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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Novel mode of ligand recognition by the erbin pdz domain.
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Authors
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G.Birrane,
J.Chung,
J.A.Ladias.
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Ref.
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J Biol Chem, 2003,
278,
1399-1402.
[DOI no: ]
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PubMed id
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Abstract
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Erbin contains a class I PDZ domain that binds to the C-terminal region of the
receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the
human Erbin PDZ bound to the peptide EYLGLDVPV corresponding to the C-terminal
residues 1247-1255 of human ErbB2 has been determined at 1.25-A resolution. The
Erbin PDZ deviates from the canonical PDZ fold in that it contains a single
alpha-helix. The isopropyl group of valine at position -2 of the ErbB2 peptide
interacts with the Erbin Val(1351) and displaces the peptide backbone away from
the alpha-helix, elucidating the molecular basis of class II ligand recognition
by a class I PDZ domain. Strikingly, the phenolic ring of tyrosine -7 enters
into a pocket formed by the extended beta 2-beta 3 loop of the Erbin PDZ.
Phosphorylation of tyrosine -7 abolishes this interaction but does not affect
the binding of the four C-terminal peptidic residues to PDZ, as revealed by the
crystal structure of the Erbin PDZ complexed with a phosphotyrosine-containing
ErbB2 peptide. Since phosphorylation of tyrosine -7 plays a critical role in
ErbB2 function, the selective binding and sequestration of this residue in its
unphosphorylated state by the Erbin PDZ provides a novel mechanism for
regulation of the ErbB2-mediated signaling and oncogenicity.
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Figure 1.
Fig. 1. Structure of the Erbin PDZ bound to the
unphosphorylated ErbB2 peptide. A, sequence comparison of
selected class I PDZ domains. Identical residues in four or more
domains are shown as white letters on blue background. Hyphens
represent gaps inserted for optimum alignment. The secondary
structure of the Erbin PDZ is indicated at the top. Residues
forming a short  -helix in
PDZs with known structures are enclosed in a red box. B, stereo
view of the Erbin PDZ bound to the peptide EYLGLDVPV. The figure
was made using BOBSCRIPT (30) and POV-Ray (www.povray.org). C,
surface topology of the Erbin PDZ bound to the ErbB2 peptide.
The figure was made using GRASP (31). D, two-dimensional
representation of the interactions between Erbin PDZ residues
(orange) and the peptide (purple). Water molecules (W) are shown
as cyan spheres, hydrogen bonds as dashed lines, and hydrophobic
interactions as arcs with radial spokes. The figure was made
using LIGPLOT (32). E, stereo view of a weighted 2F[obs]  F[calc]
electron density map at the P[2] pocket calculated at 1.25
Å and contoured at 2.5  .
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Figure 2.
Fig. 2. Structure of the Erbin PDZ bound to the
phosphorylated ErbB2 peptide. A, stereo view of the Erbin PDZ
bound to the peptide EpYLGLDVPV. A weighted 2F[obs] F[calc]
electron density map calculated at 1.88-Å resolution and
contoured at 1.0 is
superimposed on the ErbB2 peptide. B, superposition of the C
backbone
traces of Erbin PDZ-peptide (pink), Erbin PDZ-phosphopeptide
(blue), and PSD-95 PDZ3-peptide (yellow) (Protein Data Bank code
1BE9). Side chains of the peptidic residues, Erbin His1347 and
Val1351, and PSD-95 His372 are shown as stick models.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2003,
278,
1399-1402)
copyright 2003.
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Secondary reference #1
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Title
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Erbin: a basolateral pdz protein that interacts with the mammalian erbb2/her2 receptor.
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Authors
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J.P.Borg,
S.Marchetto,
A.Le bivic,
V.Ollendorff,
F.Jaulin-Bastard,
H.Saito,
E.Fournier,
J.Adélaïde,
B.Margolis,
D.Birnbaum.
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Ref.
