Interaction between SAP97 and PSD-95,

Two Maguk Proteins Involved in Synaptic Trafficking

of AMPA Receptors

Received for publication,May 31, 2005, and in revised form, October 26, 2005 Published, JBC Papers in Press, December 6, 2005Chunlin Cai, Hong Li, Claudio Rivera, and Kari Keinänen

From the Department of Biological and Environmental Sciences, Division of Biochemistry and Division of Physiology and the

Institute of Biotechnology, Viikki Biocenter, University of Helsinki.

Ceccaldi BenoîtDe la Crompe Brice

Master 2 Neurosciences 2011 BordeauxUE Cellular and Molecular Neurobiology

Plan INTRODUCTION

EXPERIMENTAL PROCEDURES Experimental design GST Pull-down assay

MATERIALS AND METHODS – RESULTS Part 1: Protein interaction

SAP97 Associates with PSD-95: in Vivo and in Vitro Mapping the PSD-95 Binding Site in SAP97 Mapping the SAP97 Binding Site in PSD-95 GST Pull-down Analysis of SAP97-PSD-95 Interaction

Part 2: Study of SAP97-PSD-95 Interaction in Cultured Neurons

DISCUSSION Proteins interaction Role of PSD-95/SAP97 in GluR-A-containing AMPAR synaptic clustering Authors’ hypothesis on trafficking GluR-A-containing AMPAR

INTRODUCTION

SAP97 and PSD-95 are two Maguk proteins (membrane-associated guanylate kinase homologs) implicated in the synaptic targeting and anchoring of AMPA receptors:

SAP97 bind directly to the C-terminus of the GluR-A subunit. SAP97 overexpression promote the synaptic delivery of GluR-A-containing AMPAR

PSD-95 bind indirectly via stargazin and TARPs to GluR-A. PSD-95 overexpression trigger the synaptic trafficking GluR-A-containing AMPAR in synaptic spines

Experiment using RNAi Knock down of SAP97 and PSD-95 inhibit the clustering of GluR-A

AMPA receptor

PDZ of SAP97 directly interacts with the C-terminal part of GluR-A

PSD-95 associates with AMPA-R via stargazin and TARPs

MAGUKs (Membrane-Associated Guanylate Kinase homologs)

PSD-95 (PostSynaptic Density-95)

SAP97 (Synapse-Associated Protein 97)

Others: PSD-93/Chapsyn-110 , SAP102

L27

The oligomeric nature of Maguks oriented the authors to examinate the potentiality for SAP97 and PSD-95 to form heteromeric complexe

Experimental questions:

Interaction studing of the interaction between these two proteins (domains interaction)

Impact studing of this interaction into the synaptic transport of GluR-A-containing AMPAR

EXPERIMENTAL PROCEDURES

Experimental designPart 1: Proteins interactionAuthors use coimmunoprecipitation experiments to study interaction between PSD-95 and SAP97. Firstable they analyse the formation of a complex in vivo (rat brain lysat) and in vitro (transfected HEK293 cells)

Then they show domains involved in the interaction.

They validate results by using GST-pull down assay in HEK293 cells

Part 2: study of SAP97-PSD-95 Interaction in Cultured Neurons They study in hippocampal neurons (mouse embryos, E17) the effect of an overexpression of PSD-95 on the synaptic clustering of SAP97.

After they analyse the impact of the complex formation on the GluR-A –containing AMPAr synaptic clustering.

GST Pull-down assay

•Pull-down assay is an in vitro experiment use to determine physical interaction between 2 or more proteins.

•GST fusion proteins is transfected and expressed in Esherichia coli BL21.

•Then they are purified by interaction of glutathione-Sepharose with GST-protein.

•After centrifugation we obtain only the fusion protein in solution.

•In the time we obtain a other solution by lysing the cell wich contain the prey protein.

•After, to allow the formation of the prey/bait complex, we mix the two solutions.

•The solution is wash and elute in SDS buffer then submit to electrophoresis and western blotting.

Materials and methods - Results

Part 1: Protein interaction

SAP97 Associates with PSD-95 in Vivo Materials and Methods

Detergent extract of cerebella from adulte Wistar rats were submitted to co-immunoprecipitation using SAP97 N antiserum, the corresponding preimmune serum or a PSD-95-specific antibody.

The obtained supernatants are subjected to immunoblotting with SAP97 N and anti-PSD-95.

SAP97 Associates with PSD-95 in Vivo Results

Fig 1A: Coimmunoprecipitation of SAP97 and PSD-95 in Rat brain detergent extract.

