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First published online 5 October 2004
doi: 10.1242/jcs.01379

Journal of Cell Science 117, 5367-5379 (2004)
Published by The Company of Biologists 2004
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New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4(a) receptor splice variant: roles in receptor targeting

Lara Joubert1, Brendon Hanson2, Gaël Barthet1, Michèle Sebben1, Sylvie Claeysen1, Wanjin Hong2, Philippe Marin1, Aline Dumuis1 and Joël Bockaert1,*

1 Laboratoire de Génomique Fonctionnelle, CNRS UPR2580, CCIPE, 141 rue de la Cardonille, 34094 Montpellier CEDEX 05, France
2 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Rep. of Singapore



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  		Fig. 1. C-Terminal sequences of the mouse 5-HT4(a), 5-HT4(b) and 5-HT4(e) receptor splice variants. Residues in grey represent the peptide baits used in the affinity-chromatography step of the proteomic screen [C-t(a), C-t(b) and C-t(e) for the 5-HT4(a)R, 5-HT4(b)R and 5-HT4(e)R variant, respectively]. The boxes show the canonical PDZ ligands expressed in the C-termini of 5-HT4(a)R and 5-HT4(e)R.

 


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  		Fig. 2. Two-dimensional analysis of the proteins interacting with the C-termini of mouse 5-HT4R splice variants. Proteins that bind to the C-terminus of the 5-HT4(a)R and 5-HT4(e)R variants were purified by affinity chromatography, separated by 2D electrophoresis and stained with silver. (A) 2D gels obtained with the C-t(a)wt and the C-t(e)wt peptides are illustrated. Arrows indicate the position of spots or trains of spots that were specifically retained by the C-t(a)wt and the C-t(b)wt peptides but not truncated peptides lacking their PDZ ligand [C-t(a){Delta}SCF and C-t(e){Delta}PVPV, respectively]. (B,C) Proteins recruited by affinity chromatography using the C-t(a)wt, C-t(b)wt, C-t(e)wt, C-t(a){Delta}SCF or C-t(e){Delta}PVPV peptides were separated by 2D electrophoresis and stained with silver. (B, top) Areas of interest of 2D gels showing the specific recruitment of ten spots by the 5-HT4(a)R C-terminus [C-t(a)wt peptide] via a PDZ-based mechanism. (C, top) Areas of interest of 2D gels showing the specific recruitment of three spots by the 5-HT4(e)R C-terminus [C-t(e)wt peptide] via a PDZ-based mechanism. The data are representative of four experiments performed independently.

 


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  		Fig. 3. Analysis of the interaction of 5-HT4R C-termini with specific sets of proteins by immunoblotting. C-t(a), C-t(b) and C-t(e) peptide baits were incubated with protein extracts from either whole mouse brain (A) or mouse colliculi neurons in primary culture (B). Proteins retained by affinity were separated by SDS-PAGE and transferred electrophoretically onto nitrocellulose sheets. Immunoblotting was performed with antibodies raised against the indicated proteins (anti-Ulip2, 1:2,000; anti-NHERF, 1:500; anti-Veli1-3, 1:200; anti-nNOS, 1:500). For each protein, the immunoreactive signals were found at molecular weights identical to those observed in silver-stained 2D gels. Input represents 5% of the total protein amount used in pull-down experiments. The data illustrated are representative of three experiments.

 


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  		Fig. 4. Interaction of the 5-HT4(a)R variant with both SNX27a and SNX27b in a PDZ-dependent manner. HEK-293 cells were co-transfected with the indicated cMyc-tagged-5-HT4R, with or without HA-tagged-SNX27 constructs using LipofectamineTM 2000. 24 hours after transfection, total cell lysates were immunoprecipitated with anti-Myc cross-linked protein-A/Sepharose. Immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting using the anti-HA antibody (top). Input (bottom) represents 5% of the total protein used in immunoprecipitation. The data illustrated are representative of three experiments.

