Signal regulation of protease-activated receptor-2 and structural determinants of Gαq-dependent activation and deactivation of phospholipase C-β
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Ricks, Tiffany K. Signal Regulation of Protease-activated Receptor-2 and Structural Determinants of Gαq-dependent Activation and Deactivation of Phospholipase C-β. Chapel Hill, NC: University of North Carolina at Chapel Hill, 2010. https://doi.org/10.17615/scnt-xr15APA
Ricks, T. (2010). Signal regulation of protease-activated receptor-2 and structural determinants of Gαq-dependent activation and deactivation of phospholipase C-β. Chapel Hill, NC: University of North Carolina at Chapel Hill. https://doi.org/10.17615/scnt-xr15Chicago
Ricks, Tiffany K. 2010. Signal Regulation of Protease-Activated Receptor-2 and Structural Determinants of Gαq-Dependent Activation and Deactivation of Phospholipase C-Β. Chapel Hill, NC: University of North Carolina at Chapel Hill. https://doi.org/10.17615/scnt-xr15- Alternate title
- Signal regulation of protease-activated receptor-2 and structural determinants of G[alpha]q-dependent activation and deactivation of phospholipase C-[beta]
- Last Modified
- March 22, 2019
- Creator
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Ricks, Tiffany K.
- Affiliation: School of Medicine, Department of Pharmacology
- Abstract
- Cells respond to changes in their environment by relaying information from extracellular cues to intracellular compartments, and receptors play an important role in the transmission of these signals. We examined signal transduction of G protein-coupled protease-activated receptor-2 (PAR2), a cell surface receptor for serine proteases. Unlike most GPCRs, PAR2 is irreversibly activated by proteolytic cleavage, and the mechanisms regulating desensitization and trafficking are essential for the fidelity of PAR2 signaling. Most activated GPCRs are rapidly desensitized and internalized following phosphorylation and β-arrestin binding. However, the role of phosphorylation in signaling and trafficking of PAR2 is unknown. We found that PAR2 phosphorylation is required for receptor desensitization and β-arrestin binding. Phosphorylated PAR2 internalized through a canonical dynamin-, clathrin-, and β-arrestin-dependent pathway. In contrast, phosphorylation-deficient PAR2 constitutively internalized through a dynamin-dependent but clathrin- and β-arrestin-independent pathway. Collectively, we show that phosphorylation of PAR2 is critical for β-arrestin binding and desensitization but not endocytic trafficking. Activated PAR2 and GPCRs catalyze GTP exchange on heterotrimeric G proteins. GTP-bound Gα subunits activate protein effectors including phospholipases C- β (PLC-β) isozymes. PLC-β isozymes are stimulated by Gαq and also accelerate GTP hydrolysis of on their activating G protein. The mechanisms that regulate Gαq-dependent activation and deactivation of PLC-β are not clearly understood. Inspection of a threedimensional crystal structure of the PLC-β3⋅Gαq complex revealed three novel contacts within the binding interface. A small region following the C2 domain of PLC-β3 inserts into the effector binding pocket of Gαq. Gαq also makes electrostatic interactions with a region preceding the C2 domain of PLC-β3. In addition, a loop between the third and fourth EF hands of PLC-β3 contacts the nucleotide binding pocket of Gαq. Mutation of this loop abrogated PLC-β3 GAP activity, and a GAP-deficient PLC-β3 mutant displayed a much slower rate of deactivation. Consequently, PLC activity was largely unchanged compared to rapid termination of wildtype PLC-β3. Our studies define the important domains within the PLC-β3/Gαq binding interface that are required for activation and deactivation of PLC-β isozymes. The studies presented herein describe the mechanisms regulating PAR2 and GPCRs at the cell surface and controlling PLC-β isozymes within intracellular compartments.
- Date of publication
- May 2010
- DOI
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- Rights statement
- In Copyright
- Note
- "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology."
- Advisor
- Trejo, JoAnn
- Harden, T. Kendall
- Degree granting institution
- University of North Carolina at Chapel Hill
- Language
- Publisher
- Place of publication
- Chapel Hill, NC
- Access right
- Open access
- Date uploaded
- March 18, 2013
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