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SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors.

Temkin P, Lauffer B, Jäger S, Cimermancic P, Krogan NJ, von Zastrow M - Nat. Cell Biol. (2011)

Bottom Line: Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4.Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule.The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of California at San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

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SNX27 serves as an adapter for β2AR, sorting it into the retromer tubule(a) Comparative localization of β2AR (red) with the trans-Golgi marker GalT-GFP (green) and CIMPR (blue). Cells were fed anti-FLAG antibody in the presence of isoproterenol for 25 minutes prior to agonist washout with alprenelol for 45 minutes. Successful knockdown of SNX27 was judged by failure of β2AR to recycle to the plasma membrane. The scale bar represents 20 μm. (b) CIMPR surface immunoreactivity was quantified by fluorescence flow cytometry (n=6) to assess the integrity of CIMPR trafficking when SNX27 is depleted with siRNA. Successful depletion of SNX27 was confirmed by looking at visual recycling assays of β2AR as in (a). (c) Representative confocal image from live cell imaging showing an endosome from a SNX27-4 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (d) Confocal image of transiently transfected SNX27-HA (red) and VPS29-GFP (green) in fixed cells. The scale bar represents 20 μm. (e) Representative immunoblot showing co-immunoprecipitation of endogenous VPS35 with transiently transfected SNX27-HA. Lanes 1 and 3 show 2% whole cell lysate for the mock IP at left and the experiment at right. (f) SNX27 mediates plasma membrane recycling of PDZ motif containing cargo by linking to the retromer through an interaction with the WASH complex. SNX27 also interacts with the endosome directly through its lipid binding PX domain. (g) Isoproterenol or forskolin induced cAMP formation was measured in HEK 293 cells stably expressing β2AR, in the absence of IBMX, 20 minutes after agonist addition (n=6). Data points are the mean ± SEM.
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Figure 5: SNX27 serves as an adapter for β2AR, sorting it into the retromer tubule(a) Comparative localization of β2AR (red) with the trans-Golgi marker GalT-GFP (green) and CIMPR (blue). Cells were fed anti-FLAG antibody in the presence of isoproterenol for 25 minutes prior to agonist washout with alprenelol for 45 minutes. Successful knockdown of SNX27 was judged by failure of β2AR to recycle to the plasma membrane. The scale bar represents 20 μm. (b) CIMPR surface immunoreactivity was quantified by fluorescence flow cytometry (n=6) to assess the integrity of CIMPR trafficking when SNX27 is depleted with siRNA. Successful depletion of SNX27 was confirmed by looking at visual recycling assays of β2AR as in (a). (c) Representative confocal image from live cell imaging showing an endosome from a SNX27-4 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (d) Confocal image of transiently transfected SNX27-HA (red) and VPS29-GFP (green) in fixed cells. The scale bar represents 20 μm. (e) Representative immunoblot showing co-immunoprecipitation of endogenous VPS35 with transiently transfected SNX27-HA. Lanes 1 and 3 show 2% whole cell lysate for the mock IP at left and the experiment at right. (f) SNX27 mediates plasma membrane recycling of PDZ motif containing cargo by linking to the retromer through an interaction with the WASH complex. SNX27 also interacts with the endosome directly through its lipid binding PX domain. (g) Isoproterenol or forskolin induced cAMP formation was measured in HEK 293 cells stably expressing β2AR, in the absence of IBMX, 20 minutes after agonist addition (n=6). Data points are the mean ± SEM.

Mentions: We next investigated the mechanistic basis for the role of retromer in β2AR recycling. For CIMPR, it is proposed that a direct interaction between the cytoplasmic tail and retromer complex is required for proper trafficking29. β2AR trafficking is dependent on a PDZ motif present in the cytoplasmic tail that is both necessary and sufficient to mediate its plasma membrane recycling, but the core retromer complex is devoid of any recognizable PDZ domain. SNX27 contains a PDZ domain that binds the β2AR tail, and has recently been shown to be essential for PDZ-directed recycling of the β2AR30. Verifying this, knockdown of SNX27 robustly inhibited recycling of β2ARs (Fig 5a). Despite this pronounced decrease in β2AR recycling, CIMPR distribution in the same cells appeared unaffected. Furthermore, SNX27 depletion did not alter CIMPR surface expression (Fig 5b) or CIMPR turnover in the presence of cyclohexamide (data not shown).


SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors.

