From the Department of Biological Sciences, Korea
Advanced Institute of Science and Technology, Daejeon 305-701, Korea,
the § Molecular Neurobiology Laboratory, Institute of
Molecular Biology and Genetics, School of Biological Sciences, College
of Natural Sciences, Seoul National University, Seoul 151-742, Korea,
the ¶ Department of Neurochemistry and Molecular Biology, Leibniz
Institute for Neurobiology, Brenneckestr. G, D-39118 Magdeburg,
Germany, the
Department of Psychiatry and Behavioral
Sciences, Nancy Pritzker Laboratory, Stanford University, Stanford,
California 94305-5485, and the ** Department of Medicine
(Gastroenterology), Duke University Medical Center, Durham, North
Carolina 27710
Received for publication, December 3, 2002
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ABSTRACT |
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The cytoskeletal matrix assembled at active zones
(CAZ) is implicated in defining neurotransmitter release sites.
However, little is known about the molecular mechanisms by which the
CAZ is organized. Here we report a novel interaction between Piccolo, a
core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for
ADP-ribosylation factor small GTPases. A small region (~150 amino
acid residues) in Piccolo, which is not conserved in the closely
related CAZ protein Bassoon, mediates a direct interaction with the
Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize
at synaptic sites in cultured neurons. In brain, Piccolo forms a
complex with GIT1 and various GIT-associated proteins, including
The active zone is a specialized presynaptic plasma membrane
region where synaptic vesicles dock and fuse (1). The cytoskeletal matrix (cytomatrix) assembled at active zones
(CAZ)1 is a complex
proteinaceous structure implicated in organizing the site of
neurotransmitter release (2, 3). Recent studies have identified several
core CAZ components involved in orchestrating the formation and
functions of the CAZ: Piccolo/aczonin (4-6), Bassoon (7), RIM (8),
Munc13 (9), and CAST/ERC (10, 11).
Piccolo is a large (~530 kDa) CAZ protein that is spatially
restricted to active zones within the nerve terminal (4-6). Through its two zinc fingers, Piccolo interacts with the prenylated Rab acceptor 1 (5), a small (185 aa) soluble protein known to bind regulators of endo- and exocytosis, including Rab3, Rab5, and VAMP2/synaptobrevin II (12). A proline-rich sequence of Piccolo binds
the actin cytoskeleton regulator profilin (6). In addition, the
C2A domain of Piccolo mediates homodimerization and
heterodimerization with RIM (13). Intriguingly, Piccolo associates with
an ~80-nm dense core granulated vesicle (termed Piccolo transport
vesicle) that contains other active zone components, suggesting that
Piccolo and related active zone components are transported to nascent synapses as a preassembled package for the rapid and efficient formation of active zones (14). Although Piccolo's size, domain structure, and association with an active zone precursor vesicle suggest that it may be an important organizer of the CAZ, little is
known about the mechanism by which this organization is carried out.
GIT1 was originally isolated as a protein interacting with
G-protein-coupled receptor kinases (15). The GIT family of proteins contains two known members, GIT1/Cat-1/p95-APP1 and
GIT2/Cat-2/PKL/p95-APP2/KIAA0148 (15-22). GIT proteins contain a
GTPase-activating protein (GAP) domain for ADP-ribosylation factors
(ARFs), small GTP-binding proteins implicated in the regulation of
membrane traffic, and the actin cytoskeleton (23). In addition,
GIT proteins contain various domains for protein interactions,
including ankyrin repeats, the Spa2 homology domain (SHD), and the
G-protein-coupled receptor kinase-binding domain (GRKBD). GIT proteins
regulate endocytosis of various membrane proteins (15, 24) and regulate
the assembly of focal adhesion complexes by interacting with the
Rho-type guanine nucleotide exchange factor (GEF) Here we report that Piccolo directly interacts with GIT and various
GIT-associated proteins. Moreover, GIT proteins form homo- and
heteromultimers, which enable GIT proteins to form a ternary complex
with Piccolo and Yeast Two-hybrid Assay--
A yeast two-hybrid assay was
performed as previously described (26). HIS3 growth and
GST Pull-down Assay--
For GST fusion protein constructs, the
following regions of Piccolo and GIT1 were subcloned into
BamHI-EcoRI site of pGEX-4T-1 (Amersham
Biosciences): Piccolo (aa 2011-2350); GIT1, GAP-SHD (aa 1-374), CC
(aa 405-485), SP (aa 486-645), and PBS (aa 646-770). GIT1 GRKBD (aa
375-770) was subcloned into the EcoRI site of pGEX-4T-1. For H6 fusion protein constructs, GIT1 GAP-SHD (aa 1-374) was subcloned into pRSET A (Invitrogen) digested with BamHI and
EcoRI, and GIT1 GRKBD (aa 375-770) and GIT1 full-length (aa
1-770) into the EcoRI site of pRSET B (Invitrogen).
