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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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Knockdown of retromer by RNAi inhibits β2AR recycling and misroutes internalized β2ARs to lysosomes(a) Representative images from a visual assay for β2AR trafficking are shown. Stably transfected HEK 293 cells expressing FLAG-β2AR were transfected with either control siRNA or siRNA targeting the retromer component VPS35. In the “Agonist” condition, cells were incubated in the presence of the β2AR agonist isoproterenol (10 μM) and Alexa-conjugated M1 anti-FLAG for 25 min. In the “Agonist → Antagonist” condition, cells were incubated with isoproterenol for 25 min and then for an additional 45 min in the absence of isoproterenol (and in the presence of 10 μM of the β2AR antagonist alprenelol to prevent effects of any residual agonist). The scale bar represents 20 μm. (b) Flow cytometric analysis of β2AR recycling by uptake and efflux of bound M1 anti-FLAG antibody (n=4). (c) A time-course of recycling is shown for β2AR. The experiment was performed as in (b) but the duration after agonist washout was varied (n=4). (d) Representative confocal image from live cell imaging showing an endosome from a VPS35-1 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (e) A representative immunoblot assay of agonist induced FLAG-β2AR degradation. Detergent extracts were prepared from HEK 293 cells expressing FLAG-β2AR incubated in the absence of agonist or in the continuous presence of 10 μM isoproterenol for 4 h. Knockdown was verified by VPS35 immunoblot of the same lysates (middle panel), and equal loading was verified by immunoblotting for GAPDH (bottom panel). (f) FLAG-β2AR immunoblots were quantified by scanning densitometry across multiple experiments (n=3), and the percent receptor remaining after 4 h isoproterenol exposure was calculated. (g) Representative immunoblot showing isoproterenol induced β2AR degradation in cells depleted of VPS35 and treated with the vehicle dimethyl sulfoxide (DMSO), the lysosomal cathepsin inhibitor N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), or the proteosomal inhibitor epoxomicin (EPOX) (n=3). Data points are the mean ± SEM.
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Figure 2: Knockdown of retromer by RNAi inhibits β2AR recycling and misroutes internalized β2ARs to lysosomes(a) Representative images from a visual assay for β2AR trafficking are shown. Stably transfected HEK 293 cells expressing FLAG-β2AR were transfected with either control siRNA or siRNA targeting the retromer component VPS35. In the “Agonist” condition, cells were incubated in the presence of the β2AR agonist isoproterenol (10 μM) and Alexa-conjugated M1 anti-FLAG for 25 min. In the “Agonist → Antagonist” condition, cells were incubated with isoproterenol for 25 min and then for an additional 45 min in the absence of isoproterenol (and in the presence of 10 μM of the β2AR antagonist alprenelol to prevent effects of any residual agonist). The scale bar represents 20 μm. (b) Flow cytometric analysis of β2AR recycling by uptake and efflux of bound M1 anti-FLAG antibody (n=4). (c) A time-course of recycling is shown for β2AR. The experiment was performed as in (b) but the duration after agonist washout was varied (n=4). (d) Representative confocal image from live cell imaging showing an endosome from a VPS35-1 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (e) A representative immunoblot assay of agonist induced FLAG-β2AR degradation. Detergent extracts were prepared from HEK 293 cells expressing FLAG-β2AR incubated in the absence of agonist or in the continuous presence of 10 μM isoproterenol for 4 h. Knockdown was verified by VPS35 immunoblot of the same lysates (middle panel), and equal loading was verified by immunoblotting for GAPDH (bottom panel). (f) FLAG-β2AR immunoblots were quantified by scanning densitometry across multiple experiments (n=3), and the percent receptor remaining after 4 h isoproterenol exposure was calculated. (g) Representative immunoblot showing isoproterenol induced β2AR degradation in cells depleted of VPS35 and treated with the vehicle dimethyl sulfoxide (DMSO), the lysosomal cathepsin inhibitor N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), or the proteosomal inhibitor epoxomicin (EPOX) (n=3). Data points are the mean ± SEM.

Mentions: After treatment with agonist such as isoproterenol, β2ARs trigger a signaling cascade and undergo clathrin mediated endocytosis. β2ARs are then rapidly recycled from the early endosome antigen 1 (EEA1) compartment (Fig 1a, b, 4a) to the plasma membrane (Fig 2c), resensitizing the cell5. Internalized transmembrane proteins are generally thought to leave the endosome through tubules6. In the case of β2AR, receptor-containing tubular endosomal protrusions can be visualized in living cells (Fig 1a, c)7. β2AR recycling is sequence-dependent, requiring a C-terminal PDZ ligand2. When this ligand is occluded by a HA tag (β2AR-HA), mutant receptors fail to recycle efficiently and are not seen in endosomal tubules (Fig 1c)2. Therefore, these tubules likely represent the structure responsible for sequence-dependent recycling of β2AR.


