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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Neuropharmacology and Signaling Study Section (MNPS)
Program Officer
Dunsmore, Sarah
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Diego
Schools of Medicine
La Jolla
United States
Zip Code
Kawamura, Tetsuya; Stephens, Bryan; Qin, Ling et al. (2014) A general method for site specific fluorescent labeling of recombinant chemokines. PLoS One 9:e81454
Grimsey, Neil; Lin, Huilan; Trejo, JoAnn (2014) Endosomal signaling by protease-activated receptors. Methods Enzymol 535:389-401
Dores, Michael R; Trejo, JoAnn (2014) Atypical regulation of G protein-coupled receptor intracellular trafficking by ubiquitination. Curr Opin Cell Biol 27:44-50
Chen, Buxin; Siderovski, David P; Neubig, Richard R et al. (2014) Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins. J Biol Chem 289:1580-91
Lin, Huilan; Liu, Allen P; Smith, Thomas H et al. (2013) Cofactoring and dimerization of proteinase-activated receptors. Pharmacol Rev 65:1198-213
Gilliland, C Taylor; Salanga, Catherina L; Kawamura, Tetsuya et al. (2013) The chemokine receptor CCR1 is constitutively active, which leads to G protein-independent, *-arrestin-mediated internalization. J Biol Chem 288:32194-210
Lin, Huilan; Trejo, JoAnn (2013) Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits ?-arrestin-mediated endosomal signaling. J Biol Chem 288:11203-15
Canto, Isabel; Trejo, JoAnn (2013) Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting. J Biol Chem 288:15900-12
Dores, Michael R; Chen, Buxin; Lin, Huilan et al. (2012) ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting. J Cell Biol 197:407-19
Grimsey, Neil; Soto, Antonio G; Trejo, JoAnn (2011) Regulation of protease-activated receptor signaling by post-translational modifications. IUBMB Life 63:403-11

Showing the most recent 10 out of 11 publications