EGFP-GIT1 deletion constructs were generated by subcloning the
following regions of GIT1 into pEGFP-C1 (Clontech):
GAP (aa 1-129), ankyrin repeats (aa 130-254), SHD (aa 255-374), and
GAP-SHD (aa 1-374) (EcoRI and BamHI site); GRKBD
(aa 375-770) and GIT1 full-length (aa 1-770) (EcoRI site). FLAG-tagged GIT1, GIT2 long, GIT2 short, and PAP Surface Plasmon Resonance Measurements--
H6-GIT1 GAP-SHD and
H6-Piccolo were used as ligand and analyte, respectively. For
H6-Piccolo, Piccolo (aa 2011-2350) was subcloned into the
BamHI and EcoRI site of pRSET A (Invitrogen). 177 resonance units (RU) of H6-GIT1 GAP-SHD was immobilized on a CM5 sensor chip (BIAcore) by amine coupling. H6-Piccolo at different
concentrations was injected at a flow rate of 10 µl/min. Surface
plasmon resonance was monitored by a BIAcore X instrument (BIAcore).
HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% (v/v) surfactant P20) was used as running
buffer. The surface of the sensor chips was regenerated by 1-min
injections of 25 mM NaOH at 10 µl/min. All experiments
were performed at 25 °C. Data were analyzed using BIAevaluation
software 3.1 (BIAcore).
Coimmunoprecipitation Assay--
EGFP-Piccolo
was constructed by subcloning aa
2011-2350 of Piccolo into pcDNA3 (Invitrogen) digested with
BamHI and EcoRI, followed by elution of the
insert with KpnI and XbaI enzymes and subcloning
into pEGFP-C1 (Clontech). GIT1 Sucrose Density Gradient Sedimentation Analysis--
H6-GIT1
full-length and H6-GIT1 GAP-SHD fusion proteins were purified and
dialyzed against HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100). 200 µl of each fusion
protein or molecular weight standards were laid on top of 4 ml of
15-30% linear sucrose density gradient prepared in HEPES buffer
containing 10% glycerol and 1 mM dithiothreitol, and
centrifuged at 40,000 rpm in an SW41 rotor (Beckman) at 4 °C for
20 h. Molecular mass standards included bovine serum
albumin (67 kDa, Sigma), catalase (232 kDa, Sigma), and thyroglobulin
(670 kDa, Sigma). After centrifugation, fractions were collected from
the top and analyzed by immunoblotting with His antibodies. Molecular
weight standards were visualized by Coomassie Brilliant Blue staining.
