial and temporal control of receptor signaling and dysregulated trafficking contributes to aberrant signaling and disease. Thus, understanding the mechanisms that control trafficking of GPCRs, and developing the ability to manipulate it, may provide new strategies for the prevention and treatment of a wide range of human diseases.