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Subcellular sorting of the G-protein coupled mouse somatostatin receptor 5 by a network of PDZ-domain containing proteins.

Bauch C, Koliwer J, Buck F, Hönck HH, Kreienkamp HJ - PLoS ONE (2014)

Bottom Line: PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes.PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5.Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

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SNX27 is involved in recycling of SSTR5.A. 293 cells were transfected with expression constructs coding for mRFP-tagged wt and mutant SSTR5, and GFP-SNX27. Cells were plated on cover slips and imaged by fluorescence microscopy using mRFP and GFP specific settings. B. mRFP-SSTR5 expressing cells were transfected with SNX27 specific siRNA or control non-specific siRNA; 72 hours after transfection, cells were lyzed and analyzed by Western blotting using mRFP, SNX27 and GAPDH specific antibodies. Reduction of SNX27 levels to 19% of control led to a reduction of SSTR5 levels to 88%. C. SNX27 siRNA treated cells expressing the wt SSTR5 were analyzed for agonist dependent endocytosis as in Fig. 2A and B. The fraction of receptors which have been endocytosed after 15 min of agonist treatment is depicted. No differences in endocytosis efficiency was observed. D. The degradation of endocytosed wt SSTR5 after agonist dependent endocytosis was analyzed by surface biotinylation. Data are represented as the percentage of receptors remaining after the time indicated (*, significantly different, p<0.05, t-test; n = 6). E,F. SNX27 expression was knocked down in cells expressing the wt SSTR5 (E), or the SSTR5ΔCT mutant (F). Recycling of the receptor after agonist-induced internalization was analyzed as described in Fig. 2 by surface biotinylation and stripping of remaining surface receptors with glutathione. Data are presented as the fraction of the internalized receptors which return to the cell surface at a given time point (*, significantly different, p<0.05, Mann-Whitney U-test; n = 4).
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pone-0088529-g004: SNX27 is involved in recycling of SSTR5.A. 293 cells were transfected with expression constructs coding for mRFP-tagged wt and mutant SSTR5, and GFP-SNX27. Cells were plated on cover slips and imaged by fluorescence microscopy using mRFP and GFP specific settings. B. mRFP-SSTR5 expressing cells were transfected with SNX27 specific siRNA or control non-specific siRNA; 72 hours after transfection, cells were lyzed and analyzed by Western blotting using mRFP, SNX27 and GAPDH specific antibodies. Reduction of SNX27 levels to 19% of control led to a reduction of SSTR5 levels to 88%. C. SNX27 siRNA treated cells expressing the wt SSTR5 were analyzed for agonist dependent endocytosis as in Fig. 2A and B. The fraction of receptors which have been endocytosed after 15 min of agonist treatment is depicted. No differences in endocytosis efficiency was observed. D. The degradation of endocytosed wt SSTR5 after agonist dependent endocytosis was analyzed by surface biotinylation. Data are represented as the percentage of receptors remaining after the time indicated (*, significantly different, p<0.05, t-test; n = 6). E,F. SNX27 expression was knocked down in cells expressing the wt SSTR5 (E), or the SSTR5ΔCT mutant (F). Recycling of the receptor after agonist-induced internalization was analyzed as described in Fig. 2 by surface biotinylation and stripping of remaining surface receptors with glutathione. Data are presented as the fraction of the internalized receptors which return to the cell surface at a given time point (*, significantly different, p<0.05, Mann-Whitney U-test; n = 4).

Mentions: Immunocytochemical analysis indicated that SSTR5 and SNX27 colocalize mostly in intracellular vesicular structures which are likely to be endosomes, based on the known localization of SNX27 [10] (Fig. 4A; see also Figure S3 in File S1). To determine the role of SNX27 in SSTR5 trafficking, we established an efficient siRNA based knockdown for the endogenous SNX27 in HEK293 cells (SNX27 protein levels reduced to 19 +/− 3% of control values). This treatment slightly, but significantly reduced the total cellular levels of the receptor (Fig. 4B). SNX27 has been reported to accelerate endocytosis of GIRK/Kir3 channels [9], [10]; however, the rate of agonist dependent endocytosis of the SSTR5 was not affected by loss of SNX27 (Fig. 4C). Instead, knockdown of SNX27 significantly delayed recycling of the SSTR5 after agonist-dependent endocytosis, while recycling of the SSTR5ΔCT mutant was not affected (Fig. 4D,E). In addition, using assays established in Fig. 1, we observed enhanced degradation of endocytosed SSTR5 upon SNX27 knockdown (Fig. 4F). These data are in agreement with previous observations made for the β2-adrenergic receptor, [4], [9], suggesting that interaction of SNX27 with the PDZ ligand motif switches trafficking of the receptor from lysosomal targeting to a recycling mode.


