Interaction between SAP97 and PSD-95, Two Maguk Proteins Involved in Synaptic Trafficking of AMPA Receptors

1
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, 2005
• Chunlin 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 Benoît De la Crompe Brice
Master 2 Neurosciences 2011 Bordeaux UE Cellular
and Molecular Neurobiology
2
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

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INTRODUCTION
4

• 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

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• 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

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• MAGUKs (Membrane-Associated Guanylate Kinase
  homologs)
• PSD-95 (PostSynaptic Density-95)
• SAP97 (Synapse-Associated Protein 97)
• Others PSD-93/Chapsyn-110 , SAP102

L27
7

• 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

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EXPERIMENTAL PROCEDURES
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Experimental design

• Part 1 Proteins interaction
• Authors 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.

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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.

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Materials and methods - Results
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Part 1 Protein interaction
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SAP97 Associates with PSD-95 in Vivo Materials
and Methods

• Detergent extract of cerebella from adulte
  Wistar rats were submitted to co-immunoprecipitati
  on 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.

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SAP97 Associates with PSD-95 in Vivo Results

• 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
  .
• gt In rat brain, PSD-95 and SAP97 interacting to
  form a complex.

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

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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
• gt interaction between this two tagged proteins
  is direct.
• gt Authors verified the presence of endogenous
  SAP97 or PSD-95 (not published data). HEK293
  cells express endogenous SAP97.

Fig 1B and1C Coimmunoprecipitation of SAP97 and
PSD-95 in HEK293 detergent extract.
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SAP97 Associates with PSD-95 in Vitro Results(2/2)

• Coimmunoprecipitation of GFP-PSD-95 and
  endogenous SAP97
• gt GFP-PSD-95 interacting directly to form a
  complex with endogenous SAP97.
• gt Because HEK293 cells express SAP97, we cannot
  exclude the possibility that they can express
  other MAGUK proteins.
• gt 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.
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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.

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Mapping the PSD-95 Binding Site in SAP97 Results

• Coimmunoprecipitation of His-PSD-95 with
• Myc-SAP97
• Myc-?SH3-GK
• Myc-NTD

• NTD is unique common domain which Co-IP with
  PSD-95.
• gt The binding site of PSD-95 in SAP97 is the
  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.
20
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.

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Mapping the SAP97 Binding Site in PSD-95 Results

• Coimmunoprecipitation of GFP-SAP97 with
• Myc-PSD-95
• Myc-?N-PSD-95
• Myc-SH3-PSD-95
• Myc-SH3-GK-PSD-95 (fig B)

• SH3 is unique common domain which Co-IP with
  SAP97.
• gt The binding site of SAP97 in PSD-95 is the SH3
  domain.
• gt Why in first experiment, the SH3-GK-PSD-95
  domain did not bind to SAP97?

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.
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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.
• SAP97?NTD 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.

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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
• gt The domain of GST-SAP97 that binds to
  GFP-PSD-95 is NTD of SAP97.
• gt GST-PDZ1-3 may constitue a second binding
  site.
• Fig B Interaction between GFP-PSD-95-SH3 and
  GST-SAP97-NTD.
• gt 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).
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Part 2 Study of SAP97-PSD-95 Interaction in
Cultured Neurons
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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.

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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.
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Distribution of SAP97 and PSD-95 in cultured
hippocampal neurons
SAP97-PSD-95 Interaction in Cultured Neurons -
Results (2/4)

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

• 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.

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).
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 (plt0.05) NA, not applicable.
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SAP97-PSD-95 Interaction in Cultured Neurons -
Results (3/4)
PSD-95-induced clustering of SAP97 to synaptic
spines is inhibited by PSD-95SH3

• Overexpression of PSD-95 trigger the spine
  clustering of SAP97.
• The PSD-95SH3 inhibits the clustering of SAP97

Number of SAP97 immunoreactive spines in
transfected neurons
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.
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DISCUSSION
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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.

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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.

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Authors hypothesis on trafficking
GluR-A-containing AMPAR

• 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.

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Thank you
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Yeast two hybrid system
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GKAP
C-terminal GluR-A-containing
AMPAR
PDZ SAP97
NTD SAP97
SH3 PSD-95
GK PSD-95