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Nat Cell Biol, 2000,
2,
407-414.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. ERBIN interacts preferentially with the non-activated
HER1/2 receptor. a, HER1/2 was transiently co-expressed with
Myc-tagged ERBIN, SAP97 and PSD-95 in COS-1 cells. HER1/2 was
immunoprecipitated (IP) with anti-EGFR antibody (clone 108) and
bound proteins were revealed by immunoblotting (IB) with
anti-Myc and anti-ERBB2 antibodies, respectively. Only ERBIN is
associated with HER1/2. b, HER1/2 and EGFR were transiently
co-expressed with ERBIN in COS-1 cells. Equal amounts of ERBIN
and receptors are found in the lysates (data not shown).
Receptors were immunoprecipitated by anti-EGFR antibody, bound
proteins were western blotted and revealed with anti-ERBIN and
anti-receptor antibodies. Only HER1/2 interacts with ERBIN. As a
control, p52 SHC protein is increasingly co-immunoprecipitated
with HER1/2 and EGFR after EGF stimulation. c, HER1/2 and EGFR
were transiently expressed in COS-1 cells. GST pull down assays
were performed using purified GST -SHC PTB or GST -ERBIN PDZ
domains bound on agarose beads. Precipitated proteins were
revealed by western blot analysis using anti-PY antibody (upper
panels). After stripping, membranes were revealed with
anti-ERBB2 and anti-EGFR antibodies, respectively (anti-RTK).
While only phosphorylated receptors bind to the SHC PTB domain,
unphosphorylated HER1/2 interacts with the ERBIN PDZ domain. d,
HER1/2 containing a mutation of the C-terminal valine (mutant
VA) or a kinase-dead HER1/2 (mutant KA) were expressed in COS-1
cells and interaction with GST fusion proteins was tested as
described in c. e, Myc-tagged ERBIN was co-expressed with HER1/2
in COS cells. After lysis, anti-Myc or anti-SHC antibodies were
used for immunoprecipitation. After western blotting, the
membrane was successively probed with anti-ERBB2, anti-PY and
anti-Myc antibodies, respectively. The white arrow shows
unphosphorylated HER1/2 receptor co-immunoprecipitated with
ERBIN in the top panel. The arrowheads point to phosphorylated
HER1/2 interacting with SHC proteins in the top and middle
panels. Note the slower migration of phosphorylated HER1/2
(arrowhead) compared to the unphosphorylated HER1/2 in the upper
panel. In the middle and bottom panels, an asterisk indicates
the position of Myc-ERBIN. SHC proteins (p52 and p46 SHC) are
indicated at the bottom of the gel. ERBIN is phosphorylated by
HER1/2 but interacts preferentially with the unphosphorylated
receptor, in contrast to SHC proteins. f, As in e but a
kinase-dead HER1/2 (HER1/2. KA) was also co-expressed with
Myc-ERBIN. ERBIN is tyrosine phosphorylated when co-expressed
with HER1/2 but not HER1/2. KA (bottom panel). Lysates were also
run on SDS -PAGE, transferred to nitrocellulose and revealed
with anti-Myc (top panel) and anti-ERBB2 (middle panel)
antibodies. g, HER1/2 was co-expressed with Myc-tagged wild-type
ERBIN or ERBIN lacking its PDZ domain (  PDZ).
Deletion of the PDZ domain inhibits interaction with HER1/2 and
decreases ERBIN tyrosine phosphorylation.
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Figure 4.
Figure 4. Basolateral localization of ERBIN and ERBB2 in human
colon. a, Semi-thin frozen sections of human colon were
double-stained with antibodies against ERBIN and an apical
marker, CEA, or a basolateral marker, Ag525. Apical (AP) and
basolateral (Bl) membranes are indicated by arrowheads and
arrows respectively. The location of the basement membrane (BM)
is also indicated. ERBIN co-localizes with Ag525 on the
basolateral membrane. b, Semi-thin frozen sections of human
colon were double-labelled with antibodies against ERBIN and
ERBB2. Both proteins are localized on the basolateral membrane
(Bl) and are absent from the apical membrane (AP). Scale bars
represent 5 µm. c, Cell fractioning was carried out on Caco-2
cells and cytosolic (Cyt) and membrane (Mbr) fractions were
subjected to western blot analysis with anti-ERBIN and anti-SHC
antibodies. Whereas SHC proteins are mainly cytosolic
(arrowhead), ERBIN is found in the membrane fraction (asterisk).
