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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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Interaction of the SSTR5 with SNX27 and NHERF1.A. Cells were transfected with expression vectors coding for mRFP-tagged wt SSTR5 or the truncation mutant SSTR5ΔCT, and a GFP-fusion of SNX27. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and SNX27 specific antibodies. The positions of endogenous and GFP-tagged recombinant SNX27 are indicated. B. A GFP-fusion of NHERF1 was coexpressed with either of the two receptor variants. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and GFP specific antibodies. C. mRFP-tagged SSTR5 was coexpressed with GFP-tagged PDZ proteins; cells lysates were subjected to immunoprecipitation using GFP-trap matrix. Input and precipitate samples were analyzed by Western blotting. For A–C, a typical of three experiments is shown in each case. D. GFP-tagged PDZ domain proteins from cell lysates of transfected 293 cells (input) were precipitated with control NHS-sepharose, or sepharose conjugated with the SSTR5 C-terminal peptide carrying the PDZ ligand sequence. Input and precipitate samples were analyzed by Western blotting using anti-GFP antibody. For quantitation, the ratio of precipitate to input signal intensity was determined. *,#, significantly different from PIST and NHERF1, respectively (p<0.05; Anova (p = 0.015), followed by t-test with Bonferroni correction; n = 4).
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pone-0088529-g003: Interaction of the SSTR5 with SNX27 and NHERF1.A. Cells were transfected with expression vectors coding for mRFP-tagged wt SSTR5 or the truncation mutant SSTR5ΔCT, and a GFP-fusion of SNX27. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and SNX27 specific antibodies. The positions of endogenous and GFP-tagged recombinant SNX27 are indicated. B. A GFP-fusion of NHERF1 was coexpressed with either of the two receptor variants. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and GFP specific antibodies. C. mRFP-tagged SSTR5 was coexpressed with GFP-tagged PDZ proteins; cells lysates were subjected to immunoprecipitation using GFP-trap matrix. Input and precipitate samples were analyzed by Western blotting. For A–C, a typical of three experiments is shown in each case. D. GFP-tagged PDZ domain proteins from cell lysates of transfected 293 cells (input) were precipitated with control NHS-sepharose, or sepharose conjugated with the SSTR5 C-terminal peptide carrying the PDZ ligand sequence. Input and precipitate samples were analyzed by Western blotting using anti-GFP antibody. For quantitation, the ratio of precipitate to input signal intensity was determined. *,#, significantly different from PIST and NHERF1, respectively (p<0.05; Anova (p = 0.015), followed by t-test with Bonferroni correction; n = 4).

Mentions: Based on the differential effects of the PDZ ligand motif we assumed that additional PDZ proteins might be involved in receptor sorting. Therefore we performed peptide affinity chromatography on HEK293 cell lysates, using a synthetic peptide corresponding to the C-terminal tail of the SSTR5, including the PDZ ligand motif (supporting information; Figure S1 in File S1). We identified sorting nexin 27 (SNX27) as a new interactor which contains one Phox domain and, similar to PIST, a single PDZ domain (Figure S1 in File S1). In addition, we had previously shown that PDZ-K1/NHERF3 binds to the SSTR5 C-terminus [8]. NHERF3 is expressed in HEK293 cells at low levels whereas its homologue, NHERF1, has been shown to be present in this cell line [20]. Coexpression/coimmunoprecipitation assays showed that both SNX27 and NHERF1 strongly interact with the SSTR5, whereas the SSTR5ΔCT mutant does not (Fig. 3A,B). In further experiments, the SSTR5 specifically interacted with PIST, SNX27, NHERF1 and NHERF3 but not with PSD-95 carrying three PDZ domains (Fig. 3C). In peptide pulldown experiments using the immobilized C-terminal PDZ ligand we observed that SNX27 interacts less efficiently when compared with PIST and NHERF1, suggesting that the affinity of the SNX27 PDZ domain is lower than that of PIST or NHERF1 (Fig. 3D).


