The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR2), which is required for post-endocytic sorting of PAR2 to lysosomes, where degradation arrests signaling. The mechanisms of post-endocytic sorting of ubiquitinated receptors are incompletely understood. Here, we investigated the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), in post-endocytic sorting and signaling of PAR2. In HEK-PAR2 cells, PAR2 activating peptide (PAR2-AP) induced PAR2 trafficking from the cell-surface to early endosomes containing endogenous Hrs, and then to lysosomes. Hrs overexpression or knockdown with siRNA caused formation of enlarged Hrs-positive endosomes, where activated PAR2 and c-Cbl accumulated, and PAR2 failed to traffic to lysosomes. Overexpression of Hrs prevented PAR2-AP-induced degradation of PAR2, as determined by Western blotting. Overexpression of Hrs mutant lacking an ubiquitin-binding motif similarly caused retention of PAR2 in enlarged endosomes. Moreover, Hrs overexpression or knockdown caused retention of ubiquitin-resistant PAR2 14K/R in enlarged Hrs-containing endosomes, preventing recycling and resensitization of PAR2 14K/R. Hrs overexpression or knockdown similarly prevented lysosomal trafficking and recycling of calcitonin receptor-like receptor, a non-ubiquitinated receptor that traffics to lysosomes after sustained activation and recycles after transient activation. Thus, Hrs plays a critically important role in the post-endocytic sorting of single receptors, PAR2 and CLR, to both degradative and recycling pathways. This sorting role for Hrs is independent of its ubiquitin-interacting motif, and it can regulate trafficking of both ubiquitinated and non-ubiquitinated PAR2 and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur down-stream from Hrs.
Hasdemir, B., Bunnett, N., & Cottrell, G. (2007). Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor. The Journal of Biological Chemistry, 282(40), 29646 - 29657. https://doi.org/10.1074/jbc.M702974200