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J. Biol. Chem., Vol. 282, Issue 40, 29646-29657, October 5, 2007

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

From the Departments of Surgery and Physiology, University of California, San Francisco, California 94143-0660

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 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 PAR₂14K/R in enlarged HRS-containing endosomes, preventing recycling and resensitization of PAR₂14K/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.

Received for publication, April 9, 2007 , and in revised form, July 6, 2007.

^* This work was supported by National Institutes of Health Grants DK43207, DK57840, and DK39957 (to N. W. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 513 Parnassus Ave., San Francisco CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936; E-mail: nigel.bunnett{at}ucsf.edu.

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