Antibodies--
Rabbit polyclonal GIT1 antibodies (1176) were
raised using H6-GIT1 (aa 375-770) as immunogen.2
Polyclonal Piccolo antibodies were generated in guinea pigs (1203) with
H6-Piccolo (aa 2011-2350). The following antibodies have been
previously described: rabbit polyclonal GIT1 antiserum (du139) (15),
EGFP (1167) (29), liprin- Primary Neuron Culture and Immunostaining--
Primary
hippocampal neurons were prepared from E18 embryonic rat brains as
previously described (31) and maintained in the neurobasal medium
(Invitrogen) supplemented with B27 (Invitrogen), 0.5 mM
glutamine, and 12.5 µM glutamate. At 21 days in
vitro (DIV), hippocampal neurons were fixed and permeabilized with
precooled methanol at Characterization of the Interaction between Piccolo and the GIT
Family Proteins in the Yeast Two-hybrid Assay--
We identified the
interaction between Piccolo and GIT1 by yeast two-hybrid screen of a
rat brain cDNA library using the full-length GIT1 as bait. One of
the positive clones was a fragment of Piccolo (aa 2011-2468). By
deletion analysis, a minimal GIT-binding domain in Piccolo was narrowed
down to the middle third of the positive clone (aa 2197-2350, ~150
aa residues), excluding the C-terminal proline-rich region (Fig.
1A). Conversely, a minimal
Piccolo-binding region in GIT1 was defined as the SHD (Fig.
1B), a domain known to mediate the association of GIT1 with
Characterization of the Interaction between Piccolo and GIT
Proteins by Pull-down Assays and Surface Plasmon Resonance
Measurements--
As an independent assay to characterize the
interaction of Piccolo with GIT proteins, we performed GST pull-down
assays using GST-Piccolo (aa 2011-2350) and HEK293T cell lysates
transfected with EGFP-tagged GIT1 deletions (Fig. 2, A and
B). Consistent with the yeast-two hybrid results (Fig.
1B), GST-Piccolo, but not GST alone, selectively pulled down
EGFP-tagged GIT1 deletions containing the SHD (Fig. 2B),
indicating that the SHD of GIT1 mediates its interaction with Piccolo.
In addition, GST-Piccolo selectively pulled down H6-GIT1 GAP-SHD (aa
1-374) but not H6-GIT1 GRKBD (aa 375-770) fusion proteins (Fig.
2C), indicating that Piccolo directly interacts with GIT1.
We further performed pull-down assays on HEK293T cell lysates
transfected with FLAG-tagged full-length GITs (GIT1-FLAG, GIT2
long-FLAG, and GIT2 short-FLAG) and an unrelated protein with the ARF
GAP domain, PAP
To determine the affinity and stoichiometry of the interaction between
Piccolo and GIT1, we performed surface plasmon resonance experiments.
H6-Piccolo (analyte) in running buffer specifically bound to the
H6-GIT1 GAP-SHD (ligand) immobilized on the sensor chip. The plot of
Req (response at the steady state) against the concentration of H6-Piccolo gave a dissociation constant of 14.5 µM and an Rmax (maximum binding
capacity of the surface ligand) of 142 RU (Fig. 2E).
Considering that the RU of immobilized H6-GIT1 GAP-SHD is 177, the
molar ratio of H6-Piccolo to H6-GIT1 GAP-SHD is ~0.9, indicating that
the GIT1 SHD has one binding site for Piccolo. The relatively low
affinity of the Piccolo-GIT interaction in this assay may result from,
in contrast to full-length GIT1, the GAP-SHD region of GIT1 showing a
relatively low apparent affinity for Piccolo in the yeast two-hybrid
assay (Fig. 1B) and pull-down assay (Fig. 2B).
Another explanation may be that immobilization of H6-GIT1 GAP-SHD on
the CM5 sensor chip by amine coupling might have modified arginine
residues in the GIT1 SHD that appear to be important for binding to
Piccolo (see Fig. 5 for further details).
Piccolo and GIT1 Colocalize at Synaptic Sites in Cultured
Neurons--
To determine whether Piccolo and GIT1 colocalize in
subcellular regions of neurons, we performed double-label
immunofluorescence staining on cultured hippocampal neurons after 21 days in vitro (DIV) (Fig. 3).
Consistent with the reported presynaptic localization of Piccolo (5),
Piccolo was primarily localized at discrete punctate structures located
along neurites (Fig. 3B). GIT1 displayed a much wider
distribution than Piccolo, and the immunoreactivity was found
associated with a number of small intracellular structures scattered
throughout the neurons (Fig. 3A). Importantly, however, several of the GIT1-positive structures, presumably representing synaptic GIT1 proteins, colocalized with the Piccolo puncta. The GIT1
puncta that do not overlap with Piccolo appear to be extrasynaptic GIT1
proteins, as evidenced by immunohistochemical and biochemical results.2 These results suggest that GIT1 proteins are
widely distributed to both synaptic as well as non-synaptic sites and
that presynaptic GIT1 proteins colocalize with Piccolo.
