Signal regulation of protease-activated receptor-2 and structural determinants of Galpha(q)-dependent activation and deactivation of phospholipase C-beta.

摘要

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 beta-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 beta-arrestin binding. Phosphorylated PAR2 internalized through a canonical dynamin-, clathrin-, and beta-arrestin-dependent pathway. In contrast, phosphorylation-deficient PAR2 constitutively internalized through a dynamin-dependent but clathrin- and beta-arrestin-independent pathway. Collectively, we show that phosphorylation of PAR2 is critical for beta-arrestin binding and desensitization but not endocytic trafficking.;Activated PAR2 and GPCRs catalyze GTP exchange on heterotrimeric G proteins. GTP-bound Galpha subunits activate protein effectors including phospholipases C-beta (PLC-beta) isozymes. PLC-beta isozymes are stimulated by Galpha q and also accelerate GTP hydrolysis of on their activating G protein. The mechanisms that regulate Galphaq-dependent activation and deactivation of PLC-beta are not clearly understood. Inspection of a three-dimensional crystal structure of the PLC-beta3.Galphaq complex revealed three novel contacts within the binding interface. A small region following the C2 domain of PLC-beta3 inserts into the effector binding pocket of Galpha q. Galphaq also makes electrostatic interactions with a region preceding the C2 domain of PLC-beta3. In addition, a loop between the third and fourth EF hands of PLC-beta3 contacts the nucleotide binding pocket of Galphaq. Mutation of this loop abrogated PLC-beta3 GAP activity, and a GAP-deficient PLC-beta3 mutant displayed a much slower rate of deactivation. Consequently, PLC activity was largely unchanged compared to rapid termination of wildtype PLC-beta3. Our studies define the important domains within the PLC-beta3/Galphaq binding interface that are required for activation and deactivation of PLC-beta isozymes. The studies presented herein describe the mechanisms regulating PAR2 and GPCRs at the cell surface and controlling PLC-beta isozymes within intracellular compartments.