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Internalization of Protease‐Activated Receptor 4 is AP‐2 Dependent and Utilizes a Non‐canonical Sorting Motif
Author(s) -
Smith Thomas Horace,
Coronel Luisa,
Nieman Marvin,
Trejo JoAnn
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.710.5
Subject(s) - clathrin , dynamin , microbiology and biotechnology , endocytosis , endocytic cycle , internalization , endosome , g protein coupled receptor , signal transduction , biology , proteases , intracellular , receptor , chemistry , biochemistry , enzyme
Protease‐activated receptor‐4 (PAR4) is a GPCR that is activated by proteolytic cleavage of its N‐terminus by thrombin and other proteases. Due to this irreversible proteolytic mechanism of activation, the mechanisms that control signaling by PAR4 are important for regulating the magnitude and duration of the signaling response. While the processes that control signaling of PAR1, a related GPCR activated by thrombin, have been extensively characterized, the mechanisms that regulate PAR4 signaling remain poorly understood. Unlike PAR1, activated PAR4 is not phosphorylated and exhibits prolonged signaling. These findings raised the question of whether PAR4 signaling is regulated by intracellular trafficking, a process which remains poorly defined for the receptor. The focus of this study is to delineate the mechanisms that control PAR4 intracellular trafficking. To examine the mechanisms that control PAR4 endocytosis, we expressed FLAG‐PAR4 in HeLa cells, a cell model system used extensively to characterize clathrin‐ and dynamin‐mediated endocytosis. We showed that upon activation with the agonist peptide AYPGKF, ~25% of PAR4 internalized from the cell surface over one hour. We next employed immunofluorescence confocal microscopy to visualize the endocytic sorting of activated PAR4 and found that the majority of activated and internalized PAR4 localized to early endosomes by 30 minutes and then sorted to lysosomes between 60 and 90 minutes. We utilized siRNA knockdown of clathrin and immunofluorescence microscopy to show that PAR4 undergoes clathrin‐mediated endocytosis following activation. Consistent with clathrin‐mediated endocytosis, expressing a dominant‐negative dynamin K44A mutant also blocked activated PAR4 internalization. Interestingly, however, unlike most GPCRs, PAR4 internalization occurred independently of both β‐arrestin 1 and 2 and the C‐tail domain. Rather, we identified a highly conserved potential AP‐2 binding motif in the receptor's third intracellular loop, and thus hypothesized that PAR4 internalization was mediated by the clathrin adapter protein AP‐2. Using siRNA knockdown of AP‐2, we confirmed that the adapter protein was necessary for PAR4 endocytosis. We also observed that a PAR4 mutant lacking the putative AP‐2 binding motif exhibited a defect in agonist‐induced internalization. To confirm our findings we examined endogenous PAR4 internalization in the Dami megakaryoblastic cell line. In ongoing studies, we are investigating the effect of dysregulated PAR4 trafficking on signal regulation. Support or Funding Information Funding for this project was provided by NIH/NHLBI F31 HL116187 (T. Smith).