Input lane verifies the presence of protein in cells.

The two Co-IP constitute there reciprocal controls.

In rat brain, PSD-95 and SAP97 coimmunoprecipitate.

=> In rat brain, PSD-95 and SAP97 interacting to form a complex.

Non palmitoylatedPalmitoylated

SAP97 Associates with PSD-95 in Vitro Materials and Methods In first, HEK293 cells were transfected (Calcium-

Phosphate coprecipitation) with plasmids containing fusion tagged-proteins: Myc-SAP97 or GFP-PSD-95.

Myc tag was added on N-Terminal part of proteins

Green fluorescent protein was added on C-terminal

Detergent extract of HEK293 cells were submitted to co-immunoprecipitation using Myc or GFP-specific antibody.

The obtained supernatants were subjected to immunoblotting with anti-Myc and anti-GFP.

Fig 1B and1C: Coimmunoprecipitation of SAP97 and PSD-95 in HEK293 detergent extract.

SAP97 Associates with PSD-95 in Vitro Results(1/2)

In cotransfected HEK293 cells, Myc-SAP97 and GFP-PSD-95 coimmunoprecipitate.

The Co-IP does not allow to show a direct proteins interaction.

But, HEK293 cells are non-neurals so the authors conclude that:

=> interaction between this two tagged proteins is direct.

=> Authors verified the presence of endogenous SAP97 or PSD-95 (not published data). HEK293 cells express endogenous SAP97.

SAP97 Associates with PSD-95 in Vitro Results(2/2)

Coimmunoprecipitation of GFP-PSD-95 and endogenous SAP97

=> GFP-PSD-95 interacting directly to form a complex with endogenous SAP97.

=> Because HEK293 cells express SAP97, we cannot exclude the possibility that they can express other MAGUK proteins.

=> To validate the direct interaction, we purpose to use other methods like Yeast double hybrid method or

Fig 1A: Coimmunoprecipitation of endogenous SAP97 and GFP-PSD-95 in HEK293 cell detergent extract.

Mapping the PSD-95 Binding Site in SAP97 Materials and Methods In first, HEK293 cells were transfected (Calcium-Phosphate

coprecipitation) with plasmids containing fusion tagged-proteins: His-PSD-95 (full length, C-term) and differents Myc tagged domains of SAP97 (N-term).

His tag was added on C-terminal The same precedent protocol of Co-IP was used.

Mapping the PSD-95 Binding Site in SAP97 Results

Coimmunoprecipitation of His-PSD-95 with:

Myc-SAP97 Myc-ΔSH3-GK Myc-NTD

Fig 2A and 2B: Mapping the PSD-95 binding domain in SAP97. HEK293 cells were transfected for expression of His-tagged full-length PSD-95 together with the indicated Myc-tagged SAP97 domains. The arrows indicate the position of the immunoglobulin heavy chain band. Molecular size markers are shown on the left. Un., untransfected cells; Neg., no cotransfection with SAP97.

NTD is unique common domain which Co-IP with PSD-95.

=> The binding site of PSD-95 in SAP97 is the NTD.

Mapping the SAP97 Binding Site in PSD-95 Materials and Methods• In first, HEK293 cells were transfected (Calcium-Phosphate

coprecipitation) with plasmids containing fusion tagged-proteins: GFP-SAP97 (full length, N-terminal tag) and differents Myc tagged domains of PSD-95 (N-term).

• The same precedent protocol of Co-IP was used.

Mapping the SAP97 Binding Site in PSD-95 Results

Coimmunoprecipitation of GFP-SAP97 with:

Myc-PSD-95 Myc-ΔN-PSD-95 Myc-SH3-PSD-95 Myc-SH3-GK-PSD-95 (fig B)

Fig 3A and 3B: Mapping the SAP97 binding domain in PSD-95. HEK293 cells were transfected for the expression of GFP-tagged full-length SAP97 together with the indicated Myc-tagged PSD-95 domains. The arrows point to the immunoglobulin heavy chain band. Molecular size markers are shown on the left. Un., untransfected cells.

SH3 is unique common domain which Co-IP with SAP97.

=> The binding site of SAP97 in PSD-95 is the SH3 domain.

=> Why in first experiment, the SH3-GK-PSD-95 domain did not bind to SAP97?

GST Pull-down analysis of SAP97-PSD-95 interaction - Materials and Methods GST fusion protein were expressed in E.coli BL21 and

purified with gluthatione-Sepharose bead. In first experiment:

HEK293 cells were transfected by C-terminal tagged GFP-PSD-95. All SAP97 fragments were fusioned with GST then expressed and purified in

E.coli.

