1. Volume 9, Issue 6, Pages 1215-1225 (June 2002)
PIP2-PDZ Domain Binding Controls the Association of Syntenin with the Plasma Membrane 
Pascale Zimmermann, Kris Meerschaert, Gunter Reekmans, Iris Leenaerts, J.Victor Small, Joël Vandekerckhove, Guido David, Jan Gettemans 
Molecular Cell 
Volume 9, Issue 6, Pages (June 2002)
DOI: /S (02)00549-X

2. Figure 1
The PDZ Domains of Syntenin, PTP-BL, CASK, and Tiam-1 Bind PIP2 Micelles
(A) Schematic representation of the proteins and their PDZ domains used in this study.
(B) Superdex-200 gel filtration of GST (10 μM) preincubated with 100 μM PIP2.
(C) Superdex-200 gel filtration of 10 μM GST-PDZ1-syntenin in the absence of PIP2 (curve 1), preincubated with 20 μM (curve 2) or 100 μM (curve 3) PIP2. Markers are indicated: Ov., ovalbumine (45 kDa); IgG, immunoglobulin G (158 kDa); Thyr, Thyroglobulin (660 kDa). A.U., absorption units.
(D) Binding (mean ± range, n = 2) to PIP2 micelles of GST-PDZ domains as a function of PIP2 concentration, based on gel filtration experiments. GST-PDZ2-syntenin and GST-PDZ-Tiam-1 (data not shown) are similar to GST-PDZ1-syntenin and GST-PDZ5-PTP-BL, respectively.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

3. Figure 2
The Syntenin PDZ Domains Interact with Bis- and Trisphosphate Phosphoinositide-Containing Lipid Layers in Biosensor Analysis
(A) The binding of the isolated PDZ1 domain of syntenin (100 μg/ml) to lipid layers increases as the PIP2 concentration increases.
(B) Interaction of GST and of GST fused to the PDZ1 domain of syntenin, to the tandem PDZ domains of syntenin (I103–V298), to full-length syntenin, or to the PH domain of PLCδ (all at 25 μg/ml) with lipid layers containing 10% PIP2.
(C–E) Interaction of GST fused to the tandem PDZ domains of syntenin or to the PH domains of Grp1, DAPP1, or PLCδ as controls, with lipid layers containing 10% phosphatidylinositol 3,4-bisphosphate (C), phosphatidylinositol 3,5-bisphosphate (D), or phosphatidylinositol 3,4,5-trisphosphate (E). All proteins were used at 1 μM.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

4. Figure 3
Localization of the Tandem PDZ Domains of Syntenin in PIP2-Rich Plasma Membrane Regions and Ionomycin-Induced Cytosolic Relocation of These PDZ Domains
(A–C) Confocal images of MDCK cells transiently overexpressing EGFP-PH-PLCδ (green), simultaneously stained for endogenous syntenin (red), show that syntenin and the PIP2 probe are enriched at various plasma membrane contact regions (yellow).
(D–F) Epifluorescence micrographs of MCF-7 cells simultaneously overexpressing EGFP fused to the tandem PDZ domains of syntenin (green) and DsRed-PH-PLCδ (red). The merged picture (yellow) shows colocalization between both polypeptides.
(G–O) Time-lapse microscopy of MCF-7 cells overexpressing EGFP-PH-PLCδ (G–I), EGFP fused to the tandem PDZ domains of syntenin (EGFP-tandem PDZ-Synt, [J–L]), or farnesylated EGFP (EGFP-F, [M–O]) before (G, J, and M), or 50 s (H, K, and N) and 100 s (I, L, and O) following ionomyin-induced hydrolysis of plasma membrane PIP2.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

5. Figure 4
PIP2 Binding to PDZ Domains Inhibits Association with the Cytosolic Part of Transmembrane Proteins in Pull-Down Experiments
(A) Binding of isolated PDZ domains to PIP2 micelles. (a) Binding of GST-PDZ2-syntenin to the C-terminal peptide of ephrin-B progressively decreases in the presence of increasing PIP2 concentrations. (b and c) Similar results were obtained with GST-PDZ-CASK and GST-PDZ2-syntenin with respect to syndecan-2 binding. (d) Phosphatidylinositol (PI) has no effect on the association between GST-PDZ2-syntenin and syndecan-2, even at the highest concentrations tested (100 μM).
(B) Incubation of GST-PDZ2-syntenin with increasing concentrations of inositol 1,4,5-trisphosphate (IP3) does not affect binding to syndecan-2 peptide.
(C) Vesicles containing 10% PIP2 (20 μM final concentration of PIP2) inhibit the binding of the tandem PDZ domains of syntenin but not the binding of single PDZ domains to syndecan-2 peptide. GST-fusion proteins containing the tandem PDZ domains of syntenin or only PDZ1 or PDZ2 were preincubated with 20 μl PC/PE/PS vesicles (1 mM) containing 10% PI (lane 2) or 10% PIP2 (lane 3) in a total volume of 100 μl before addition to syndecan-2 peptide Sepharose. Lane 1 in each case represents PDZ binding in the absence of lipid vesicles. Gels were stained with Coomassie.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