Subcellular sorting of the G-protein coupled mouse somatostatin receptor 5 by a network of PDZ-domain containing proteins.

Bauch C, Koliwer J, Buck F, Hönck HH, Kreienkamp HJ - PLoS ONE (2014)

SNX27 is involved in recycling of SSTR5.A. 293 cells were transfected with expression constructs coding for mRFP-tagged wt and mutant SSTR5, and GFP-SNX27. Cells were plated on cover slips and imaged by fluorescence microscopy using mRFP and GFP specific settings. B. mRFP-SSTR5 expressing cells were transfected with SNX27 specific siRNA or control non-specific siRNA; 72 hours after transfection, cells were lyzed and analyzed by Western blotting using mRFP, SNX27 and GAPDH specific antibodies. Reduction of SNX27 levels to 19% of control led to a reduction of SSTR5 levels to 88%. C. SNX27 siRNA treated cells expressing the wt SSTR5 were analyzed for agonist dependent endocytosis as in Fig. 2A and B. The fraction of receptors which have been endocytosed after 15 min of agonist treatment is depicted. No differences in endocytosis efficiency was observed. D. The degradation of endocytosed wt SSTR5 after agonist dependent endocytosis was analyzed by surface biotinylation. Data are represented as the percentage of receptors remaining after the time indicated (*, significantly different, p<0.05, t-test; n = 6). E,F. SNX27 expression was knocked down in cells expressing the wt SSTR5 (E), or the SSTR5ΔCT mutant (F). Recycling of the receptor after agonist-induced internalization was analyzed as described in Fig. 2 by surface biotinylation and stripping of remaining surface receptors with glutathione. Data are presented as the fraction of the internalized receptors which return to the cell surface at a given time point (*, significantly different, p<0.05, Mann-Whitney U-test; n = 4).
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pone-0088529-g004: SNX27 is involved in recycling of SSTR5.A. 293 cells were transfected with expression constructs coding for mRFP-tagged wt and mutant SSTR5, and GFP-SNX27. Cells were plated on cover slips and imaged by fluorescence microscopy using mRFP and GFP specific settings. B. mRFP-SSTR5 expressing cells were transfected with SNX27 specific siRNA or control non-specific siRNA; 72 hours after transfection, cells were lyzed and analyzed by Western blotting using mRFP, SNX27 and GAPDH specific antibodies. Reduction of SNX27 levels to 19% of control led to a reduction of SSTR5 levels to 88%. C. SNX27 siRNA treated cells expressing the wt SSTR5 were analyzed for agonist dependent endocytosis as in Fig. 2A and B. The fraction of receptors which have been endocytosed after 15 min of agonist treatment is depicted. No differences in endocytosis efficiency was observed. D. The degradation of endocytosed wt SSTR5 after agonist dependent endocytosis was analyzed by surface biotinylation. Data are represented as the percentage of receptors remaining after the time indicated (*, significantly different, p<0.05, t-test; n = 6). E,F. SNX27 expression was knocked down in cells expressing the wt SSTR5 (E), or the SSTR5ΔCT mutant (F). Recycling of the receptor after agonist-induced internalization was analyzed as described in Fig. 2 by surface biotinylation and stripping of remaining surface receptors with glutathione. Data are presented as the fraction of the internalized receptors which return to the cell surface at a given time point (*, significantly different, p<0.05, Mann-Whitney U-test; n = 4).
Mentions: Immunocytochemical analysis indicated that SSTR5 and SNX27 colocalize mostly in intracellular vesicular structures which are likely to be endosomes, based on the known localization of SNX27 [10] (Fig. 4A; see also Figure S3 in File S1). To determine the role of SNX27 in SSTR5 trafficking, we established an efficient siRNA based knockdown for the endogenous SNX27 in HEK293 cells (SNX27 protein levels reduced to 19 +/− 3% of control values). This treatment slightly, but significantly reduced the total cellular levels of the receptor (Fig. 4B). SNX27 has been reported to accelerate endocytosis of GIRK/Kir3 channels [9], [10]; however, the rate of agonist dependent endocytosis of the SSTR5 was not affected by loss of SNX27 (Fig. 4C). Instead, knockdown of SNX27 significantly delayed recycling of the SSTR5 after agonist-dependent endocytosis, while recycling of the SSTR5ΔCT mutant was not affected (Fig. 4D,E). In addition, using assays established in Fig. 1, we observed enhanced degradation of endocytosed SSTR5 upon SNX27 knockdown (Fig. 4F). These data are in agreement with previous observations made for the β2-adrenergic receptor, [4], [9], suggesting that interaction of SNX27 with the PDZ ligand motif switches trafficking of the receptor from lysosomal targeting to a recycling mode.

Bottom Line: PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes.PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5.Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

View Article: PubMed Central - PubMed

Affiliation: Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

Show MeSH
Related in: MedlinePlus