Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR ₂), 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 PAR ₂. 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 small interfering RNA 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 PAR₂Δ14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR₂Δ 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 downstream from HRS.