Piccolo and GIT1 Form a Complex in Heterologous Cells and
Brain--
We then examined in a series of coimmunoprecipitation
experiments whether interaction between Piccolo and GIT1 occurs on a cellular level (Fig. 4, A and
B). Incubation of HEK293T cell lysates doubly transfected
with EGFP-tagged Piccolo (EGFP-Piccolo, aa 2011-2350) and
GIT1-FLAG (full-length) with FLAG antibodies immunoprecipitated GIT1-FLAG and coprecipitated EGFP-Piccolo (Fig. 4A). In
control experiments, FLAG antibodies did not bring down EGFP-Piccolo
from singly transfected cells. In inversely designed
coimmunoprecipitation experiments Piccolo antibodies
immunoprecipitated EGFP-Piccolo and coprecipitated GIT1-FLAG
(Fig. 4B).
To determine whether Piccolo and GIT1 form a complex in brain, we
performed coimmunoprecipitation experiments on rat brain extracts (Fig.
4C). Immunoprecipitation on detergent lysates of the crude
synaptosomal fraction of adult rat brain with Piccolo antibodies
precipitated Piccolo and coprecipitated GIT1 and various proteins that
are known to associate with GIT1, including Piccolo,
Multiple aa sequence alignment of the repeats in the SHD from GITs of
various species and yeast Spa2p revealed that there are four invariant
residues in each repeat; Phe, Leu, Arg, and Arg (Fig. 5A,
indicated by arrowheads). To determine whether any of these
amino acids were important for Piccolo binding, these residues in the
first repeat of the GIT1 SHD were mutated into alanines (F285A, L288A,
R298A, and R299A). When these mutations were introduced into the aa
1-374 GIT1 backbone and tested in the yeast two-hybrid assay, we found
that the L288A mutation abolished GIT1-Piccolo interactions (Fig.
5B). The other mutations also significantly reduced
GIT1-Piccolo interactions, showing effects in the order of R299A,
F285A, and R298A (the smallest effect observed for R298A).
Intriguingly, none of the four mutations appeared to affect the
GIT1-
We performed coimmunoprecipitation experiments to test whether the same
SHD mutations affected biochemical associations of GIT1 with its
specific SHD-binding partners (Piccolo, GIT Proteins Form Homo- and Heteromultimers through the C-terminal
GRKBD--
Synaptic multidomain proteins often form multimers
(36-39). This organization is thought to be a mechanism for
efficiently increasing the complexity of synaptic macromolecular
complexes. Because GIT1 is a multidomain protein localized at synaptic
sites and it interacts with Piccolo, a potential active zone organizer, we tested whether GIT proteins form multimers. When HEK293T cells doubly transfected with EGFP-GIT1 and GIT1-FLAG or GIT2-FLAG were immunoprecipitated with FLAG antibodies, EGFP-GIT1 coimmunoprecipitated with GIT1-FLAG and GIT2-FLAG (Fig.
6A), indicating that GIT
proteins can form both homo- and heteromultimers. FLAG antibodies did
not bring down EGFP-GIT1 expressed alone (Fig. 6A). In the
yeast two-hybrid assay, GIT1 and GIT2 interacted with both GIT1 and
GIT2 (Fig. 6B), confirming the coimmunoprecipitation results
(Fig. 6A) and indicating that both GIT1 and GIT2 form
homo- and heteromultimers.