In second experiment: HEK293 cells were transfected by C-terminal tagged GFP-PSD-95-SH3. SAP97ΔNTD and SAP97NTD were fusioned with GST then expressed and

purified in E.coli.

Then GST-Protein-containing solution were mix with cells lysat.

Finally, the purified solution was submitted to western blot.

GST Pull-down analysis of SAP97-PSD-95 interaction - Results

Fig A: Interaction between GFP-PSD-95 and: GST-SAP97 GST-NTD-SAP97 GST-PDZ1-3-SAP97

=> The domain of GST-SAP97 that binds to GFP-PSD-95 is NTD of SAP97.

=> GST-PDZ1-3 may constitue a second binding site.

Fig B: Interaction between GFP-PSD-95-SH3 and GST-SAP97-NTD.

=> The SH3 domain of GFP-PSD-95 bind directly to the GST-SAP97 NTD.

Fig 4: GST pull-down analysis of SAP97-PSD-95 interaction A, binding of GFP-PSD-95 to SAP97 domains (transfection of HEK293 cells with GFP-PSD-95 and GST-SAP97 fusion proteins).B, binding of GFP-PSD-95 SH3 to SAP97 domains (transfection of HEK293 cells with GFP-PSD-95 and GST-SAP97 fusion proteins).

Part 2: Study of SAP97-PSD-95 Interaction in

Cultured Neurons

SAP97-PSD-95 Interaction in Cultured Neurons Materials and Methods Hippocampal neurons were fixed.

The fixed hippocampal neurons were permeabilized.

An overnight incubation with the primary Antibodies/antiserum was realized (SAP97N antiserum, anti-PSD-95 monoclonal antibody, and/or GluR-ACTD antiserum).

After washing, the secondary antibodies conjugated to Cyanine 3 (red) or Alexa fluor 488 (green) were added.

Immunostaining was visualized via fluorescence microscope.

SAP97-PSD-95 Interaction in Cultured Neurons - Results (1/4) Both endogenous proteins

are distributed in whole cell.

We can see that PSD-95 (red) is also localized in synaptic spines unlike SAP97 (green).

In colocalization study, SAP97 can be present in spines but always with PSD-95 (white arrows in merge photo).

Distribution of SAP97 and PSD-95 in cultured hippocampal neurons

A, immunofluorescence localization of endogenous SAP97 and PSD-95. Mouse hippocampal neurons (E17; kept for 17 days in vitro) were fixed and stained with rabbit anti-SAP97 N and mouse PSD-95 antibodies followed by Alexa Fluor-488-conjugated anti-rabbit and Cy3-conjugated anti-mouse IgGs, respectively. Individual immunofluorescence stainings for SAP97 (green) and PSD-95 (red) are shown in the upper panel, whereas the lower panel represents a merged picture of the individual immunostainings showing overlapping staining in yellow (Merge; Zoom for an enlarged view). The arrows indicate spines containing both SAP97 and PSD-95.

Distribution of SAP97 and PSD-95 in cultured hippocampal neurons

The overexpression of PSD-95 trigger the spine clustering of SAP97

To verifying this observations, the authors conducted the following experiment.

Induction of SAP97 clustering by overexpression of PSD-95. Cultured neurons transfected for expression of GFP-tagged PSD-95 were fixed and stained with anti-SAP97 N followed by Cy3-conjugated anti-mouse IgG. A merged image of the Cy3 (red) and GFP (green) fluorescence is shown. Individual fluorescence images of the boxed dendritic area are shown on the right.

SAP97-PSD-95 Interaction in Cultured Neurons - Results (2/4)

PSD-95-induced clustering of GluR-A.Cultured hippocampal neurons transfected with GFP-PSD-95 plasmid alone (A) or together with Myc-tagged SAP97 NTD (B) or PSD-95 SH3 (C) constructs were stained with GluR-A CTD antiserum followed by Cy3-con-jugated anti-rabbit IgG and then visualized for GluR-A immunoreactivity (red) and for GFP fluorescence (green). The panels on the right show enlarged images of GluR-A clusters along dendrites of untransfected cells (Un; blue-lined boxed area in the left panel) and of GFP-positive PSD-95-overexpressing cells (white-lined boxed area in the left panel).

