From the Department of Biological Sciences, Faculty of Medicine, and the Department of Molecular and System Biology, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan
Received for publication, January 8, 2003, and in revised form, February 12, 2003
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ABSTRACT |
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Tamalin is a scaffold protein that comprises
multiple protein-interacting domains, including a 95-kDa
postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ)
domain, a leucine-zipper region, and a carboxyl-terminal PDZ
binding motif. Tamalin forms a complex with metabotropic glutamate
receptors and guanine nucleotide exchange factor cytohesins and
promotes intracellular trafficking and cell surface expression of group
1 metabotropic glutamate receptors. In the present study, using
several different approaches we have shown that tamalin interacts with
multiple neuronal proteins through its distinct protein-binding
domains. The PDZ domain of tamalin binds to the PDZ binding motifs of
SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ
binding motif of tamalin is capable of interacting with the PDZ domain
of S-SCAM. In addition, tamalin forms a complex with PSD-95 and
Mint2/X11 Multimolecular protein assembly through protein-protein
interaction is important as a general mechanism for diverse cellular functions in neuronal and other cells (reviewed in Refs. 1-5). Molecular assembly of protein complexes is built around one or more
central scaffold proteins that contain multiple domains for protein-protein interaction. The 95-kDa postsynaptic density protein (PSD-95)1/discs-large/ZO-1
(PDZ) domain is a key protein-binding domain comprised of ~90
amino acid residues and interacts with a PDZ binding motif with the
consensus sequences S/TXV/I/L (X is any amino
acid) (6, 7). In neurons, postsynaptic PDZ domain-containing scaffold
proteins, PSD-95 and S-SCAM, interact with a number of membrane and
cytoplasmic proteins, including NMDA receptors (7-9) and
SAP90/PSD-95-associated proteins (SAPAPs) (also called guanylate kinase-associated proteins/human Discs Large-associated
proteins) (10-12). PSD-95 and S-SCAM are localized at the PSD
of excitatory synapses and play an important role in functional
assembly of a postsynaptic macromolecular complex. The PDZ
domain-containing scaffold proteins are also important in subcellular
trafficking of their partner proteins (reviewed in Refs. 1, 4, and 13). LIN-2, LIN-7, and LIN-10 of Caenorhabditis elegans
are all PDZ domain-containing proteins and are important for the proper
localization of the LET-23 receptor tyrosine kinase (13). LIN-2
is the homolog of mammalian scaffold protein CASK, which was
simultaneously discovered as a protein interacting with the cell
surface protein neurexin (14, 15). The mammalian homolog of
LIN-7 (16) was cloned and named as Veli (17), mLIN-7 (18), or MALS
(19), whereas LIN-10 was found to be a homolog of mammalian scaffold
protein, Mint/X11 (20-22). CASK, Veli, and Mint form a tripartite
complex in the mammalian brain and are considered to be involved in
protein targeting in mammalian polarized cells (1, 4, 17, 23). Thus,
the PDZ domain-containing scaffold proteins participate in diverse
mechanisms underlying neuronal activity and function.
Tamalin (also termed GRP1-associated scaffold protein) is a scaffold
protein that comprises multiple protein-interacting domains (24, 25).
It possesses a PDZ domain, a leucine-zipper region, a proline-rich
region, and a carboxyl-terminal PDZ binding motif (24, 25). The PDZ
domain of tamalin interacts with the carboxyl termini of group 1 and
group 2 metabotropic glutamate receptors (mGluRs) and
GABAB2 receptor (24), whereas the leucine-zipper region
binds to the coiled coil region of guanine nucleotide exchange factor
cytohesins (24, 25). Tamalin promotes intracellular trafficking and
cell surface expression of group 1 mGluRs in COS-7 cells and cultured
hippocampal neurons through the interaction with cytohesins (24).
Because tamalin has multiple, distinct protein-interacting domains and
is enriched in the PSD fraction, we sought in this investigation to
examine a protein complex formation of tamalin with other neuronal
proteins, using several different approaches. Here we report that
tamalin exists as a key element that forms a protein complex with
several postsynaptic and protein-trafficking scaffold proteins through
its distinct protein-binding domains.