To determine the minimal region mediating the multimerization, we
tested deletions of GIT1 for their ability to form multimers. The
C-terminal GRKBD (aa 375-770), but not the N-terminal half (aa
1-374), of GIT1 interacted with full-length GIT1 and GIT2 in the yeast
two-hybrid assay (Fig. 6B), indicating that the GRKBD mediates homo- and heteromultimerization. In the pull-down assay, a GST
fusion protein containing the GRKBD of GIT1 (GST-GRKBD), but not GST
alone, brought down EGFP-tagged GIT1 containing the full-length and the
GRKBD, but not the N-terminal half (GAP-SHD) (Fig. 6C), thus
confirming the yeast two-hybrid results (Fig. 6B). The
limited pull-down of full-length GIT1 by GST-GRKBD as compared with
that of the GRKBD may indicate that full-length GIT1 proteins form
stronger homomultimers that are less competed for by the shorter
construct and, therefore, less efficiently pulled down. GST-GRKBD
pulled down H6-GIT1 GRKBD (Fig. 6D), indicating that the
interaction between GRKBDs is direct. Taken together, these results
indicate that GIT proteins form homo- and heteromultimers through a
direct interaction between GRKBDs in a tail-to-tail fashion.
The Entire GRKBD Is Involved in Multimerization--
The size of
the GRKBD is relatively large (~400 aa), and the GRKBD contains
several distinct subdomains. The N-terminal region of the GRKBD
contains a prominent coiled-coil (CC) domain (aa 432-483) with the
properties of a leucine zipper (leucine residues at every seventh aa
position). In addition, the C-terminal region of the GRKBD contains
paxillin-binding subdomain (PBS, aa 646-770) that is known to
associate with paxillin (18, 25). Given this complexity, we sought to
further narrow down the minimal region mediating the multimerization.
We generated deletions of the GRKBD containing combinations of the CC
domain, the PBS domain, and the spacer region in between (termed SP
hereafter; aa 486-645) and tested them for binding to full-length GIT1
and GIT2 in the yeast two-hybrid assay. Intriguingly, all the
deletions, including the ones containing only the CC domain, the SP
region, or the PBS domain, interacted with GIT1 and GIT2 (Fig.
7A). Similarly, GST fusion
proteins containing only the CC domain, the SP region, or the PBS
domain pulled down EGFP-tagged GIT1 GRKBD (Fig. 7B). These
results indicate that all subregions of the GRKBD participate in the
multimerization.
Sedimentation of GIT1 Proteins in a Sucrose Density Gradient
Suggests the Formation of Dimers--
To determine the stoichiometry
of GIT1 multimers, we performed sucrose density gradient sedimentation
experiments. We employed fusion proteins of H6-GIT1 full-length and
H6-GIT1 GAP-SHD (aa 1-374, negative control lacking the GRKBD). Both
proteins exhibited a single peak in their sedimentation pattern (Fig.
8, A and B). When
compared with the sedimentation of molecular weight standards (Fig.
8C), the peak of H6-GIT full-length corresponded to a
molecular mass of 138 kDa, whereas that of H6-GAP-SHD corresponded to
55 kDa, close to its monomeric size (46 kDa). Because the calculated molecular mass of H6-GIT1 full-length is 90 kDa, the size of 138 kDa
corresponds to the stoichiometry of 1.53, suggesting that GIT is likely
to form dimers. The formation of a single peak of 138 kDa, a size in
between monomer and dimer, is likely to be due to incomplete separation
monomeric and dimeric peaks.
GIT1 Multimerization Is Required for the Formation of a Ternary
Complex between Piccolo, GIT1, and
We used this deletion mutant (GIT1 The molecular mechanisms underlying the organization of the
presynaptic CAZ are largely unknown. Our results indicate that the CAZ
protein Piccolo associates with GIT proteins as well as various
GIT-associated scaffolding and signaling proteins, including liprin- Functions of the Interaction between Piccolo and the GIT Family
Proteins--
Our data indicate that Piccolo associates with GIT
proteins in vitro and in vivo. A possible
function of the Piccolo-GIT interaction can be inferred from their
subcellular localizations. Piccolo is restricted to the active zone
within the nerve terminal when characterized by immunoelectron
microscopic (immuno-EM) analysis (4, 6). Extensive quantitative
immuno-EM analysis reveals that GIT1 immunogold particles are sharply
concentrated at the active zone in addition to their localization in
the postsynaptic density.2 However, the GIT1 is not
confined to synaptic sites. It also shows a widespread distribution to
non-synaptic sites as shown by a variety of studies, including
immunostaining in cultured neurons, immuno-EM, and subcellular
fractionation analysis of rat brain samples.2 Consistently,
GIT1 proteins distribute to several distinct subcellular compartments
(adhesion-like structures, the leading edge and cytoplasmic complexes)
in non-neuronal cells (40). Thus a reasonable prediction for the
function of the Piccolo-GIT interaction based upon the clear difference
in their subcellular localizations would be that Piccolo may be
involved in the recruitment of GIT proteins to the CAZ.