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)

Knockdown of retromer by RNAi inhibits β2AR recycling and misroutes internalized β2ARs to lysosomes(a) Representative images from a visual assay for β2AR trafficking are shown. Stably transfected HEK 293 cells expressing FLAG-β2AR were transfected with either control siRNA or siRNA targeting the retromer component VPS35. In the “Agonist” condition, cells were incubated in the presence of the β2AR agonist isoproterenol (10 μM) and Alexa-conjugated M1 anti-FLAG for 25 min. In the “Agonist → Antagonist” condition, cells were incubated with isoproterenol for 25 min and then for an additional 45 min in the absence of isoproterenol (and in the presence of 10 μM of the β2AR antagonist alprenelol to prevent effects of any residual agonist). The scale bar represents 20 μm. (b) Flow cytometric analysis of β2AR recycling by uptake and efflux of bound M1 anti-FLAG antibody (n=4). (c) A time-course of recycling is shown for β2AR. The experiment was performed as in (b) but the duration after agonist washout was varied (n=4). (d) Representative confocal image from live cell imaging showing an endosome from a VPS35-1 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (e) A representative immunoblot assay of agonist induced FLAG-β2AR degradation. Detergent extracts were prepared from HEK 293 cells expressing FLAG-β2AR incubated in the absence of agonist or in the continuous presence of 10 μM isoproterenol for 4 h. Knockdown was verified by VPS35 immunoblot of the same lysates (middle panel), and equal loading was verified by immunoblotting for GAPDH (bottom panel). (f) FLAG-β2AR immunoblots were quantified by scanning densitometry across multiple experiments (n=3), and the percent receptor remaining after 4 h isoproterenol exposure was calculated. (g) Representative immunoblot showing isoproterenol induced β2AR degradation in cells depleted of VPS35 and treated with the vehicle dimethyl sulfoxide (DMSO), the lysosomal cathepsin inhibitor N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), or the proteosomal inhibitor epoxomicin (EPOX) (n=3). Data points are the mean ± SEM.
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Figure 2: Knockdown of retromer by RNAi inhibits β2AR recycling and misroutes internalized β2ARs to lysosomes(a) Representative images from a visual assay for β2AR trafficking are shown. Stably transfected HEK 293 cells expressing FLAG-β2AR were transfected with either control siRNA or siRNA targeting the retromer component VPS35. In the “Agonist” condition, cells were incubated in the presence of the β2AR agonist isoproterenol (10 μM) and Alexa-conjugated M1 anti-FLAG for 25 min. In the “Agonist → Antagonist” condition, cells were incubated with isoproterenol for 25 min and then for an additional 45 min in the absence of isoproterenol (and in the presence of 10 μM of the β2AR antagonist alprenelol to prevent effects of any residual agonist). The scale bar represents 20 μm. (b) Flow cytometric analysis of β2AR recycling by uptake and efflux of bound M1 anti-FLAG antibody (n=4). (c) A time-course of recycling is shown for β2AR. The experiment was performed as in (b) but the duration after agonist washout was varied (n=4). (d) Representative confocal image from live cell imaging showing an endosome from a VPS35-1 siRNA treated cell expressing FLAG-β2AR (red) and VPS29-GFP (green). The scale bar represents 1 μm. (e) A representative immunoblot assay of agonist induced FLAG-β2AR degradation. Detergent extracts were prepared from HEK 293 cells expressing FLAG-β2AR incubated in the absence of agonist or in the continuous presence of 10 μM isoproterenol for 4 h. Knockdown was verified by VPS35 immunoblot of the same lysates (middle panel), and equal loading was verified by immunoblotting for GAPDH (bottom panel). (f) FLAG-β2AR immunoblots were quantified by scanning densitometry across multiple experiments (n=3), and the percent receptor remaining after 4 h isoproterenol exposure was calculated. (g) Representative immunoblot showing isoproterenol induced β2AR degradation in cells depleted of VPS35 and treated with the vehicle dimethyl sulfoxide (DMSO), the lysosomal cathepsin inhibitor N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD), or the proteosomal inhibitor epoxomicin (EPOX) (n=3). Data points are the mean ± SEM.
Mentions: After treatment with agonist such as isoproterenol, β2ARs trigger a signaling cascade and undergo clathrin mediated endocytosis. β2ARs are then rapidly recycled from the early endosome antigen 1 (EEA1) compartment (Fig 1a, b, 4a) to the plasma membrane (Fig 2c), resensitizing the cell5. Internalized transmembrane proteins are generally thought to leave the endosome through tubules6. In the case of β2AR, receptor-containing tubular endosomal protrusions can be visualized in living cells (Fig 1a, c)7. β2AR recycling is sequence-dependent, requiring a C-terminal PDZ ligand2. When this ligand is occluded by a HA tag (β2AR-HA), mutant receptors fail to recycle efficiently and are not seen in endosomal tubules (Fig 1c)2. Therefore, these tubules likely represent the structure responsible for sequence-dependent recycling of β2AR.

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