Sequence Analysis--
The amino acid sequence of rat tamalin
(GenBankTM AF374272) was used as probe in a BLAST data base
search for the expressed sequence tag (EST) and nr data base at
the National Center for Biotechnology Information
(www.ncbi.nlm.nih.gov/BLAST) with the TBLASTN algorithm. The
Xenopus ESTs (GenBankTM BU906716, BU901589,
BJ033586, BF613520, BJ029054, AI880945, BJ038540, BG487268, BJ048855,
BJ043787, BJ053111, and BQ398412), the zebrafish EST
(GenBankTM AW422012), and the zebrafish genomic DNA
sequence (GenBankTM AL807377.9) were assembled to create
electronic cDNA sequences from which the amino acid sequences of
Xenopus and zebrafish tamalin were predicted. Human IMAGE
clone cDNAs 4422992 and 6151168 (GenBankTM BC035500 and
BU165906), Drosophila melanogaster RH12258 full-insert
cDNA (GenBankTM AY089654.1), and C. elegans
cDNA encoding the PDZ domain-containing protein (27.8 kDa)
(GenBankTM NM 059763.1) were also identified as the
cDNAs encoding proteins homologous to rat tamalin. Protein
sequences were analyzed by the pfscan program on the ISREC profile scan
server (hits.isb-sib.ch/cgi-bin/PFSCAN). Sequence alignment was
performed with the Clustal W software (26) and DNASIS software
(Hitachi). The phylogenetic tree was generated with Clustal W
software using the Neighbor-Joining method and displayed with NJPLOT
(27).
DNA Constructs--
The carboxyl-terminal portion of SAPAP3
(amino acid residues 853-977, GenBankTM U67139), the first
and second PDZ domains of PSD-95 (PSD-95 PDZ1 + 2) (residues 48-274,
GenBankTM M96853), the third PDZ domain of PSD-95 (PSD-95
PDZ3) (residues 266-411, GenBankTM M96853), the Src
homology 3 (SH3) domain of PSD-95 (PSD-95 SH3) (residues 394-533,
GenBankTM M96853), the guanylate kinase (GK) domain of
PSD-95 (PSD-95 GK) (residues 504-724, GenBankTM M96853),
the fifth PDZ domain of S-SCAM (S-SCAM PDZ5) (residues 1104-1277,
GenBankTM AF04863), the PDZ domain of CASK (CASK PDZ)
(residues 469-598, GenBankTM U47110), two PDZ domains of
Mint1 (Mint1 PDZ1 + 2) (residues 640-839, GenBankTM
AF029105), and the Munc18-interacting domain (MID) (residues 1-356,
GenBankTM AF029107), the phosphotyrosine-binding domain
(PTB) (residues 349-601), and two PDZ domains (residues 550-750) of
Mint2 were cloned by reverse transcriptase-mediated polymerase chain
reaction (RT-PCR) from rat brain RNA. The PDZ domain of Veli2 (residues 57-207, GenBankTM AF173082) was amplified by PCR, using
mouse brain Marathon-Ready cDNA (Clontech) as a
template. Isolated domains were subcloned in frame into pACT2
(Clontech), pGEX-4T-1/2 (Amersham Biosciences), or
pET32a (Novagen). The deletional mutants of tamalin and SAPAP3 lacking the last 3 amino acid residues were constructed by PCR techniques. To prepare the full-length cDNAs for rat Mint2 and SAPAP3, we first cloned several partial cDNAs by RT-PCR with rat brain total RNA as a template and constructed the full-length cDNAs
by connecting the partial cDNAs (28). The isolated rat Mint2
cDNA was inserted in frame into pCMV-Tag3B (Stratagene). Proper in frame insertions and the absence of any sequence errors of all PCR products were confirmed by DNA sequencing. Mammalian expression vectors for myc-tagged full-length S-SCAM and myc-tagged full-length PSD-95 were kindly provided by Dr. Yutaka Hata (9) and Dr.
