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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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Rapid recycling β2ARs selectively enter retromer-associated endosomal tubules(a) Representative images from a movie of a cell expressing β2AR (red) and VPS29-GFP (green). Below, the inset box is shown across multiple frames of the movie. The edges of the cell are shown as dotted lines in the merged image. The scale bar represents 4 μm. (b) Representative immunoblots are shown from endosomes that were immuno-purified using antibody against the early endosome component EEA1 (n=3). (c) Representative images of endosomes containing wild type β2AR or recycling-defective β2AR-HA receptors (red) and SNX1-GFP or VPS29-GFP (green). Images were acquired by confocal microscopy of living cells after stimulating receptor endocytosis with isoproterenol. Wild type β2ARs, but not recycling-defective β2AR-HA mutant receptors, were visible in the VPS29-associated tubule extending from the endosome body. The scale bar represents 1 μm. (d) Fluorescence intensity tracing of labeled β2AR (black squares) and VPS29 (red triangles) around the edge of the endosome. Each point represents average fluorescence over a six degree arc of the endosome circumference. The four points of greatest VPS29-GFP fluorescence (open triangles) were used to mark the tubule. β2AR fluorescence values were background-corrected and normalized to the average fluorescence of a portion (240o) of the endosome, excluding the 120o of circumference centered at the tubule base. The scale bar represents 1 μm. (e) Relative receptor enrichment (average of open squares in panel b) of β2AR (red bar) or β2AR-HA (blue bar) at the tubule base. 20 endosomes (4 independent experiments), extending a single retromer-associated tubule, per receptor type were analyzed. Data points are the mean ± standard error of the mean (SEM).
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Figure 1: Rapid recycling β2ARs selectively enter retromer-associated endosomal tubules(a) Representative images from a movie of a cell expressing β2AR (red) and VPS29-GFP (green). Below, the inset box is shown across multiple frames of the movie. The edges of the cell are shown as dotted lines in the merged image. The scale bar represents 4 μm. (b) Representative immunoblots are shown from endosomes that were immuno-purified using antibody against the early endosome component EEA1 (n=3). (c) Representative images of endosomes containing wild type β2AR or recycling-defective β2AR-HA receptors (red) and SNX1-GFP or VPS29-GFP (green). Images were acquired by confocal microscopy of living cells after stimulating receptor endocytosis with isoproterenol. Wild type β2ARs, but not recycling-defective β2AR-HA mutant receptors, were visible in the VPS29-associated tubule extending from the endosome body. The scale bar represents 1 μm. (d) Fluorescence intensity tracing of labeled β2AR (black squares) and VPS29 (red triangles) around the edge of the endosome. Each point represents average fluorescence over a six degree arc of the endosome circumference. The four points of greatest VPS29-GFP fluorescence (open triangles) were used to mark the tubule. β2AR fluorescence values were background-corrected and normalized to the average fluorescence of a portion (240o) of the endosome, excluding the 120o of circumference centered at the tubule base. The scale bar represents 1 μm. (e) Relative receptor enrichment (average of open squares in panel b) of β2AR (red bar) or β2AR-HA (blue bar) at the tubule base. 20 endosomes (4 independent experiments), extending a single retromer-associated tubule, per receptor type were analyzed. Data points are the mean ± standard error of 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)

Rapid recycling β2ARs selectively enter retromer-associated endosomal tubules(a) Representative images from a movie of a cell expressing β2AR (red) and VPS29-GFP (green). Below, the inset box is shown across multiple frames of the movie. The edges of the cell are shown as dotted lines in the merged image. The scale bar represents 4 μm. (b) Representative immunoblots are shown from endosomes that were immuno-purified using antibody against the early endosome component EEA1 (n=3). (c) Representative images of endosomes containing wild type β2AR or recycling-defective β2AR-HA receptors (red) and SNX1-GFP or VPS29-GFP (green). Images were acquired by confocal microscopy of living cells after stimulating receptor endocytosis with isoproterenol. Wild type β2ARs, but not recycling-defective β2AR-HA mutant receptors, were visible in the VPS29-associated tubule extending from the endosome body. The scale bar represents 1 μm. (d) Fluorescence intensity tracing of labeled β2AR (black squares) and VPS29 (red triangles) around the edge of the endosome. Each point represents average fluorescence over a six degree arc of the endosome circumference. The four points of greatest VPS29-GFP fluorescence (open triangles) were used to mark the tubule. β2AR fluorescence values were background-corrected and normalized to the average fluorescence of a portion (240o) of the endosome, excluding the 120o of circumference centered at the tubule base. The scale bar represents 1 μm. (e) Relative receptor enrichment (average of open squares in panel b) of β2AR (red bar) or β2AR-HA (blue bar) at the tubule base. 20 endosomes (4 independent experiments), extending a single retromer-associated tubule, per receptor type were analyzed. Data points are the mean ± standard error of the mean (SEM).
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Related In: Results  -  Collection

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Figure 1: Rapid recycling β2ARs selectively enter retromer-associated endosomal tubules(a) Representative images from a movie of a cell expressing β2AR (red) and VPS29-GFP (green). Below, the inset box is shown across multiple frames of the movie. The edges of the cell are shown as dotted lines in the merged image. The scale bar represents 4 μm. (b) Representative immunoblots are shown from endosomes that were immuno-purified using antibody against the early endosome component EEA1 (n=3). (c) Representative images of endosomes containing wild type β2AR or recycling-defective β2AR-HA receptors (red) and SNX1-GFP or VPS29-GFP (green). Images were acquired by confocal microscopy of living cells after stimulating receptor endocytosis with isoproterenol. Wild type β2ARs, but not recycling-defective β2AR-HA mutant receptors, were visible in the VPS29-associated tubule extending from the endosome body. The scale bar represents 1 μm. (d) Fluorescence intensity tracing of labeled β2AR (black squares) and VPS29 (red triangles) around the edge of the endosome. Each point represents average fluorescence over a six degree arc of the endosome circumference. The four points of greatest VPS29-GFP fluorescence (open triangles) were used to mark the tubule. β2AR fluorescence values were background-corrected and normalized to the average fluorescence of a portion (240o) of the endosome, excluding the 120o of circumference centered at the tubule base. The scale bar represents 1 μm. (e) Relative receptor enrichment (average of open squares in panel b) of β2AR (red bar) or β2AR-HA (blue bar) at the tubule base. 20 endosomes (4 independent experiments), extending a single retromer-associated tubule, per receptor type were analyzed. Data points are the mean ± standard error of 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