A possible argument against this hypothesis is that Piccolo and GIT1
may form a complex in the preassembled Piccolo transport vesicle and
are inserted together into the plasma membrane of nascent synapses.
Zhai et al. (2001) showed that Piccolo and other constituents of the active zone such as Bassoon and N-cadherin colocalize in discrete puncta in axonal growth cones of immature cultured neurons (DIV 4). However, we found that Piccolo and GIT1 showed a minimal colocalization in growth cones of immature cultured neurons, whereas we could confirm the reported colocalization between
Piccolo and N-cadherin. Thus, it appears that GIT proteins are
minimally associated with the preassembled Piccolo transport vesicle
and instead associate with Piccolo after the initial formation of the
CAZ.
Functions of the GIT Family Proteins in Active Zones--
GIT
proteins contain various domains for protein interactions as well as an
ARF GAP domain. A possible function of GIT proteins would be to bring
various GIT-associated proteins to the vicinity of Piccolo. Among the
GIT-interacting proteins that we have shown to form a complex with
Piccolo (Fig. 4C), a protein of particular interest is the
multidomain protein liprin-
We demonstrated that the GIT family proteins form homo- and
heteromultimers through the GRKBD. GIT multimerization is expected to
increase the number and diversity of GIT-based docking sites at the
active zone. Importantly, Piccolo can from a ternary complex with
GIT proteins may exert various presynaptic functions through the
modulation of ARF small GTPases. GIT1 regulates endocytosis of various
membrane proteins that are internalized by the clathrin-coated pit
pathway in a
In conclusion, our results suggest that GIT proteins, through their
multimerization and association with Piccolo, contribute to the
formation of a Piccolo-based protein network at the active zone. The
next step would be to determine how the multifunctional GIT family
proteins regulate presynaptic organization and integrate various
GIT-associated signaling pathways.
PIX, focal adhesion kinase, liprin-
, and paxillin. Point
mutations in the SHD of GIT1 differentially interfere with the
association of GIT1 with Piccolo,
PIX, and focal adhesion kinase,
suggesting that these proteins bind to the SHD by different mechanisms.
Intriguingly, GIT proteins form homo- and heteromultimers through their
C-terminal G-protein-coupled receptor kinase-binding domain in a
tail-to-tail fashion. This multimerization enables GIT1 to
simultaneously interact with multiple SHD-binding proteins including
Piccolo and
PIX. These results suggest that, through their
multimerization and interaction with Piccolo, the GIT family proteins
are involved in the organization of the CAZ.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PIX, focal
adhesion kinase (FAK), and the focal adhesion adaptor protein paxillin
(16, 18, 25). However, neuronal functions of GIT proteins, despite
their widespread tissue expression, including the brain (15, 19),
remained unknown.
PIX. These results suggest that GIT proteins
participate in the organization of the CAZ.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity were measured semi-quantitatively. For yeast
two-hybrid screen, the full-length GIT1 (aa 1-770) was amplified by
PCR from pBK(
) GIT1 (15) and subcloned into pBHA (a bait vector
containing LexA DNA-binding domain) digested with EcoRI.
