Hepatocyte Growth Factor-regulated Tyrosine Kinase Substrate (HRS) Mediates Post-endocytic Trafficking of Protease-activated Receptor 2 and Calcitonin Receptor-like Receptor

Burcu Hasdemir,Nigel W Bunnett,Graeme S Cottrell

doi:10.1074/jbc.m702974200

Burcu Hasdemir, Nigel W Bunnett + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m702974200

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Abstract

The E3 ligase c-Cbl ubiquitinates protease-activated receptor 2 (PAR(2)), which is required for post-endocytic sorting of PAR(2) 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-PAR(2) cells, PAR(2) activating peptide (PAR(2)-AP) induced PAR(2) 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 PAR(2) and c-Cbl accumulated, and PAR(2) failed to traffic to lysosomes. Overexpression of HRS prevented PAR(2)-AP-induced degradation of PAR(2), as determined by Western blotting. Overexpression of HRS mutant lacking an ubiquitin-binding motif similarly caused retention of PAR(2) in enlarged endosomes. Moreover, HRS overexpression or knockdown caused retention of ubiquitin-resistant PAR(2)Delta14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR(2)Delta14K/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, PAR(2) 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 PAR(2) and non-ubiquitinated CLR. The ultimate sorting decision to degradative or recycling pathways appears to occur downstream from HRS.

Highlights

Endocytic trafficking depends on the receptor and the nature of the stimulus
We found that in cells not overexpressing Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endogenous immunoreactive HRS was detected in multiple small vesicles containing early endosomal antigen-1 (EEA1), which are early endosomes (Fig. 1A)
We observed that in unstimulated cells, protease-activated receptor 2 (PAR2) was detected at the plasma membrane and in a perinuclear location, which we have previously identified as the Golgi apparatus [30, 31], whereas HRS was restricted to endosomes [21] (Fig. 1C)

Summary

Introduction

Endocytic trafficking depends on the receptor and the nature of the stimulus. Some GPCRs are sorted to lysosomes or proteasomes, where degradation irrevocably inactivates internalized receptors and prevents uncontrolled signaling during chronic stimulation [1,2,3,4]. PAR2 was localized to the plasma membrane and present in enlarged endosomes containing HRS (Fig. 2A). In unstimulated HRS-overexpressing cells, PAR2⌬14K/R was at the plasma membrane and in enlarged HRS-containing endosomes (Fig. 5B), suggesting constitutive endocytosis.

Results

Conclusion