Morgen Sheng (29), respectively. Other plasmids were described
previously (24).
Glutathione-S-transferase (GST) Pull-down Assay,
Immunoprecipitation, and Yeast Two-hybrid Screening--
Protein
purification and pull-down assay were performed as described previously
(24, 30). For pull-down assay, 10 µg of GST fusion proteins were
immobilized on glutathione-Sepharose 4B beads (25 µl) and incubated
with either supernatants (100 µg) containing recombinant tamalin
transiently expressed in COS-7 cells or the purified recombinant
thioredoxin fusion proteins (10 µg). Yeast two-hybrid screening and
immunoprecipitation were performed as described previously (24).
Antibodies--
Anti-tamalin antisera were raised in rabbits
against GST·N-tam (24) emulsified with TiterMax Gold Adjuvand
(Funakoshi) and used for immunoprecipitation. For
immunoprecipitation from brain lysates, the affinity-purified
anti-tamalin peptide antibody was used as described previously (24).
Polyclonal rabbit anti-S-SCAM and anti-Veli1/2/3 antibodies were gifts
from Dr. Yutaka Hata (9, 31). Other primary antibodies were obtained
from the following sources: mouse monoclonal anti-Mint1, anti-Mint2,
anti-CASK, and anti-PSD-95 antibodies from BD Transduction
Laboratories; mouse monoclonal anti-SAPAP1 antibody from StressGen;
mouse monoclonal anti-hexahistidine tag and anti-myc tag antibodies
from Clontech. The secondary antibodies were
described previously (24).
Northern Blotting--
Mouse Brain Aging Blot (20 µg of total
RNA/lane, Seegene) was probed with 32P-labeled
cDNA fragments under high stringency conditions. The cDNA
fragment used corresponded to amino acid residues 10-200 of mouse
tamalin. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe was
purchased from Clontech and used as a control.
Evolutionary Conservation of the Domain Structures of
Tamalin--
Since we had previously reported the sequences of rat and
mouse tamalin (24), the DNA data base of C. elegans,
Drosophila, and human became available. We first addressed,
by BLAST data base search of the above species as well as
Xenopus and zebrafish with the rat tamalin sequence as a
probe, whether the characteristic structure of tamalin has been
conserved during evolution. This analysis showed the existence of
homologs of tamalin in all species analyzed (Fig.
1). The deduced amino acid sequences of
tamalin of rat, mouse, human, Xenopus, zebrafish,
Drosophila, and C. elegans are composed of 394, 392, 395, 353, 379, 706, and 245 amino acid residues, respectively
(Fig. 1A). They all possess a PDZ domain, a leucine-zipper
region, and a carboxyl-terminal PDZ binding motif in common (Fig.
1A). Although the amino-terminal position of the PDZ domain
is deleted in the Drosophila tamalin homolog and the carboxyl-terminal portion between a leucine-zipper region and a
carboxyl-terminal PDZ binding motif diverges between vertebrates and
invertebrates, the amino acid sequences of tamalin are highly conserved
over the PDZ domain, the leucine-zipper region, and the PDZ binding
motif (Fig. 1, B-D). The result indicated that the multiple
protein-interacting domains of tamalin are conserved during evolution
and may serve as scaffold for other proteins in cell function.
Interaction between Tamalin and SAPAP3--
To identify
tamalin-interacting proteins, we extended yeast two-hybrid screening of
a rat brain cDNA library, using rat tamalin as bait. In addition to
cytohesin-2 reported previously (24), 65 positive clones were isolated,
in which 19 different cDNA species were included; 7 of them
possessed a typical PDZ binding motif at their carboxyl termini (Table
I). Four independent cDNA clones isolated encoded SAPAP3, which represents a postsynaptic scaffold protein containing the conserved carboxyl-terminal PDZ binding motif
(11). The interaction between tamalin and SAPAP3 was analyzed in more
detail with yeast two-hybrid assay by constructing a prey plasmid
containing the intact carboxyl-terminal portion of SAPAP3 (SAPAP3-ct)
or the truncated mutant of SAPAP3-ct lacking the last 3 amino acids
(SAPAP3-ct del3). This analysis showed that tamalin bound to SAPAP3-ct,
but not to SAPAP3-ct del3 (Fig.