Regions of Piccolo (aa 2197-2350) and
PIX (aa 440-646) were
subcloned into the EcoRI-BamHI site of pBHA. The
EcoRI site of GIT2 long and GIT2 short in pBK(
) GIT2 long
and pBK(
) GIT2 short (KIAA0148) was removed by site-directed mutagenesis without changing amino acids, and GIT2 long and GIT2 short
were subcloned into the EcoRI site of pBHA. The following regions of GIT1 and Piccolo were subcloned into the
BamHI-EcoRI site of pGAD10: GIT1, aa
1-129, 1-244, 1-336, 1-374, 130-244, 130-374, 245-374,
375-528, 375-645, 405-485, 486-645, 486-770, and 646-770;
Piccolo, aa 2011-2196, 2011-2350, 2197-2350, 2197-2468, and
2344-2468. Full-length GIT1 (aa 1-770) and GIT1 GRKBD (aa 375-770)
were subcloned into the EcoRI site of pGAD10. Full-length GIT2 long (aa 1-759) was subcloned into the EcoRI site of
pGAD10. Bassoon (aa 1730-2237) was amplified by PCR from pCMV2-RBB29
(aa 1630-3264 of rat Bassoon) and subcloned into pGAD10 digested with BamHI and EcoRI. For pGAD10 GIT1 aa 1-374
(273-303), GIT1 aa 1-272 and 304-374 were amplified by PCR, digested
with BamHI-KpnI and
KpnI-EcoRI, respectively, and subcloned into the
BamHI-EcoRI site of pGAD10. GIT1 (aa 1-374 and
1-770) with mutations in the SHD (F285A, L288A, R298A, and R299A) were
amplified by PCR from pBK(
) GIT1 mutants and subcloned into the
BamHI-EcoRI (for aa 1-374) or EcoRI
(for aa 1-770) sites of pGAD10.
in pBK(
)
were previously described (15, 19). The GIT-binding domain (GBD, aa
486-566) of
PIX-a was subcloned into the
EcoRI-BamHI site of pEGFP-C1
(Clontech). For pull-down assay, transfected
HEK293T cells were extracted with binding buffer (phosphate-buffered
saline with 1% Triton X-100) containing protease inhibitors at
4 °C for 30 min. After centrifugation, the extracts were mixed with
GST fusion proteins and incubated for 30 min, followed by precipitation with glutathione-Sepharose 4B resin (Amersham Biosciences). H6-GIT1 fusion proteins were pulled down by the same method. The precipitates were analyzed by immunoblotting with EGFP and His antibodies.
PBS (aa 1-645)
was subcloned into the BamHI-EcoRI site of
pBK(
). For GIT1
CC-FLAG, GIT1 aa 1-431 and GIT1 aa 484-770 were
amplified by PCR, digested with BamHI-KpnI and
KpnI-EcoRI, respectively, and subcloned into pBK(
) digested with BamHI and EcoRI.
Myc-tagged
PIX has been described previously (27). Expression
constructs of full-length GIT1 (pBK(
) GIT1-FLAG) with point
mutations in the SHD (F285A, L288A, R298A, and R299A) were generated
using a QuikChange site-directed mutagenesis kit (Stratagene). For
immunoprecipitation, transfected HEK293T cells were extracted with
binding buffer and incubated with M2 FLAG-agarose (Sigma) at 4 °C
for 4 h or antibodies against Piccolo (1203, 1:250) or Myc (2 µg/ml) followed by protein A-Sepharose (Amersham Biosciences)
precipitation. In vivo coimmunoprecipitation was performed
as previously described (28). Briefly, the crude synaptosomal fraction
of adult rat brain was solubilized with DOC buffer (50 mM
Tris-HCl, 1% sodium deoxycholate, pH 9.0), dialyzed against
binding/dialysis buffer (50 mM Tris-HCl, 0.1% Triton
X-100, pH 7.4), and centrifuged. The supernatant was incubated with
Piccolo antibodies (1203, 1:250) or boiled antibodies (negative
control) at 4 °C for 2 h, with protein A-Sepharose for
additional 2 h, followed by immunoblot analysis of the
precipitates with GIT1 (du139, 1:2000), Piccolo (1203, 1 µg/ml),
PIX (1:1000), FAK (1:200), paxillin (1:1000), liprin-
(1120, 0.5 µg/ml), GRIP1 (43-8, 1:1000), vinculin (1:1000), and synaptophysin
(1:1000) antibodies.
