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

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)29646-29657
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number40
DOIs
StatePublished - Oct 5 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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