c-Cbl mediates ubiquitination, degradation, and down-regulation of human protease-activated receptor 2

Abstract

Mechanisms that arrest G-protein-coupled receptor (GPCR) signaling prevent uncontrolled stimulation that could cause disease. Although uncoupling from heterotrimeric G-proteins, which transiently arrests signaling, is well described, little is known about the mechanisms that permanently arrest signaling. Here we reported on the mechanisms that terminate signaling by protease-activated receptor 2 (PAR2), which mediated the proinflammatory and nociceptive actions of proteases. Given its irreversible mechanism of proteolytic activation, PAR2 is a model to study the permanent arrest of GPCR signaling. By immunoprecipitation and immunoblotting, we observed that activated PAR2 was mono-ubiqttitinated. Immunofluorescence indicated that activated PAR2 translocated from the plasma membrane to early endosomes and lysosomes where it was degraded, as determined by immunoblotting. Mutant PAR2 lacking intracellular lysine residues (PAR2Δ14K/R) was expressed at the plasma membrane and signaled normally but was not ubiquitinated. Activated PAR 2Δ14K/R internalized but was retained in early endosomes and avoided lysosomal degradation. Activation of wild type PAR2 stimulated tyrosine phosphorylation of the ubiquitin-protein isopeptide ligase c-Cbl and promoted its interaction with PAR2 at the plasma membrane and in endosomes in an Src-dependent manner. Dominant negative c-Cbl lacking the ring finger domain inhibited PAR2 ubiquitination and induced, retention in early endosomes, thereby impeding lysosomal degradation. Although wild type PAR2 was degraded, and recovery of agonist responses required synthesis of new receptors, lysine mutation and dominant negative c-Cbl impeded receptor ubiquitination and degradation and allowed PAR2 to recycle and continue to signal. Thus, c-Cbl mediated ubiquitination and lysosomal degradation of PAR2 to irrevocably terminate signaling by this and perhaps other GPCRs. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.