The overexpression of GFP-PSD-95 (green) induces the spine clustering of GluR-A (red). (cf fig A)

The cotransfection with PSD-95-SH3 inhibits the GluR-A clustering induces by PSD-95. (cf fig B)

The cotransfection with SAP97-NTD inhibits the GluR-A clustering induces by PSD-95. (cf fig C)

The overexpression of SAP97-NTD alone does not induce the clustering of GluR-A.

The overexpression of PSD-95-SH3 alone does not induce the clustering of GluR-A.

SAP97-PSD-95 Interaction in Cultured Neurons - Results (4/4)

Intensities of GluR-A immunoreactive clusters in transfected neurons. Clustering of GluR-A was analyzed by immunofluorescence and quantified as described under “Experimental Procedures.” Cluster intensities in GFP-positive cells are indicated as percentage of control using cluster intensities measured from neighboring untransfected cells as a 100% reference including a total of 127 clusters in 20 neurons; 100 +/- 8.4%). Statistical significance of the results was analyzed by using unpaired t test to calculate the indicated two-tailed p values. NS, not significant (p<0.05); NA, not applicable.

Overexpression of PSD-95 trigger the spine clustering of SAP97.

The PSD-95SH3 inhibits the clustering of SAP97

Cultured hippocampal neurons were transfected for expression of GFP-tagged PSD-95 alone or together with Myc-tagged PSD-95 SH3 as indicated and then stained with anti-SAP97 N followed by Cy3-conjugated anti-mouse IgG. Merged images of the Cy3 (red) and GFP (green) fluorescence are shown on the left. The right panels show an enlarged view of the SAP97 immunofluorescence of the boxed areas. Dendrites of untransfected and transfected cells are indicated by the arrows and arrowheads, respectively.

SAP97-PSD-95 Interaction in Cultured Neurons - Results (3/4)PSD-95-induced clustering of SAP97 to

synaptic spines is inhibited by PSD-95SH3

Number of SAP97 immunoreactive spines in transfected neurons

DISCUSSION

Proteins interaction

In rat brain and in transfected cells, the SAP97 and PSD-95 can be associate directly as heteromeric complex.

Known mechanisms involving interaction of PSD-95 domains with other Maguks are PSD-95/Chapsyn110 via NTD and PSD-95/SAP102 via SH3-GK domains.

Authors found a new interaction mechanism between Dlg proteins: an association of SH3 (PSD-95) and NTD (SAP97).

SAP97 does exist in two states because of intramolecular interactions: In the closed complex, SH3 and GK interacting. This association prevents the GKAP binding

on GK domain.

In the second state, NTD interacting with SH3 liberate GK domain.

Similary configuration were found in PSD-95.

The authors hypothetis: The binding of NTD(SAP97) with SH3(PSD-95) promotes the complex anchoring via PSD-95-GK/GKAP interaction. GKAP interacts with Shank proteins which is a central actor for anchoring of Glutamate receptor in PSD.

Implication of PSD-95/SAP97 in GluR-A-containing AMPAR synaptic clustering In this study authors found:

GFP-PSD-95 overexpression trigger synaptic clustering of SAP97.

The inhibition induced by cotransfection of PSD-95-SH3 suggest the major role of PSD95/SAP97 in synaptic clustering of SAP97.

Overexpression of GFP-PSD-95 induces the synaptic clustering of GluR-A.

The inhibition triggered by co-transfection with SAP97-NTD and PSD-95-SH3 suggest the importance of SAP97/PSD-95 interaction in GluR-A clustering.

Precedent study has demonstrated that:

Association between GluR-A-containing AMPAR and PSD-95 is mediate by SAP97.

An endocytosis mechanism of synaptic GluR-A-containing AMPAR is based on the trimeric complex formation of a GluR-A/SAP97/MyosineVI.

Myosine VI is a motor protein implicated in clathrin mediated endocytosis of GluR-A.

Myosine VI can bind to NTD SAP97 like PSD-95.

competition between PSD-95 and MyoVI.

The PSD-95 binding on the NTD-SAP97 may prevent the binding of MyoVI. This interaction can cause the stabilization of GluR-A-containing AMPAR clustering.

This clustering may be mediate by the interaction of GK PSD-95 domain with GKAP.

Authors « tentavely » suggest that SAP97-PSD-95 interaction serves an essential role in synaptic accumulation of GluR-A-containing AMPAR, possibly through stabilization of receptor cluster.

Authors’ hypothesis on trafficking GluR-A-containing AMPAR

Thank you

Yeast two hybrid system

C-terminal GluR-A-containing AMPAR

PDZ SAP97

NTD SAP97

SH3 PSD-95

GKAP

GK PSD-95