 


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  		Fig. 5. Recruitment of the 5-HT4(a)R by SNX27a to early endosomes in A431 cells. (A) A431 cells transiently transfected using EFFECTENE with the cMyc-tagged-5-HT4(a)R together with HA-tagged SNX27a (a-d) or HA-SNX27a{Delta}PDZ (e-h) and the cMyc-tagged-5-HT4(a)R{Delta}SCF receptor together with HA-SNX27a (i-l) were processed for indirect immunofluorescence using the polyclonal anti-Myc antibody (visualized in green using goat anti-rabbit IgG conjugated to FITC) (a,e,i) and the monoclonal anti HA (visualized in red using goat anti-mouse IgG conjugated to AlexaFluor 555) (b,f,j). Merge images (c,g,k) were further magnified to show detail (d,h,l). Yellow staining highlights colocalization between 5-HT4(a)R and SNX27a. (B) Cells transfected as described in A were processed for indirect immunofluorescence, initially using the polyclonal anti-Myc antibody visualized in green (a,e,i) and the monoclonal anti-EEA1 antibody (visualized in blue using goat anti-mouse IgG conjugated to AlexaFluor647) (b,f,j), followed by incubation with a biotin-labelled monoclonal anti-HA antibody (visualized in red using streptavidin conjugated to Texas Red) (a,e,i). Overlaid images (c,g,k) were further magnified to show detail (d,h,l). Yellow staining highlights colocalization of the 5-HT4(a)R and SNX27a, whereas purple indicates colocalization of SNX27a and EEA1, and cyan indicates colocalization of 5-HT4(a)R and EEA1. White staining indicates colocalization between the 5-HT4(a)R, SNX27a and EEA1. Bars, 10 µm.

 


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  		Fig. 6. Colocalization of NHERF with the 5-HT4(a)R, but not the 5-HT4(b)R splice variant in transiently transfected NIH-3T3 cells. (A) Cells were transfected by electroporation with constructs encoding HA-tagged NHERF (1), the Rho-tagged 5-HT4(a)R (2) or the Rho-tagged 5-HT4(b)R (3) alone. They were permeabilized, immunostained with the anti-HA (1:400) or the anti-RhoTag (1:100) antibody and analysed by confocal laser scanning microscopy. Mid-nuclear slices from representative cells are shown. (B,C) Cells were transfected with either the HA-NHERF-RhoTag 5-HT4(a)R/pIRES2 (B) or the HA-NHERF-RhoTag-5-HT4(b)R/pIRES2 (C) construct, which ensure efficient co-expression in the same cells. Confocal slices are shown from the mid-nuclear level (top) or close to the cell surface (bottom). Left, medium and right panels show HA staining, RhoTag staining and merged images, respectively. Bars, 10 µm.

 


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  		Fig. 7. Colocalization of endogenous ezrin with the 5-HT4(a) but not the 5-HT4(b) splice variant in the presence of NHERF in NIH-3T3 cells. Cells were transfected by electroporation with plasmids encoding either HA-NHERF-RhoTag-5-HT4(a)R/pIRES2 (A, top, B) or the HA-NHERF-RhoTag-5-HT4(b)R/pIRES2 (A, bottom, C) construct. They were permeabilized and examined by confocal microscopy. Left, medium and right panels illustrate endogenous ezrin staining (anti-ezrin antibody 1:1,000), RhoTag staining (anti-RhoTag antibody 1:100) and merged images, respectively. Mid-nuclear slices (inlay: zoom) and vertical scans in the z axis (z) (along the black lines in the x-y scans) are depicted. Representative cells are represented. Bars, 10 µm.

 


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  		Fig. 8. Interaction of CIPP with the 5-HT4(e)R in transiently transfected COS-7 and NIH-3T3 cells. (A) COS-7 cells were transfected with Flag-tagged CIPP (1), the cMyc-5-HT4(e)R (2), the cMyc-5-HT4(b)R (3), the cMyc-5-HT4(e) {Delta}PVPV R (4) or Flag-tagged CIPP together with each 5-HT4R construct (5-13) using LipofectamineTM 2000. Cells were examined by confocal microscopy. (left) Flag staining (1:1,000); (middle) Myc staining (1:400); (right) merged images. (B) NIH-3T3 cells were transfected with the same constructs using Lipofectamine 2000. Merged images obtained after Flag and Myc staining of cells co-transfected with Flag-tagged CIPP and the cMyc-5-HT4(e)R (14), the cMyc-5-HT4(b)R (15) or the cMyc-5-HT4(e) {Delta}PVPV R (16) are depicted. Bars, 10 µm.

 

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© The Company of Biologists Ltd 2004