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

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.