Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2002 Jun;13(6):1965-76.
doi: 10.1091/mbc.e01-11-0131.

Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1

Yingjie Wang  1 , Yixing Zhou, Katalin Szabo, Carol Renfrew Haft, JoAnn Trejo
Affiliations

Down-regulation of protease-activated receptor-1 is regulated by sorting nexin 1

Yingjie Wang et al. Mol Biol Cell. 2002 Jun.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Agonist-induced colocalization of PAR1 with endogenous SNX1 in HeLa cells. HeLa cells stably expressing PAR1 were incubated in the absence (Control) or presence of 50 μM SFLLRN for 10 min at 37°C. Cells were fixed, permeabilized, and immunostained for PAR1 (green) and endogenous SNX1 (red) and were examined by confocal microscopy. These images are representative of many cells examined in three separate experiments. Note the prominent agonist-induced colocalization (yellow) of PAR1 and SNX1 in the merged image. Scale bar represents 10 μm.
Figure 2
Figure 2
SNX1 associates with PAR1 in vitro. (A) GST-SNX1 fusion protein or GST protein alone absorbed to glutathione-Sepharose beads was incubated with equal amounts of membranes prepared from PAR1-transfected HeLa cells or untransfected (UT) control cells. Bound proteins were eluted, resolved by SDS-PAGE, and immunoblotted with anti-PAR1 antibody (upper panel). An aliquot of membranes prepared from PAR1-expressing HeLa cells or UT control cells representing 10% of input is shown in lanes 5 and 6, respectively. The nonspecific bands detected in pull-downs of UT control cells are shown in lanes 3 and 4. Similar findings were observed in four separate experiments. Note the detection of one prominent transfection-dependent ∼68 kDa protein in GST-SNX1 pull-down shown in lane 2. (B) GST-SNX1 or GST protein alone absorbed to glutathione-Sepharose beads was incubated with membranes prepared from HeLa cells expressing HA-tagged P2Y2 receptor or UT control cells. Bound proteins were eluted and immunoblotted with anti-HA antibody (upper panel). An aliquot of membranes representing 10% of input is shown in lane 5 (upper panel). Similar results were observed in three separate experiments. Note the failure of GST-SNX1 to bind the P2Y2 receptor. The total amount of GST-SNX1 and GST protein loaded in various lanes was visualized by Ponceau S staining (bottom panels).
Figure 3
Figure 3
Coimmunoprecipitation of SNX1 with activated PAR1 in HeLa cells. HeLa cells transiently cotransfected with FLAG-tagged PAR1 and myc-SNX1 were treated in the absence (−) or presence (+) of 50 μM SFLLRN for 10 min at 37°C. Cells were lysed and equal amounts of lysates were immunoprecipitated with M2 anti-FLAG antibody or isotype-matched control IgG. Immunoprecipitates were immunoblotted for SNX1 using anti-myc-peroxidase antibody (upper panel) or for PAR1 using 1809 antibody (middle panel). SNX1 expression in total cell lysates representing 12% of input and was detected with anti-myc antibody (bottom panel). Similar results were observed in six independent experiments. Note the prominent agonist-induced association of SNX1 with PAR1.
Figure 4
Figure 4
Overexpression of SNX C terminus markedly inhibits agonist-induced degradation of PAR1. (A) Domain structure model of SNX1 and various deletion mutants. The phox homology domain is represented as “PX” and the coiled-coiled region is shown as “CC.” (B) HeLa cells transiently cotransfected with PAR1 and SNX1, SNX1 mutants, or pcDNA vector were incubated in the absence (−) or presence (+) of 50 μM SFLLRN for 60 min at 37°C. Cells were lysed, immunoprecipitated with anti-FLAG antibody, and the amount of PAR1 remaining was detected by immunoblot with anti-PAR1 antibody and quantitated by Fluor-S Imager (upper panel). The expression of SNX1 and various deletion mutants in cell lysates was detected with anti-myc anti-body (lower panel). Results in the bar graph are expressed as a percentage of PAR1 measured in untreated control lysates and were determined for each transfection condition. The data are represented as the mean ± SEM of three separate experiments in which duplicate determinations were made. Note the marked inhibition of agonist-induced PAR1 degradation in cells expressing SNX1 C-terminal coiled-coil domain.
Figure 5
Figure 5
SNX1 C-terminal coiled-coil domain associates with full-length SNX1. HeLa cells transiently cotransfected with HA-SNX1 and myc-SNX1 deletion mutants were lysed and immunoprecipitated with anti-HA antibody. Immunoprecipitates were immunoblotted for full-length myc-SNX1 or deletion mutants using anti-myc-peroxidase antibody (upper panel). The expression of HA-SNX1 and myc-SNX1 deletion mutants in total cell lysates is shown in the middle and lower panels, respectively. Note the prominent association of SNX1 C-terminal coiled-coil domain with full-length SNX1, lane 6. The data are representative of two separate experiments.