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)

Interaction of the SSTR5 with SNX27 and NHERF1.A. Cells were transfected with expression vectors coding for mRFP-tagged wt SSTR5 or the truncation mutant SSTR5ΔCT, and a GFP-fusion of SNX27. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and SNX27 specific antibodies. The positions of endogenous and GFP-tagged recombinant SNX27 are indicated. B. A GFP-fusion of NHERF1 was coexpressed with either of the two receptor variants. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and GFP specific antibodies. C. mRFP-tagged SSTR5 was coexpressed with GFP-tagged PDZ proteins; cells lysates were subjected to immunoprecipitation using GFP-trap matrix. Input and precipitate samples were analyzed by Western blotting. For A–C, a typical of three experiments is shown in each case. D. GFP-tagged PDZ domain proteins from cell lysates of transfected 293 cells (input) were precipitated with control NHS-sepharose, or sepharose conjugated with the SSTR5 C-terminal peptide carrying the PDZ ligand sequence. Input and precipitate samples were analyzed by Western blotting using anti-GFP antibody. For quantitation, the ratio of precipitate to input signal intensity was determined. *,#, significantly different from PIST and NHERF1, respectively (p<0.05; Anova (p = 0.015), followed by t-test with Bonferroni correction; n = 4).
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Related In: Results  -  Collection

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pone-0088529-g003: Interaction of the SSTR5 with SNX27 and NHERF1.A. Cells were transfected with expression vectors coding for mRFP-tagged wt SSTR5 or the truncation mutant SSTR5ΔCT, and a GFP-fusion of SNX27. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and SNX27 specific antibodies. The positions of endogenous and GFP-tagged recombinant SNX27 are indicated. B. A GFP-fusion of NHERF1 was coexpressed with either of the two receptor variants. After cell lysis, receptors were precipitated using RFP-trap matrix. Input and precipitate (IP) samples were analyzed by Western Blot using mRFP and GFP specific antibodies. C. mRFP-tagged SSTR5 was coexpressed with GFP-tagged PDZ proteins; cells lysates were subjected to immunoprecipitation using GFP-trap matrix. Input and precipitate samples were analyzed by Western blotting. For A–C, a typical of three experiments is shown in each case. D. GFP-tagged PDZ domain proteins from cell lysates of transfected 293 cells (input) were precipitated with control NHS-sepharose, or sepharose conjugated with the SSTR5 C-terminal peptide carrying the PDZ ligand sequence. Input and precipitate samples were analyzed by Western blotting using anti-GFP antibody. For quantitation, the ratio of precipitate to input signal intensity was determined. *,#, significantly different from PIST and NHERF1, respectively (p<0.05; Anova (p = 0.015), followed by t-test with Bonferroni correction; n = 4).
Mentions: Based on the differential effects of the PDZ ligand motif we assumed that additional PDZ proteins might be involved in receptor sorting. Therefore we performed peptide affinity chromatography on HEK293 cell lysates, using a synthetic peptide corresponding to the C-terminal tail of the SSTR5, including the PDZ ligand motif (supporting information; Figure S1 in File S1). We identified sorting nexin 27 (SNX27) as a new interactor which contains one Phox domain and, similar to PIST, a single PDZ domain (Figure S1 in File S1). In addition, we had previously shown that PDZ-K1/NHERF3 binds to the SSTR5 C-terminus [8]. NHERF3 is expressed in HEK293 cells at low levels whereas its homologue, NHERF1, has been shown to be present in this cell line [20]. Coexpression/coimmunoprecipitation assays showed that both SNX27 and NHERF1 strongly interact with the SSTR5, whereas the SSTR5ΔCT mutant does not (Fig. 3A,B). In further experiments, the SSTR5 specifically interacted with PIST, SNX27, NHERF1 and NHERF3 but not with PSD-95 carrying three PDZ domains (Fig. 3C). In peptide pulldown experiments using the immobilized C-terminal PDZ ligand we observed that SNX27 interacts less efficiently when compared with PIST and NHERF1, suggesting that the affinity of the SNX27 PDZ domain is lower than that of PIST or NHERF1 (Fig. 3D).

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