d, Proteins of Caco-2 cell lysates were immunoprecipitated with
anti-ERBIN (E) or preimmune serum (not shown) or a control
rabbit antibody (anti-STK-1) (C) and precipitated proteins were
resolved by western blot. Anti-ERBB2 and anti-ERBIN antibodies
were used to probe the membrane. Co-immunoprecipitation between
ERBB2 and ERBIN was evidenced only when anti-ERBIN antibody was
used for immunoprecipitation. One-tenth of the Caco-2 lysate was
run as control (TL).
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #2
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Title
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The erbin pdz domain binds with high affinity and specificity to the carboxyl termini of delta-Catenin and arvcf.
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Authors
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R.P.Laura,
A.S.Witt,
H.A.Held,
R.Gerstner,
K.Deshayes,
M.F.Koehler,
K.S.Kosik,
S.S.Sidhu,
L.A.Lasky.
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Ref.
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J Biol Chem, 2002,
277,
12906-12914.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Phage-selected peptides bind specifically to the
Erbin PDZ domain in vitro, as do protein ligands predicted from
their consensus sequences. A, the Erbin PDZ domain and six
control PDZ domains were expressed in HEK 293 cells as GFP
(indicated by asterisks) or GST fusions, and extracts were made
from each. Biotinylated versions of peptides phage-selected for
high affinity binding to either the MAGI-3 PDZ2 domain
(ATQITWV-COOH) or the Erbin PDZ domain (TGWETWV-COOH) were
tested for their ability to precipitate the various PDZ domains.
Each peptide specifically precipitated only the PDZ domain
against which it was selected. A single substitution of Glu for
Ile at position 3 of the
MAGI-3 PDZ2 domain-specific peptide produced a peptide
(ATQETWV-COOH) that no longer precipitated the MAGI-3 PDZ2
domain, but specifically precipitated only the Erbin PDZ domain
(right lane). Peptide concentrations were 10 µM, and
readout was by immunoblotting with either anti-GFP or anti-GST
antibodies. B, the indicated peptides (10 µM) were assayed
for their ability to precipitate Erbin from Caco-2 cell
extracts. Immunoblot (IB) analysis with the indicated antibodies
showed that Erbin was precipitated with a peptide ending in
ETWV, but not ITWV. Similar to Erbin, LIN7-3 has a single PDZ
domain, but was not precipitated by either peptide. C, potential
protein ligands for the Erbin PDZ domain were expressed in HEK
293 cells and assayed for their ability to be precipitated by 3
µg of E. coli cell-expressed GST-Erbin PDZ domain fusion
protein. The expressed proteins were as follows: ARVCF and  -catenin (
-cat), the
previously reported putative Erbin PDZ ligand HER2, and the
negative controls -catenin
 c6 and  -catenin.
For immunoblot analysis with the indicated antibodies, 1% of the
precipitated extracts or 30% of the precipitated protein was
used. D, precipitation of -catenin or
HER2 by 6 µg of E. coli cell-expressed GST-Erbin PDZ
domain fusion protein could be efficiently blocked by
co-incubation with a phage-selected peptide (TGWETWV-COOH) that
binds the Erbin PDZ domain with high affinity, but not by a
control peptide (ATQITWV-COOH). Note that 1 µM peptide was
approximately a 3-fold molar excess over the Erbin PDZ domain.
Immunoblot analysis showed the relative amounts of -catenin or
HER2 precipitated by the GST-Erbin PDZ domain fusion protein in
the absence or presence of varying concentrations of the
indicated peptide.
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Figure 3.