2A). The recognition of the
PDZ binding motif of SAPAP3 occurred through the PDZ domain of tamalin,
because in the yeast two-hybrid assay SAPAP3-ct bound to the
amino-terminal portion containing the PDZ domain of tamalin (N-tam),
but not to the carboxyl-terminal portion lacking the PDZ domain (C-tam)
(Fig. 2B). The interaction of tamalin with SAPAP3 was
further confirmed by immunoprecipitation assay using COS-7 cells
cotransfected with the full-length tamalin and myc-tagged full-length
SAPAP3. Cell lysates were immunoprecipitated with anti-tamalin
antibody, followed by immunoblotting with anti-myc antibody. Myc-SAPAP3
coimmunoprecipitated from cotransfected cells with anti-tamalin
antibody (Fig. 2C, lane 6) but not with
nonimmunized serum (Fig. 2C, lane 8). In control,
immunoprecipitation of myc-SAPAP3 was not seen in cells transfected
with either myc-SAPAP3 or tamalin alone (Fig. 2C,
lanes 5 and 7). The result indicated that tamalin and SAPAP3 form a protein complex in mammalian cells.
Tamalin Forms a Complex with Several Neuronal Scaffold Proteins in
the Brain--
We next addressed whether tamalin forms a protein
complex with scaffold proteins involved in postsynaptic protein
assembly or subcellular protein transport in the brain. Solubilized rat brain membrane fractions were immunoprecipitated with anti-tamalin antibody and immunoblotted with antibodies against candidate scaffold proteins (Fig. 3, A and
B). S-SCAM, PSD-95, SAPAP1 were coimmunoprecipitated from
rat brain membrane fractions with anti-tamalin antibody (Fig. 3A). In addition, Mint2 and CASK were immunoprecipitated,
but neither Mint1 nor Veli1/2/3 was precipitated with anti-tamalin antibody (Fig. 3B). Because the last 13 amino acids covering
the carboxyl-terminal PDZ binding motif are identical between SAPAP1 and SAPAP3 (11), it is likely that SAPAP1, like SAPAP3 (Fig. 2),
directly binds to the tamalin PDZ domain. That there was no coimmunoprecipitation of either Veli1/2/3 or Mint1 was rather unexpected, because CASK is known to form a ternary complex with Veli1/2/3 and Mint1. CASK/Mint1/Velis may not always exist as a
tripartite complex form.
Direct Interaction of Tamalin with S-SCAM, Mint2, and
PSD-95--
We next addressed whether tamalin directly or indirectly
interacts with S-SCAM, Mint2, PSD-95, and CASK in transfected
heterologous cells. The myc-tagged full-length cDNA encoding either
S-SCAM, Mint2, or PSD-95 was transiently co-transfected with the
tamalin cDNA in COS-7 cells. Cell lysates were immunoprecipitated
with anti-tamalin antibody and immunoblotted with the respective
antibody. Anti-tamalin antibody immunoprecipitated all these scaffold
proteins, whereas no such immunoprecipitate was detected in cells
untransfected with the tamalin cDNA (Fig.
4, A-C). The result indicated
that tamalin interacts with S-SCAM, Mint2, and PSD-95 in mammalian cells. CASK was found to be endogenously highly expressed in COS-7 cells (Fig. 4D). Only tamalin was exogenously transfected
into COS-7 cells, and anti-tamalin immunoprecipitates were
immunoblotted with anti-CASK antibody. No immunoblotting band of CASK
was detected in anti-tamalin immunoprecipitates (Fig. 4D),
suggesting that no direct interaction occurs between tamalin and
CASK.
