Cell Signaling by Protease-activated G Protein-coupled Receptors

总结 我们的NIGMS支持的研究计划的总体目标是描绘监管 通过蛋白酶激活受体1（PAR 1）和密切相关的家族控制信号传导的机制 正常生理和疾病的成员。我们的研究发现， PAR 1促进p38丝裂原活化蛋白激酶（MAPK）信号从核内体，而不 溶酶体降解P2 Y1受体的泛素化功能类似。这项工作进一步揭示了 PAR 1泛素化在p38介导的内皮屏障破坏中的独特功能。然后我们 发现了PAR 1的一种新的溶酶体途径，该途径也与其他G蛋白偶联受体相关 （GPCR）。与传统观点相反，我们发现某些GPCR的泛素化和典型的 泛素结合ESCRT不是受体溶酶体降解所必需的。然后我们确定了ALG- 相互作用蛋白（阿利克斯）和抑制蛋白相关结构域包含蛋白-3（ARRDC 3）作为关键介质， GPCR溶酶体分选。ALIX依赖性途径绕过了GPCR的要求 泛素化，并且不同于转运所需的典型内体分选复合物 （ESCRT）溶酶体途径。阿利克斯和ARRDC 3通路似乎在癌症中也失调。 这些研究的中心前提是，泛素化提供了新颖而多样的机制 用于GPCR生物学的调节。深入了解主要监管机构和 泛素化介导物调节GPCR信号传导和运输对于理解 疾病中的失调机制和识别新的药物靶点。拟议的研究将允许 我的研究小组掌握了我们开创的新概念的透彻理解- GPCR的泛素化调节正常生理学中的信号传导，并在疾病中被破坏。我们的研究 提出了许多令人兴奋的问题，并将进行最具变革性的研究。的p38 MAPK介导炎症，然而尚不清楚GPCR激活的、泛素驱动的p38 信号传导促进内皮屏障渗透性，这是炎症的一个关键特征，并将被确定。 对GPCR内体信号传导如何调节的理解有限，这是一个挑战。因此，在本发明中， 我们将定义调控GPCR激活的、泛素驱动的p38内体信号传导的机制。 几种GPCR通过ALIX依赖性途径而不是典型的ESCRT进行分类，然而， 将GPCR分离到不同的溶酶体途径中的关键步骤是未知的，将被描述。 乳腺癌中ARRDC 3表达缺失或被抑制，但目前尚不清楚ARRDC 3是如何在乳腺癌中表达的。 影响PAR 1功能或导致癌症进展并将进行检查。我们装备精良 鉴于我的实验室的专业知识和经验， 泛素化在正常生理和疾病中调节GPCR信号传导和运输。

Summary The overall goals of our NIGMS-supported research program have been to delineate the regulatory mechanisms that control signaling by protease-activated receptor-1 (PAR1) and closely-related family members in normal physiology and disease. Our efforts have led to the discovery that ubiquitination of PAR1 promotes p38 mitogen-activated protein kinase (MAPK) signaling from endosomes and not lysosomal degradation. Ubiquitination of the P2Y1 receptor functions similarly. This work further revealed a distinct function for ubiquitination of PAR1 in p38-mediated endothelial barrier disruption. We then discovered a novel lysosomal pathway for PAR1 that is also relevant to other G protein-coupled receptors (GPCRs). Contrary to conventional view, we found that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor lysosomal degradation. We then identified ALG- interacting protein (ALIX) and arrestin-related domain containing protein-3 (ARRDC3) as key mediators of GPCR lysosomal sorting. The ALIX-dependent pathway bypasses the requirement for GPCR ubiquitination and is distinct from the canonical endosomal sorting complexes required for transport (ESCRT) lysosomal pathway. The ALIX and ARRDC3 pathway also appears to be dysregulated in cancer. The central premise for the proposed studies is that ubiquitination offers novel and diverse mechanisms for regulation of GPCR biology. A thorough understanding of the mechanism by which key regulators and mediators of ubiquitination regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for identifying new drug targets. The proposed studies will allow my research group to grasp a thorough understanding of novel concepts that we pioneered - that ubiquitination of GPCRs regulates signaling in normal physiology and is disrupted in disease. Our studies have raised many exciting questions and the most transformative research will be pursued. The p38 MAPK mediates inflammation, however it is not known how GPCR-activated, ubiquitin-driven p38 signaling promotes endothelial barrier permeability, a key feature of inflammation, and will be determined. There is a limited understanding of how GPCR endosomal signaling is regulated and is a challenge. Thus, we will define the mechanisms that regulate GPCR-activated, ubiquitin-driven p38 endosomal signaling. Several GPCRs sort through the ALIX-dependent pathway rather than canonical ESCRT, however, the key steps that segregate GPCRs into distinct lysosomal pathways are not known and will be delineated. ARRCD3 expression is either lost or suppressed in breast cancer, however it is not clear how ARRDC3 impacts PAR1 function or contributes to cancer progression and will be examined. We are well equipped to perform these studies given my laboratory's expertise and experience delineating the function of ubiquitination in regulation of GPCR signaling and trafficking in normal physiology and disease.