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.

Public Health Relevance

The proposed research is relevant to public health because it seeks to understand the regulation of G protein-coupled receptor (GPCR) signaling. GPCRs are the largest family of signaling receptors in mammalian cells, mediate vast physiological responses, and are the largest class of drug targets for therapeutics used clinically. However, the regulation of GPCR signaling remains poorly understood and discovering new aspects of GPCR signal regulation is critical for future drug development that could ultimately lead to the identification of new drug targets relevant to a wide range of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM090689-05S1
Application #
8907624
Study Section
Program Officer
Dunsmore, Sarah
Project Start
2010-01-01
Project End
2017-12-31
Budget Start
2014-02-01
Budget End
2014-12-31
Support Year
5
Fiscal Year
2014
Total Cost
$80,000
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Grimsey, Neil J; Trejo, JoAnn (2016) Integration of endothelial protease-activated receptor-1 inflammatory signaling by ubiquitin. Curr Opin Hematol 23:274-9
Dores, Michael R; Grimsey, Neil J; Mendez, Francisco et al. (2016) ALIX Regulates the Ubiquitin-Independent Lysosomal Sorting of the P2Y1 Purinergic Receptor via a YPX3L Motif. PLoS One 11:e0157587
Smith, Thomas H; Coronel, Luisa J; Li, Julia G et al. (2016) Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins. J Biol Chem 291:18453-64
Grimsey, Neil J; Coronel, Luisa J; Cordova, Isabel Canto et al. (2016) Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins. J Biol Chem 291:2223-36
Grimsey, Neil J; Aguilar, Berenice; Smith, Thomas H et al. (2015) Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes. J Cell Biol 210:1117-31
Chen, Buxin; Soto, Antonio G; Coronel, Luisa J et al. (2015) Characterization of thrombin-bound dabigatran effects on protease-activated receptor-1 expression and signaling in vitro. Mol Pharmacol 88:95-105
Soto, Antonio G; Smith, Thomas H; Chen, Buxin et al. (2015) N-linked glycosylation of protease-activated receptor-1 at extracellular loop 2 regulates G-protein signaling bias. Proc Natl Acad Sci U S A 112:E3600-8
Dores, Michael R; Lin, Huilan; J Grimsey, Neil et al. (2015) The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting. Mol Biol Cell 26:4660-73
Stoner, Terri D; Weston, Thomas A; Trejo, JoAnn et al. (2015) Group B streptococcal infection and activation of human astrocytes. PLoS One 10:e0128431
Grimsey, Neil; Lin, Huilan; Trejo, JoAnn (2014) Endosomal signaling by protease-activated receptors. Methods Enzymol 535:389-401

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