Identification of Tamalin-binding Domains of S-SCAM, PSD-95, and
Mint2--
We performed in vitro pull-down assay to
identify tamalin-binding domains of S-SCAM, PSD-95, and Mint2. GST was
fused to the PDZ domains of the scaffold proteins analyzed in this
investigation. The resultant GST fusion proteins were expressed in
Escherichia coli and purified. The GST fusion proteins were
immobilized on glutathione-Sepharose 4B
beads and incubated with the recombinant tamalin expressed in COS-7
cells. Bound proteins were eluted and immunoblotted with anti-tamalin
antibody. This analysis showed GST·S-SCAM PDZ5 bound to tamalin (Fig.
5A). Interestingly, the PDZ domains of PSD-95 had no
ability to interact with tamalin in in vitro pull-down assay
(Fig. 5A). In addition, consistent with the above findings
of brain and COS-7 cell extracts, none of the GST proteins fused to
Veli2 PDZ, Mint1 PDZ1 + 2, or CASK PDZ showed the ability to bind to
tamalin (Fig. 5A).
To confirm that tamalin binds to S-SCAM PDZ5 through its PDZ binding
motif, we divided tamalin into N-tam and C-tam and deleted the last 3 carboxyl-terminal amino acids of C-tam (C-tam del3). S-SCAM PDZ5 was
fused to hexahistidine-tagged thioredoxin at the amino terminus
(His·S-SCAM PDZ5). Truncated or deleted tamalin fused to GST was
immobilized on glutathione-Sepharose 4B beads and incubated with
His·S-SCAM PDZ5. This pull-down assay revealed that His·S-SCAM PDZ5
bound to GST·C-tam, but not to GST·N-tam or GST·C-tam del3 (Fig.
5B). The result indicated that S-SCAM PDZ5 directly binds to
the PDZ binding motif of tamalin.
Interestingly, GST·N-tam bound to tamalin (Fig. 5A).
Further pull-down assay showed that His-tagged N-tam was retained on glutathione-Sepharose 4B beads attached with GST·C-tam, but not on
glutathione-Sepharose 4B beads attached with either GST·N-tam or
GST-C·tam del3 (Fig. 5C), indicating that tamalin forms a
homomeric complex through its PDZ domain and PDZ binding motif.
To identify the tamalin-binding domain of PSD-95, we divided the
carboxyl-terminal portion following the PDZ domains of PSD-95 into the
SH3 domain and the GK domain. These two domains were fused to GST and
their ability to bind to tamalin was determined by GST pull-down assay
(Fig. 5C). This analysis showed that tamalin bound to
GST·GK but not to other protein domains of PSD-95 (Fig. 5C), indicating that the GK domain of PSD-95 is responsible
for the specific interaction with tamalin.
We also examined the tamalin-binding domain of Mint2 by GST pull-down
assay. Mint2 was dissected into the amino-terminal MID, the middle PTB,
and the carboxyl-terminal PDZ domain, each of which was fused to GST.
GST pull-down assay showed that tamalin bound to GST·MID and
GST·PTB, but not to GST·PDZ1 + 2 (Fig. 5D). The result
indicated that Mint2 interacts with tamalin through its MID and PTB
domains rather than the PDZ domain. Collectively, these results
demonstrated that tamalin distinctly interacts with many scaffold
proteins through its multiple protein-binding domains.
Developmental Change of Tamalin Expression--
Tamalin mRNA
is expressed mainly in the telencephalic region of the mouse adult
brain (24). We examined developmental changes of tamalin mRNA
expression in the mouse brain by Northern blotting analysis. Expression
levels of tamalin mRNA were low before and at birth, continuously
increased during the postnatal period, and reached the highest level
after postnatal 2 weeks (Fig. 6, upper panel). In control, G3PDH mRNA levels remained
unchanged during the time course analyzed (Fig. 6, lower
panel). The result suggested that tamalin may play some roles in
developing and mature brain.
Tamalin is a scaffold protein possessing evolutionarily conserved
protein-protein-interacting domains including a PDZ domain, a
leucine-zipper region, and a carboxyl-terminal PDZ binding motif. The
multiple protein domains of tamalin are now revealed to interact with
many neuronal proteins in different modes (Fig.
