The classic model of G protein coupled receptor (GPCR) activation centers on ligand binding, G protein activation, and signal transduction via G protein-mediated signaling events. This paradigm has been called into question however, with the finding that some ligands ?including endogenous ligands and therapeutic agents? have a preference for beta-arrestin mediated pathways. However, the information flow from receptor activation to signaling cascades and the mechanism of beta-arrestin signaling are not well understood. This proposal will elucidate, at the molecular level, the fundamental mechanisms controlling ligand-specific induction of beta- arrestin signaling with two highly clinically relevant GPCRs, the cannabinoid 2 receptor (CB2R) and the mu opioid receptor (MOR). These human receptors bind the plant-derived cannabinoids and opioids leading to psychostimulant effects and reduction of pain. Precise control of receptor activation and signaling is critical to obtain only the desired therapeutic results, however, and not the undesired side effects such as tolerance, drug abuse and dependence. Substantial preliminary studies identified ligand-specific dwell times, i.e. the time receptors are clustered into clathrin coated pits with beta-arrestins before endocytosis, as a mechanism controlling beta-arrestin signaling. This trafficking event can be chemically and genetically modulated to selectively control beta-arrestin signaling, providing novel therapeutic strategies. This project will combine state-of-the-art live cell imaging technologies (total internal reflection fluorescence and spinning disk microscopies), and biochemical approaches to determine if ligand-specific dwell times are a general event controlling beta-arrestin signaling. Multiple ligands for these receptors will be investigated in heterologous systems and in cells endogenously expressing the receptors. Preliminary results in primary cultures strongly support our hypothesis that long dwell times correlate with beta-arrestin signaling.
The aims are to: (1) examine endocytosis of the CBR2 and MOR at the single endocytic pit level, and (2) define the impact on cellular mechanisms of CB2R and MOR mediated beta-arrestin signaling, including whether endocytic dwell times can modulate these pathways. Results will provide a physiological role for the previously described variability in endocytic dwell times. These findings may be extended to future drug discovery efforts, including for other GPCRs, to rationally design therapeutic agents with specific outcomes in areas intractable via current technology.

Public Health Relevance

G protein-coupled receptors (GPCRs) are the main transducers of external stimuli into the cell and are major therapeutic targets in many pathological disorders. Elucidating the mechanisms governing GPCR-ligand induced signaling is critical to ultimately understand how GPCRs function in health and disease. Our proposal seeks to identify beta-arrestin signaling mechanisms used by GPCRs associated with pain and drug abuse and to set the stage for alternative approaches to control outcomes via these GPCRs.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21DA040920-01A1
Application #
9176213
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Rapaka, Rao
Project Start
2016-07-01
Project End
2018-06-30
Budget Start
2016-07-01
Budget End
2017-06-30
Support Year
1
Fiscal Year
2016
Total Cost
$207,200
Indirect Cost
$52,200
Name
University of Connecticut
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
614209054
City
Storrs-Mansfield
State
CT
Country
United States
Zip Code
06269
Nogueras-Ortiz, Carlos; Roman-Vendrell, Cristina; Mateo-Semidey, Gabriel E et al. (2017) Retromer stops beta-arrestin 1-mediated signaling from internalized cannabinoid 2 receptors. Mol Biol Cell 28:3554-3561
Delgado-Peraza, Francheska; Nogueras-Ortiz, Carlos; Acevedo Canabal, Agnes M et al. (2016) Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons. Methods Cell Biol 132:25-33
Nogueras-Ortiz, Carlos; Yudowski, Guillermo A (2016) The Multiple Waves of Cannabinoid 1 Receptor Signaling. Mol Pharmacol 90:620-626
Kendall, Debra A; Yudowski, Guillermo A (2016) Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease. Front Cell Neurosci 10:294