Mechanisms Regulating GPCR Trafficking

描述(申请人提供)：G蛋白偶联受体(GPCRs)组成哺乳动物基因组中最大的细胞表面信号受体家族，介导细胞对不同刺激的反应，并控制广泛的生理反应。GPCR信号失控与多种人类病理情况有关，使该受体类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间和空间控制是至关重要的。然而，负责通过内吞系统贩运GPCRs的机制仍然知之甚少。激活的GPCRs的内化通过依赖于笼蛋白和动力素的途径发生，需要抑制素。然而，逮捕并不是所有GPCR内部化的必要条件。包括蛋白酶激活受体-1(PAR1)在内的几个GPCRs通过依赖于笼蛋白和动力素的途径内化，而不依赖于拦阻蛋白，这表明不同的GPCRs内化调控机制不同。一旦内化，GPCRs被去磷酸化，并在重新敏感的状态下循环回到细胞表面，能够再次发出信号，或者被分类到溶酶体降解途径，这是终止受体信号的关键过程。其他GPCRs仍然存在于胞内隔间，信号不依赖于G蛋白。我们之前的研究表明，与大多数GPCRs不同的是，激活的PAR1被内化并循环，直接内化、分类到溶酶体并降解；这是关闭激活的PAR1信号的关键过程。PARs的降解效率使该受体类成为研究GPCR溶酶体降解的分子基础的极佳模型系统。包括蛋白酶激活受体-2(PAR2)在内的许多GPCRs都被泛素修饰，泛素促进溶酶体通过运输所需的内体分选复合体(ESCRT)途径转运。然而，一些GPCRs分类为溶酶体，与泛素化和ESCRT复合体的某些成分无关。值得注意的是，我们发现激活的PAR1不依赖于泛素化和泛素结合ESCRT组件从内体到溶酶体。PAR1和其他GPCRs独立于泛素化而分类为溶酶体的分子机制尚不清楚。这项建议的重点是描述调控泛素非依赖的GPCRs溶酶体分选的分子机制。该建议的具体目的是：1)确定通过泛素依赖和非依赖溶酶体途径进行分选的GPCRs是否通过相同或不同的网织蛋白包裹的凹坑内化，并通过相同或不同的早期或晚期内小体隔室进行分选；2)确定新的泛素非依赖性内小体到溶酶体分选的信号和机制，以及3)描述GPCRs唯一分选不依赖泛素化和泛素结合ESCRT组分的多囊体内小泡(ILV)的分子途径。 与公共健康相关：G蛋白偶联受体(GPCRs)对不同的刺激信号作出反应，控制着广泛的生理反应，并与多种人类病理疾病有关。除了快速脱敏外，GPCR转运对于受体信号的空间和时间控制也很重要，而失调的转运会导致异常信号和疾病。因此，了解控制GPCRs贩运的机制，并发展操纵它的能力，可能会为预防和治疗各种人类疾病提供新的战略。

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.