7). The PDZ domain of tamalin binds to
the PDZ binding motifs of not only mGluR1/mGluR5 and GABAB2
receptor (24) but also SAPAP3. Anti-SAPAP3 antibody was presently not
available, and an interaction between tamalin and SAPAP3 in the brain
remained elusive, but the identical carboxyl-terminal sequences between
SAPAP1 and SAPAP3 (11) most likely drive a protein assembly between
tamalin and both SAPAP1 and SAPAP3 in vivo. The
carboxyl-terminal PDZ binding motif of tamalin directs a specific
binding with the PDZ domains of S-SCAM and tamalin. To our knowledge,
the latter is the first example indicating that the PDZ domain has the
ability to associate with its own PDZ binding motif. Mint2 and PSD-95
also bind to tamalin by mechanisms different from a PDZ-mediated
interaction. In addition, CASK forms a complex with tamalin indirectly
via unknown scaffold proteins. Thus, tamalin has the ability to
associate with many key proteins involved in neuronal function.
/X11L by mechanisms different from the PDZ-mediated
interaction. Tamalin has the ability to assemble with these proteins
in vivo; their protein complex with tamalin was verified by
coimmunoprecipitation of rat brain lysates. Interestingly, the distinct
protein-interacting domains of tamalin are evolutionarily conserved,
and mRNA expression is developmentally up-regulated at the
postnatal period. The results indicate that tamalin exists as a
key element that forms a protein complex with multiple postsynaptic and
protein-trafficking scaffold proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Evolutionary conservation of the
protein-interacting domains of tamalin. A, schematic
structures of tamalin of vertebrates (rat, mouse, human,
Xenopus, and zebrafish), C. elegans, and
Drosophila. The PDZ domain and the leucine-zipper region are
indicated by a black box and a hatched
box, respectively. The typical PDZ binding motif at the
carboxyl terminus of tamalin is also depicted. B, the
phylogenetic tree of tamalin is generated by sequence comparison of the
PDZ domains and the leucine-zipper regions. A bar indicates
0.05 amino acid substitution per site. The percentages of identical
amino acid residues between rat tamalin and other sequences are shown
in the right column. C, amino acid sequence
alignment of the PDZ domains and the leucine-zipper regions of tamalin
of different species, displaying identical amino acids in gray
boxes. The PDZ domains are underlined. Four leucine
residues and one lysine residue present every seven amino acids in the
leucine-zipper region are marked by open circles and an
asterisk, respectively. D, identical amino acids
at the carboxyl-terminal PDZ binding motifs of different species are
shown by gray boxes. Asterisks indicate the
carboxyl-terminal ends.
Clones isolated by yeast two-hybrid screening with tamalin as bait
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Fig. 2.
Interaction of the PDZ domain of tamalin with
the carboxyl-terminal PDZ binding motif of SAPAP3. A,
under a schematic structure of rat SAPAP3, truncated and deletional
constructs used for yeast two-hybrid assays are indicated; the above
four truncated clones were isolated in the initial yeast two-hybrid
screening. Positive and negative interactions as determined by filter
-galactosidase assay are indicated as + and
, respectively.
B, schematic structures of rat tamalin and truncated N-tam
and C-tam mutants are shown. Results of filter
-galactosidase assay
are shown as in part A. C, COS-7 cells were
transfected with myc-SAPAP3, tamalin, or both. Cell lysates were
immunoprecipitated with either anti-tamalin antibody (lanes
5-7) or nonimmunized control serum (lane 8) and
immunoblotted with anti-myc antibody. Inputs (lanes 1-4)
show 1/20 of cell lysates used for immunoprecipitation.
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Fig. 3.
Association of tamalin with several scaffold
proteins in the brain. Rat brain membrane fractions were
immunoprecipitated with either anti-tamalin antibody or nonimmunized
control IgG, followed by immunoblotting with anti-S-SCAM, anti-PSD-95,
anti- SAPAP1 (A), anti-Mint1, anti-Mint2, anti-CASK, and
anti-Veli1/2/3 (B) antibodies. Inputs show equivalent
amounts of extracts used for immunoprecipitation.