Fig. 3. Endogenous interaction of Erbin and -catenin in
brain. A, cultured hippocampal neurons (right panels) or a
section of rat cerebral cortex (left panels) was double-labeled
with a monoclonal antibody directed against -catenin
and polyclonal antibodies directed against Erbin to assess the
level of co-localization for these proteins. Arrowheads point
out dendritic shafts of pyramidal neurons in cortex (left
panels) and dendritic shafts of cultured hippocampal neurons
(right panels). B, E. coli cell-expressed GST-Erbin PDZ domain
fusion protein (3 µg) and a control GST-PDZ domain fusion
protein (3 µg) were assayed for the ability to
precipitate, from mouse brain extracts, endogenous -catenin
and proteins with which -catenin is
know to associate. Precipitated proteins were analyzed by
immunoblotting. C, HEK 293 cells were cotransfected or
tri-transfected with the indicated constructs, and the ability
of E. coli cell-expressed GST-Erbin PDZ domain fusion protein to
precipitate -catenin-associated
proteins was analyzed by immunoblotting (IB). The results show
that the association of E-cadherin (E-cad) with -catenin
was direct (left panels), whereas the association with -catenin (
-cat) was
indirect and required E-cadherin (left and right panels) and,
finally, that precipitation of this complex by the Erbin PDZ
domain required the C terminus of -catenin.
D, -catenin
and p120 catenin (p120^ctn) were immunoprecipitated with 5
µg of each antibody from 0.5 mg of mouse brain extract. An
equal volume of the extract (ex) and the
post-immunoprecipitation (IP) depleted extract (dex) and 50% of
the precipitated protein (p) were analyzed by immunoblotting.
Erbin co-immunoprecipitated only with -catenin,
but not with the related family member p120 catenin. E,
co-immunoprecipitation of Erbin with -catenin
was disrupted by preincubating the brain extract with the Erbin
PDZ domain-binding peptide TGWETWV-COOH, but not with the
negative control peptide TGWETWA-COOH, at the indicated
concentrations prior to immunoprecipitation. Mab, monoclonal
antibody.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #3
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Title
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Erbin is a protein concentrated at postsynaptic membranes that interacts with psd-95.
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Authors
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Y.Z.Huang,
Q.Wang,
W.C.Xiong,
L.Mei.
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Ref.
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J Biol Chem, 2001,
276,
19318-19326.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Binding of ErbB2 with Erbin in yeast. A,
interaction of Erbin with ErbB2 depended on the PDZ domain in
the C terminus. The domain structure of Erbin is shown in the
schematic diagram. Erbin965 and Erbin1254 are original clones
isolated from a yeast two-hybrid screen encoding the C termini
starting from the indicated amino acid residue. Erbin195 PDZ encoded
Erbin amino acid residues 195-1279 without the PDZ domain.
Densin-180/PDZ encoded amino acid residues 1161-1495 containing
the PDZ domain. PSD-95/PDZ contains amino acid residues 65-393
with all three PDZ domains. These constructs were fused with the
Gal4AD and cotransformed with Gal4DB/ErbB2-DVPV* in yeast.
Asterisks indicate amino acid residues prior to the stop codon.
B, dependence of the interaction between ErbB2 and Erbin on the
ErbB2 C terminus. Yeast cells were cotransformed with a vector
encoding the Gal4DB fused to different ErbB2 C-terminal
constructs and Gal4AD/Erbin. C, interaction between Erbin with C
termini of ErbBs or NR2A. Yeast cells were cotransformed with
Erbin and ErbB2 C-terminal constructs. Transformed yeast cells
were seeded in His  plates
and scored for growth and for -galactosidase
( -Gal)
activity.
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Figure 8.
Fig. 8. Erbin expression in the PSD and interaction with
ErbB2 in the central nervous system. A, expression of Erbin in
various brain regions. Homogenates (100 µg of protein)
were resolved on SDS-PAGE and subjected to Western blot using
the anti-Erbin antibody. B, expression of Erbin in PSD. Rat
brain homogenates (H) were subjected to sequential
centrifugations to yield cytosol (S2) and synaptosomes (P2).
