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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090689-04
Application #
8400892
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Dunsmore, Sarah
Project Start
2010-01-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
4
Fiscal Year
2013
Total Cost
$313,654
Indirect Cost
$110,642
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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