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 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.
Department of Surgery, University of California, San Francisco, California 94143-0660, USA.
Burcu
B
Hasdemir
Nigel W
NW
Bunnett
Graeme S
GS
Cottrell
D058286:Calcitonin Receptor-Like Protein; D002460:Cell Line; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000378:metabolism*; D018003:Receptors, Calcitonin / Q000378:metabolism*; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D018003:Receptors, Calcitonin; D025801:Ubiquitin; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D050721:Proto-Oncogene Proteins c-cbl