Mechanisms Regulating GPCR Trafficking

描述（由申请人提供）：G蛋白偶联受体（GPCR）包括哺乳动物基因组中最大的细胞表面信号传导受体家族，介导对不同刺激的细胞反应并控制大量生理反应。GPCR信号转导失调与多种人类病理学状况有关，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，GPCR运输对于受体信号传导的时间和空间控制至关重要。然而，对GPCR通过内吞系统运输的机制仍然知之甚少。激活的GPCR的内化通过网格蛋白和动力蛋白依赖性途径发生，需要抑制蛋白。然而，抑制蛋白对于所有GPCR的内化不是必需的。包括蛋白酶激活受体-1（PAR 1）在内的几种GPCR通过不依赖于抑制蛋白的网格蛋白和动力蛋白依赖性途径内化，表明不同的机制调节不同GPCR的内化。一旦内化，GPCR被去磷酸化并以重新敏化的状态再循环回到细胞表面，能够再次发出信号或分选到溶酶体降解途径，这是终止受体信号传导的关键过程。其他GPCR保留在内吞区室中，并且信号独立于G蛋白。我们之前表明，与大多数内化和再循环的GPCR不同，活化的PAR 1被内化，直接分选到溶酶体并降解;这是关闭活化的PAR 1信号传导的关键过程。PAR降解的效率使得该受体类成为研究GPCR溶酶体降解的分子基础的极好模型系统。许多GPCR包括蛋白酶激活受体-2（PAR 2）被泛素修饰，泛素促进溶酶体通过转运所需的内体分选复合物（ESCRT）途径转运。然而，几种GPCR分选溶酶体独立于泛素化和ESCRT复合物的一些组分。值得注意的是，我们发现激活的PAR 1从内体运输到溶酶体，不依赖于泛素化和泛素结合的ESCRT组分。PAR 1和其他GPCR不依赖于泛素化而分选到溶酶体的分子机制尚不清楚。该建议的重点是描绘的分子机制，调节泛素非依赖性的GPCR的溶酶体分选。该提案的具体目标是：1）确定通过泛素依赖性与非依赖性溶酶体途径分选的GPCR是否通过相同或不同的网格蛋白包被的凹点内化，并通过相同或不同的早期或晚期内体区室分选，2）定义GPCR的新型泛素非依赖性内体至溶酶体分选的分选信号和机制，以及3）描绘了GPCR独特地分选为多泡体（MVB）的腔内囊泡（ILV）的分子途径，而不依赖于泛素化和泛素结合的ESCRT组分。 公共卫生相关性：G蛋白偶联受体（GPCR）响应于不同的刺激而发出信号，控制大量的生理反应，并且与多种人类病理疾病有关。除了快速GPCR脱敏，GPCR运输对于受体信号传导的空间和时间控制是重要的，并且失调的运输有助于异常信号传导和疾病。因此，了解控制GPCR贩运的机制，并开发操纵它的能力，可能为预防和治疗各种人类疾病提供新的策略。

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.