Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1.
Degradation or "down-regulation" of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, is critical for termination of receptor signaling. Toward understanding the molecular mechanisms by which activated PAR1 is internalized, sorted to lysosomes, and degraded, we investigated whether PAR1 interacted with sorting nexin 1 (SNX1). SNX1 is a membrane-associated ... protein that functions in lysosomal sorting of the epidermal growth factor receptor. In vitro biochemical binding assays revealed a specific interaction between a glutathione S-transferase fusion of SNX1 and PAR1. In HeLa cells, activated PAR1 colocalized with endogenous SNX1 and coimmunoprecipitated SNX1. SNX1 contains a phox homology domain predicted to bind phosphatidylinositol-3-phosphate and a C-terminal coiled-coil region. To assess SNX1 function, we examined the effects of SNX1 deletion mutants on PAR1 trafficking. Neither the N terminus nor phox homology domain of SNX1 affected PAR1 trafficking. By contrast, overexpression of SNX1 C-terminal domain markedly inhibited agonist-induced degradation of PAR1, whereas internalization remained virtually intact. Immunofluorescence microscopy studies revealed substantial PAR1 accumulation in an early endosome antigen-1-positive compartment in agonist-treated cells expressing SNX1 C terminus. By contrast, lysosome-associated membrane protein-1 distribution was unperturbed. Together, these findings strongly suggest a role for SNX1 in sorting of PAR1 from early endosomes to lysosomes. Moreover, this study provides the first example of a protein involved in lysosomal sorting of a G protein-coupled receptor in mammalian cells.
Mesh Terms:
Amyloid beta-Protein Precursor, Carrier Proteins, Cloning, Molecular, Down-Regulation, Endosomes, Gene Expression Regulation, Glutathione Transferase, Hela Cells, Humans, Lysosomes, Microscopy, Confocal, Mutagenesis, Plasmids, Protein Transport, Receptor, Epidermal Growth Factor, Receptor, PAR-1, Receptors, Cell Surface, Receptors, Thrombin, Recombinant Fusion Proteins, Sequence Deletion, Serpins, Thrombin, Transfection, Vesicular Transport Proteins
Amyloid beta-Protein Precursor, Carrier Proteins, Cloning, Molecular, Down-Regulation, Endosomes, Gene Expression Regulation, Glutathione Transferase, Hela Cells, Humans, Lysosomes, Microscopy, Confocal, Mutagenesis, Plasmids, Protein Transport, Receptor, Epidermal Growth Factor, Receptor, PAR-1, Receptors, Cell Surface, Receptors, Thrombin, Recombinant Fusion Proteins, Sequence Deletion, Serpins, Thrombin, Transfection, Vesicular Transport Proteins
Mol. Biol. Cell
Date: Jun. 01, 2002
PubMed ID: 12058063
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