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Fig. 4.
Interaction between tamalin and several
scaffold proteins in COS-7 cells. A-C, COS-7
cells were transfected with myc-S-SCAM, tamalin, or both
(A), myc-Mint2, tamalin, or both (B), myc-PSD-95,
tamalin, or both (C). Cell lysates were immunoprecipitated
with either anti-tamalin antibody (lanes 5-7) or
nonimmunized control serum (lane 8) and immunoblotted with
either anti-S-SCAM antibody (A), anti-Mint2 antibody
(B), or anti-PSD-95 antibody (C). Note that
because the largest splice variant of S-SCAM was used in the
experiments of part A, its molecular size was
larger than that of the major splice variant of the brain S-SCAM shown
in Fig. 3. Inputs (lanes 1-4) show 1/20 of cell lysates
used for immunoprecipitation. D, COS-7 cells were
transfected or untransfected with tamalin. Cell lysates were
immunoprecipitated with anti-tamalin antibody (lanes 3 and
4), and endogenous CASK in immunoprecipitates was analyzed
by immunoblotting with anti-CASK antibody. Inputs (lanes 1 and 2) show 1/20 of cell lysates used for
immunoprecipitation.
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Fig. 5.
Identification of tamalin-binding domains by
pull-down assays. A, GST alone and GSTs fused to S-SCAM
PDZ5, PSD-95 PDZ1 + 2, PSD-95 PDZ3, Veli2 PDZ, Mint1 PDZ1 + 2, CASK
PDZ, and tamalin PDZ (N-tam) were immobilized on glutathione-Sepharose
4B beads and incubated with recombinant tamalin expressed in COS-7
cells. In the upper panel, binding of tamalin to
GST·S-SCAM PDZ5 and GST·N-tam was seen with anti-tamalin
immunoblotting. In the lower panel, one-third of GST or GST
fusion proteins used for immunoprecipitation were visualized with
Coomassie Brilliant Blue staining. B and C, the
ability of GST·N-tam, GST·C-tam, and GST·C-tam del3 to bind to
either His·S-SCAM PDZ5 (B) or His·N-tam (C)
was tested by pull-down assays, followed by immunoblotting with
anti-His antibody. D, The PDZ1 + 2, PDZ3, SH3, and GK
domains of PSD-95 fused to GST are indicated under a schematic
structure of rat PSD-95. The ability of these GST fusion proteins or
GST to bind to tamalin was tested by pull-down assays, followed by
immunoblotting with anti-tamalin antibody. E, the MID, PTB,
and PDZ1 + 2 domains fused to GST are indicated under a schematic
structure of rat Mint2. The ability of these GST fusion proteins or GST
to bind to tamalin was tested by pull-down assays, followed by
immunoblotting with anti-tamalin antibody. Inputs in panels
B-E show 1/10 of proteins used for pull-down assays.
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Fig. 6.
Developmental changes of tamalin mRNA
expression in the mouse brain. Mouse Brain Aging Blot (20 µg of
total RNA/lane) was probed with 32P-labeled cDNA
fragments of mouse tamalin (upper panel) and human G3PDH
(lower panel).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Tamalin-associated protein assembly.
Molecules identified in the previous and present analyses of
tamalin-associated complexes are depicted. The PDZ domain of tamalin
binds to the carboxyl termini of group 1 mGluRs, GABAB2
receptor, SAPAP3, and tamalin itself, whereas the leucine-zipper region
interacts with the coiled coil domain of cytohesin-2. Tamalin interacts
with the fifth PDZ domain of S-SCAM via its carboxyl terminus. Mint2
and PSD-95 also directly bind to tamalin, whereas CASK is present in
the tamalin-associated macromolecular complex.