Temkin P, Lauffer B, Jäger S, Cimermancic P, Krogan NJ, von Zastrow M - Nat. Cell Biol. (2011)

SNX27 serves as an adapter for β2AR, sorting it into the retromer tubule(a) Comparative localization of β2AR (red) with the trans-Golgi marker GalT-GFP (green) and CIMPR (blue). Cells were fed anti-FLAG antibody in the presence of isoproterenol for 25 minutes prior to agonist washout with alprenelol for 45 minutes. Successful knockdown of SNX27 was judged by failure of β2AR to recycle to the plasma membrane. The scale bar represents 20 μm. (b) CIMPR surface immunoreactivity was quantified by fluorescence flow cytometry (n=6) to assess the integrity of CIMPR trafficking when SNX27 is depleted with siRNA. Successful depletion of SNX27 was confirmed by looking at visual recycling assays of β2AR as in (a). (c) Representative confocal image from live cell imaging showing an endosome from a SNX27-4 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (d) Confocal image of transiently transfected SNX27-HA (red) and VPS29-GFP (green) in fixed cells. The scale bar represents 20 μm. (e) Representative immunoblot showing co-immunoprecipitation of endogenous VPS35 with transiently transfected SNX27-HA. Lanes 1 and 3 show 2% whole cell lysate for the mock IP at left and the experiment at right. (f) SNX27 mediates plasma membrane recycling of PDZ motif containing cargo by linking to the retromer through an interaction with the WASH complex. SNX27 also interacts with the endosome directly through its lipid binding PX domain. (g) Isoproterenol or forskolin induced cAMP formation was measured in HEK 293 cells stably expressing β2AR, in the absence of IBMX, 20 minutes after agonist addition (n=6). Data points are the mean ± SEM.
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Figure 5: SNX27 serves as an adapter for β2AR, sorting it into the retromer tubule(a) Comparative localization of β2AR (red) with the trans-Golgi marker GalT-GFP (green) and CIMPR (blue). Cells were fed anti-FLAG antibody in the presence of isoproterenol for 25 minutes prior to agonist washout with alprenelol for 45 minutes. Successful knockdown of SNX27 was judged by failure of β2AR to recycle to the plasma membrane. The scale bar represents 20 μm. (b) CIMPR surface immunoreactivity was quantified by fluorescence flow cytometry (n=6) to assess the integrity of CIMPR trafficking when SNX27 is depleted with siRNA. Successful depletion of SNX27 was confirmed by looking at visual recycling assays of β2AR as in (a). (c) Representative confocal image from live cell imaging showing an endosome from a SNX27-4 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (d) Confocal image of transiently transfected SNX27-HA (red) and VPS29-GFP (green) in fixed cells. The scale bar represents 20 μm. (e) Representative immunoblot showing co-immunoprecipitation of endogenous VPS35 with transiently transfected SNX27-HA. Lanes 1 and 3 show 2% whole cell lysate for the mock IP at left and the experiment at right. (f) SNX27 mediates plasma membrane recycling of PDZ motif containing cargo by linking to the retromer through an interaction with the WASH complex. SNX27 also interacts with the endosome directly through its lipid binding PX domain. (g) Isoproterenol or forskolin induced cAMP formation was measured in HEK 293 cells stably expressing β2AR, in the absence of IBMX, 20 minutes after agonist addition (n=6). Data points are the mean ± SEM.
Mentions: We next investigated the mechanistic basis for the role of retromer in β2AR recycling. For CIMPR, it is proposed that a direct interaction between the cytoplasmic tail and retromer complex is required for proper trafficking29. β2AR trafficking is dependent on a PDZ motif present in the cytoplasmic tail that is both necessary and sufficient to mediate its plasma membrane recycling, but the core retromer complex is devoid of any recognizable PDZ domain. SNX27 contains a PDZ domain that binds the β2AR tail, and has recently been shown to be essential for PDZ-directed recycling of the β2AR30. Verifying this, knockdown of SNX27 robustly inhibited recycling of β2ARs (Fig 5a). Despite this pronounced decrease in β2AR recycling, CIMPR distribution in the same cells appeared unaffected. Furthermore, SNX27 depletion did not alter CIMPR surface expression (Fig 5b) or CIMPR turnover in the presence of cyclohexamide (data not shown).

Bottom Line: Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4.Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule.The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, University of California at San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

Show MeSH
Related in: MedlinePlus