Washed synaptosomes (P3) were fractionated further by
discontinuous sucrose gradient centrifugation to generate
synaptosomal plasma membrane (SPM) which was treated with 0.4%
Triton X-100. The insoluble SPM was designated as PSD. Samples
were separated by SDS-PAGE and subjected to immunoblotting with
the respective antibodies. C, interaction between Erbin and
ErbB2 in the central nervous system. Rat brain synaptosomes were
solubilized with 1% deoxycholate. The resulting detergent
extract (Input) was incubated with preimmune serum or antibodies
against Erbin. Immunoprecipitates (IP) were resolved on SDS-PAGE
and subjected to immunoblotting (IB) for ErbB2. 10 times of
input were used for immunoprecipitations.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #4
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Title
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The erbb2/her2 receptor differentially interacts with erbin and pick1 psd-95/dlg/zo-1 domain proteins.
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Authors
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F.Jaulin-Bastard,
H.Saito,
A.Le bivic,
V.Ollendorff,
S.Marchetto,
D.Birnbaum,
J.P.Borg.
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Ref.
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J Biol Chem, 2001,
276,
15256-15263.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Binding specificity of the ERBIN PDZ domain. A,
the last 15 carboxyl-terminal amino acids found in ERBB2 (wild
type or mutants) were fused to the LexA-BD and co-transformed in
L40 yeast with GAL4-AD ERBIN (1240-1371) referred as the ERBIN
PDZ domain. Co-transformed yeast were plated on Trp-Leu-His
medium containing 10 mM 3-AT. + means growth on the selective
medium ( His) and
positive -galactosidase
activity ( gal).
Asterisks point to the residues important for ERBB2/ERBIN
interaction. Mutated residues are underlined. B, HER1/2 (chimera
EGF-R/ERBB2) wild type (WT) and mutant were transiently
expressed in COS cells and pulled down with GST-ERBIN PDZ
domain. Bound receptors were revealed with anti-ERBB2 antibody
after Western blot (upper panel). Mutation of the
carboxyl-terminal valine (VA), aspartic residue in 3 position
(mut-3), leucine in 4 position
(mut-4), tyrosine in 7 position
(mut-7), and deletion of the last 6 residues in ERBB2 ( 6) abrogate
the ERBB2/ERBIN interaction. Lysates were run, transferred on
nitrocellulose, and probed with anti-ERBB2 to show a comparable
expression of receptors (lower panel). C, direct interaction
between the ERBIN PDZ domain and ERBB2, Kv1.4, NR2C, and NR2B
peptides. The last 9 amino acids of the mentioned proteins were
fused to the GST protein, Western-blotted, and probed with
soluble 32P-labeled GST-ERBIN PDZ domain or GST (data not shown)
fusion proteins. Bound proteins were revealed by
autoradiography. No binding was found with GST alone (data not
shown). Red Ponceau staining showed that similar amounts of GST
proteins were loaded (data not shown). D, sequence of the
carboxyl terminus of four putative ERBIN-interacting proteins.
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Figure 2.
Fig. 2. Mutations within the ERBIN PDZ domain modulate
the ERBB2/ERBIN interaction. A, sequence of the mouse ERBIN PDZ
domain. An arrow points to a conserved histidine found in the
B helix of
class I PDZ domains but not in NOS and class II PDZ domains
(data not shown). Mutations of His-Gly to Tyr-Asp as in NOS
(mutant YD) and His to Leu as in class II PDZ domains (mutant
HL) were engineered by site-directed mutagenesis. B, same as in
Fig. 1A. Effects of mutations within ERBB2 and the ERBIN PDZ
domain were tested by two-hybrid in yeast. C, in the left panel,
the experiment is same as panel B using increasing amount of
3-aminotriazole. In the right panel, a pull-down assay was
performed on HER1/2 expressing lysates with GST-ERBIN PDZ wild
type (WT) and mutant (HL and YD). The interaction with HER1/2
was revealed by anti-ERBB2 antibody. Ponceau Red staining shows
that equal amounts of GST proteins were used.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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