The PSD is a highly organized signal-processing machinery composed of supramolecular protein complexes. Many proteins identified as a composite of the PSD have PDZ domains and are involved in organizing the complex protein lattice at the PSD (2-5). For example, PSD-95 and S-SCAM are specifically localized at the postsynaptic site and serve to link NMDA receptors and the SAPAP family proteins to the PSD (7-12). Tamalin is enriched in the PSD fraction and promotes cell surface expression of mGluR1 in heterologously expressed COS-7 cells (24). Interestingly, mGluR1 is present in the NMDA receptor-associated macromolecular assembly (32), but it is located at the perisynaptic site and thus differs from NMDA receptors with respect to the synaptic localization (33). Tamalin interacts with typical postsynaptic scaffold proteins, PSD-95, S-SCAM, and SAPAP1/3, and the tamalin-mediated different assembly of postsynaptic scaffold proteins may have an distinct and important role in organizing a postsynaptic signal-processing machinery specific for group 1 mGluRs.
Tamalin interacts with cytohesins, and its transfection with the tamalin-containing adenovirus promotes a neurite distribution of endogenous mGluR5 in cultured hippocampal cells (24). Cytohesins are guanine nucleotide exchange factors specific for the ADP-ribosylation factor family of small GTP binding proteins and control intracellular protein transport (34, 35). Tamalin also associates with Mint2 and CASK. Mint2 binds to munc-18 (20), a protein required for exocytosis and essential for protein transport from Golgi apparatus to cell surface in epithelial cells (36). CASK is also involved in synaptic targeting of N-type calcium channels in cultured hippocampal neurons (37). Furthermore, the orthologs of the Mint family protein (LIN-10) and CASK (LIN-2) in C. elegans are required for the normal basolateral localization of LET-23 (1, 4, 13). Moreover, LIN-10 is essential for postsynaptic localization of the glutamate receptor GLR-1 in nematode neurons (1, 4, 38). It is thus possible that the transport of group 1 mGluRs could be regulated by tamalin-associated trafficking complexes, including not only cytohesins but also Mint2 and CASK.
Several postsynaptic proteins show significant changes in expression levels during development. The expression of tamalin mRNA is regulated developmentally and is well correlated with developmental changes of tamalin-interacting proteins such as SAPAP1 (39) and PSD-95 (40, 41). Importantly, immunoreactive signals for mGluR1 show developmental changes in that they are mainly localized at soma and proximal dendrites at the first postnatal week and thereafter become concentrated at distal dendrites, preferentially at synapses (42). The developmental change of tamalin expression, in concert with other scaffold proteins, may contribute to developmentally regulated distribution of mGluR1 and other signaling proteins in neurons.
In summary, our study shows that tamalin interacts with many important
scaffold proteins involved in postsynaptic organization and protein
trafficking in neurons. This indicates that tamalin may participate in
receptor clustering, trafficking, and intracellular signaling. Further
study will help to understand regulatory mechanisms of
receptor-mediated neuronal function.
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ACKNOWLEDGEMENTS |
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We thank Yutaka Hata and Yoshimi Takai for providing anti-S-SCAM and anti-Veli1/2/3 antibodies and an expression vector for S-SCAM. We are grateful to Morgan Sheng for a gift of PSD-95 expression vector.
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FOOTNOTES |
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* This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-75-753-4437;
Fax: 81-75-753-4404; E-mail: snakanis@phy.med.kyoto-u.ac.jp.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M300184200
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ABBREVIATIONS |
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The abbreviations used are: PSD-95, 95-kDa postsynaptic density protein; PDZ, PSD-95/discs-large/ZO-1; SAPAP, SAP90/PSD-95-associated protein; Mint, munc18-interacting protein; GST, glutathione S-transferase; SH3, Src-homology 3; GK, guanylate kinase; MID, munc18-interacting domain; PTB, phosphotyrosine-binding domain; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; mGluRs, metabotropic glutamate receptors; C-tam, carboxyl-terminal portion of tamalin lacking the PDZ domain; N-tam, amino terminal portion of tamalin with the PDZ domain; NMDA, N-methyl-D-aspartate.
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