Summary The long-term goal of this proposal is to understand the regulation of G protein-coupled receptor (GPCR) signaling by endocytic trafficking. GPCRs comprise the largest family of signaling receptors expressed in the mammalian genome, mediate cellular responses to diverse stimuli and control vast physiological responses. Dysregulated GPCR signaling has been implicated in neurological disorders, cardiovascular diseases and cancer progression, making this receptor class the target of nearly half the drugs used clinically. In addition to desensitization, GPCR trafficking is crucial for the temporal and spatial control of receptor signaling. This is best exemplified by protease-activated receptor-1 (PAR1), a GPCR for the coagulant protease thrombin. PAR1 has important functions in vascular physiology, development and tumor progression and is an important drug target. Similar to most GPCRs, signaling by activated PAR1 is rapidly desensitized. We also found that activated PAR1 internalization and lysosomal sorting is critical for the fidelity of thrombin signaling and appropriate cellular responses. We further discovered that activated PAR1 trafficking is dysregulated in metastatic breast carcinoma, consequently the receptor recycles to the cell surface, signals persistently and promotes tumor progression. The mechanisms responsible for dysregulation of PAR1 trafficking are not known and important to understand. Many GPCRs are modified with ubiquitin and sorted to lysosomes through interactions with ubiquitin-binding components of the ESCRT machinery. However, not all GPCRs require direct ubiquitination for lysosomal sorting including PAR1. We recently discovered a novel lysosomal sorting pathway that bypasses the requirement for receptor ubiquitination and ubiquitin-binding ESCRTs that is mediated by ALIX. ALIX, a CHMP4/ESCRT-III interacting protein, bound to an YPX3L motif of PAR1 via its V domain and mediated PAR1 lysosomal sorting. We also identified a subset of class A GPCRs containing YPXnL motifs, suggesting that this pathway may be applicable to other GPCRs. Our preliminary studies further indicate that the ALIX-interacting protein arrestin-domain containing protein -3 (ARRDC3) regulates PAR1 degradation. ARRDC3 appears to function as a tumor suppressor and its expression is lost in invasive breast carcinoma that exhibit dysregulated PAR1 trafficking. Moreover, ectopic expression of ARRDC3 promoted lysosomal sorting of PAR1 in invasive breast carcinoma. The proposed studies will advance our understanding of how ARRDC3 and ALIX function to regulate GPCR intracellular trafficking and signaling in normal and cancer cells. The specific aims of the proposal are to: 1) determine whether ARRDC3 regulates GPCR sorting to late endosomes/multivesicular bodies, 2) delineate the regulatory mechanisms of ALIX function in lysosomal sorting of GPCRs, and 3) examine the role of ARRDC3 in dysregulated GPCR trafficking in cancer.