Figure 6
Figure 6
Effect of SNX1 C-terminal domain on agonist-triggered PAR1 and P2Y2 receptor internalization. (A) HeLa cells transiently cotransfected with FLAG-tagged PAR1 and either SNX1 C terminus or pcDNA vector were incubated in the absence or presence of 50 μM SFLLRN for various times at 37°C. (B) HeLa cells transiently cotransfected with P2Y2 receptor and either SNX1 C terminus or pcDNA vector were treated in the absence or presence of 100 μM ATP for various times at 37°C. Cells were then fixed and the amount of PAR1 or P2Y2 receptor remaining on the cell surface was detected by cell surface ELISA. The initial level of PAR1 or P2Y2 receptor expressed on the cell surface before incubation at 37°C (0-min time point) was similar for each transfection condition. Results are expressed as a fraction of total antibody bound to untreated controls. The data (mean ± SEM) are representative of three separate experiments in which triplicate determinations were made.
Figure 7
Figure 7
Agonist-induced PAR1 accumulation in endosomes in SNX1 C terminus transfected cells. (A) PAR1-expressing HeLa cells transiently transfected with SNX1 or SNX1 C terminus were treated in the absence (Control) or presence of 50 μM SFLLRN for either 10 or 30 min at 37°C. Cells were fixed, immunostained for PAR1 and SNX1 or SNX1 C terminus, and imaged by confocal microscopy. Note the presence of PAR1-positive endosomes in SNX1 C terminus-transfected cells even after 30 min of agonist exposure (k and l), but the absence of such endosomes in SNX1 transfected cells (i and j). (B) The results of quantitative analysis are expressed as a percentage of cells that showed PAR1-positive endosomes and costained for either SNX1 or SNX1 C terminus. All cells containing PAR1-positive endosomes were counted in pcDNA vector-transfected controls. The data (mean ± SEM) are representative of three separate experiments in which at least 25 determinations were made. Scale bar represents 10 μm.
Figure 8
Figure 8
Effect of SNX1 C terminus overexpression on lamp1 distribution. HeLa cells were transiently transfected with myc-tagged SNX1 C terminus or pcDNA vector. At ∼48 h after transfection, cells were fixed, immunostained for SNX1 C terminus (red) and lamp1 (green), and examined by confocal microscopy. Note the similar distribution of lamp1 in SNX1 C terminus and pcDNA-transfected cells shown in b and d. The images are representative of many cells examined in two separate experiments. Scale bar denotes 10 μm.
Figure 9
Figure 9
PAR1 and endogenous SNX1 colocalize with EEA1-positive early endosomes. (A) HeLa cells expressing PAR1 were treated in the absence (Control) or presence of 50 μM SFLLRN for 10 or 30 min at 37°C. Cells were fixed, immunostained for PAR1 (green) and EEA1 (red), and imaged by confocal microscopy. Note the prominent agonist-induced colocalization (yellow) of PAR1 and EEA1 after 10 min of agonist exposure shown in the merged image and the loss of PAR1-positive endosomes after 30 min of agonist exposure. (B) HeLa cells were immunostained for endogenous SNX1 (green) and EEA1 (red) and were examined by confocal microscopy. Endogenous SNX1 and EEA1 colocalization (yellow) is shown in the merged image. The cells imaged are representative of many cells examined in four separate experiments. Scale bar denotes 10 μm.
Figure 10
Figure 10
Agonist-induced PAR1 accumulation in EEA1-positive early endosomes in SNX1 C terminus-expressing cells. PAR1-expressing HeLa cells were treated in the absence (Control) or presence of 50 μM SFLLRN for either 10 or 30 min at 37°C. Cells were fixed and immunostained for PAR1 (green), EEA1 (red), and SNX1 C terminus (blue), and were imaged by confocal microscopy. Note the prominent colocalization (yellow) of PAR1 and EEA1 even after 30 min of agonist exposure in SNX1 C terminus-expressing cells (blue). The imaged cells are representative of many cells examined in three separate experiments. The scale bar represents 10 μm.
See this image and copyright information in PMC

References

    1. Andersson S, Bishop RW, Russell DW. Expression cloning and regulation of steroid 5 α-reductase, an enzyme essential for male sexual differentiation. J Biol Chem. 1989;264:16249–16255. - PMC - PubMed
    1. Burkhard P, Stetfeld J, Strelkov SV. Coiled-coils: a highly versatile protein folding motif. Trends Cell Biol. 2001;11:82–87. - PubMed
    1. Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M. A kinase-regulated PDZ-domain interaction controls endocytic sorting of the β2-adrenergic receptor. Nature. 1999;401:286–290. - PubMed
    1. Christoforidis S, McBride H, Burgoyne R, Zerial M. The Rab5 effector EEA1 is a core component of endosome docking. Nature. 1999;397:621–625. - PubMed
    1. Coughlin SR. Thrombin signaling and protease-activated receptors. Nature. 2000;407:258–264. - PubMed

Publication types

MeSH terms

LinkOut - more resources