6. Figure 5
Membrane Anchoring of the Tandem PDZ Domains of Syntenin by Syndecan-2 and PIP2
Overexpression of wild-type (WT) syndecan-2 but not of syndecan-2 truncated for the PDZ binding site (C30) overcomes plasma membrane release of the tandem PDZ domains of syntenin to the cytosol upon PIP2 breakdown. MCF-7 cells transiently express syndecan-2 C30 (A–F) or syndecan-2 WT (G–L), together with ECFP-PH-PLCδ and EYFP-tandem PDZ domains of syntenin (EYFP-tandem PDZ-Synt). The localizations of the three molecules are shown before (A–C and G–I) or 1 min after the addition of ionomycin (D–F and J–L). Redistribution of ECFP-PH-PLCδ to the cytosol occurs in both syndecan-2 WT (K) and C30 (E) transfectants, illustrating the complete breakdown of PIP2 from the plasma membrane. On the opposite, the EYFP-tandem PDZ domains of syntenin are partially retained at the plasma membrane in syndecan-2 WT transfectants (L). Images are representative of six independent experiments.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

7. Figure 6 Syndecan-2 and PIP2 Cooperate in Binding Syntenin
Binding of GST-syntenin-myc to spots containing increasing amounts of PIP2 alone (middle lane, amounts as indicated at the bottom of the histogram) or increasing amounts of PIP2 mixed with a constant amount of 100 picomoles of scrambled peptide (upper lane) or 100 picomoles of syndecan-2 peptide (lower lane and histogram). Note that binding to 100 picomoles of peptide alone is illustrated by the first signal in the lower lane.
(B) Peptide (100 picomoles) and PIP2 (amounts as indicated) do not cooperate in the binding of the isolated PDZ2 domain of syntenin. The binding of GST-syntenin-myc (upper panel) is compared to the binding of GST-PDZ2-syntenin-myc (middle and lower panels). Binding of GST-PDZ2-syntenin-myc to scrambled peptide (100 picomoles)/PIP2 mixtures is given as control (middle panel).
(C) Model illustrating the anchoring of syntenin to the plasma membrane via its PDZ domains. Three different modes seem possible in the cell: sole binding to PIP2, sole binding to dimers of transmembrane receptors, or binding of one PDZ domain (most likely PDZ1) to PIP2 and one PDZ domain (most likely PDZ2) to one transmembrane receptor.
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)

8. Figure 7
Relative Contributions of the Syntenin PDZ1 and PDZ2 Domains in Binding to PIP2 Containing Lipid Layers and Plasma Membrane Targeting in MCF-7 Cells
(A) Mutating amino acids in PDZ1 and PDZ2 necessary for syndecan binding also reduces PIP2 interaction in Biacore analysis. The wild-type tandem PDZ domains of syntenin interact with reconstituted membranes containing PIP2. A (WT PDZ1 + mutant PDZ2) tandem shows reduced binding, whereas (mutant PDZ1 + WT PDZ2) and (mutant PDZ1 + mutant PDZ2) tandems fail to bind membranes containing PIP2. Note that while PDZ2 binds syndecan-2 with higher affinity than PDZ1 (Grootjans et al., 2000), PDZ1 binds PIP2 with higher affinity than PDZ2.
(B) Localization of EGFP-tagged WT and mutant tandem PDZ constructs in MCF-7 cells. EGFP-(WT PDZ1 + WT PDZ2) (a) and EGFP-(WT PDZ1 + mutant PDZ2) (b) are enriched at the plasma membrane, unlike EGFP-(mutant-PDZ1 + WT PDZ2) (c) or the double mutant tandem (d).
(C) Syndecan-2 overexpression partially rescues membrane localization of EGFP-[mutant PDZ1 + WT PDZ2] in an ionomycin insensitive way but does not impair cytosolic redistribution of EGFP-[WT PDZ1 + mutant PDZ2] upon ionomycin treatment. Time-lapse images are shown before (a, b, e, and f) or 1 min after (c, d, g, and h) ionomycin addition to MCF-7 cells transfected with WT syndecan-2 receptor (shown in [a], [c], [e], and [g]) and EGFP-(mutant PDZ1 + WT PDZ2) (a–d) or EGFP-(WT PDZ1 + mutant PDZ2) (e–f). Punctate membrane concentration of EGFP-[mutant PDZ1 + WT PDZ2] is indicated by arrowheads in (b) and (d). Note that ionomycin causes complete redistribution of EGFP-(WT PDZ1 + mutant PDZ2) (compare [h] with [f]).
Molecular Cell 2002 9, DOI: ( /S (02)00549-X)