(1120),2 and
PIX (27).
Guinea pig polyclonal Piccolo antibodies (gp-
-44a-GST-affi) were
affinity-purified as described (30). GRIP1 43-8 antibody was a gift
from Dr. Pann-Ghill Suh (Pohang University of Science and Technology,
Korea). The following antibodies were purchased from commercial
sources: FLAG (M2, Sigma), vinculin (Sigma), His (His-probe, H-15,
Santa Cruz Biotechnology), paxillin (Transduction Laboratories), FAK
(clone 4.47, Upstate Biotechnology), synaptophysin (Sigma),
synaptotagmin (Sigma), Myc (9E10, Santa Cruz Biotechnology), and
HA (clone 12CA5, Roche Applied Science).
20 °C for 20 min and incubated with primary
antibodies, Piccolo (gp-
-44a-GST-affi, 1:1000) and GIT1 (1176, 2 µg/ml), followed by Cy3- or fluorescein isothiocyanate-conjugated
secondary antibodies (1:250 and 1:100, respectively, Jackson
ImmunoResearch). Images were captured by confocal laser scanning
microscopy (LSM510, Zeiss).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PIX and FAK (25). In addition to GIT1, Piccolo interacted with GIT2
long (full-length), which shares the same domain structure with GIT1
(19), as well as GIT2 short, a splice variant of GIT2 that lacks most
of the C-terminal GRKBD but contains the SHD (19) (Fig. 1A)
(see Fig. 2D for the
structural organization of GIT2 splice variants). In contrast, a region
of Bassoon (aa 1730-2237) that corresponds to the Piccolo prey clone
(aa 2011-2468) showed no significant homology to the minimal
GIT-binding domain of Piccolo (aa 2197-2350), and did not interact
with GIT1, GIT2 long, or GIT2 short (data not shown). This indicates
that the GIT proteins selectively interact with Piccolo and suggests
that Piccolo and Bassoon, two closely related CAZ proteins known to
colocalize in synapses (5), may have differential functions.
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Fig. 1.
Characterization of the interaction between
Piccolo and the GIT family in the yeast two-hybrid assay.
A, deletions of the Piccolo-positive clone in the pGAD10
prey vector were tested for binding to GIT1 (full-length), GIT2 long,
and GIT2 short in the pBHA bait vector in a yeast two-hybrid assay. The
Piccolo Bassoon homology (PBH) domains are indicated by the
numbers underneath the diagram of full-length
Piccolo. Q, glutamine heptad repeats; Zn, Zinc
finger; CC, coiled coil; PDZ, PSD-95/Dlg/ZO-1
domain; C2, C2 domain. Small numbers refer to aa
residues at the boundaries of the domains or full-length proteins.
P-rich, proline-rich region. HIS3 activity: +++ (>60%), ++
(30-60%), + (10-30%), (no significant growth);
-galactosidase: +++ (<45 min), ++ (45-90 min), + (90-240 min),
(no significant
-galactosidase activity). B, deletions of
GIT1 in pGAD10 were tested for their binding against Piccolo (aa
2197-2350). The SHD of GIT1 mediates the association with Piccolo.
GAP, ARF GAP domain; ANK, ankyrin repeats;
SHD, Spa2 homology domain; GRKBD,
G-protein-coupled receptor kinase-binding domain; CC, coiled
coil; PBS, paxillin-binding subdomain.
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Fig. 2.
Characterization of the interaction between
Piccolo and GIT proteins by the pull-down assay and surface plasmon
resonance measurements. A and B, HEK293T
cell lysates transiently transfected with EGFP-tagged deletions of GIT1
were pulled down by equal amounts (5 µg) of GST-Piccolo (aa
2011-2350) or GST alone, and analyzed by immunoblotting with EGFP
antibodies. GST fusion proteins used in the pull-down assay were
visualized by Coomassie Brilliant Blue staining (A).
C, H6-GIT1 GAP-SHD (aa 1-374) and H6-GIT1 GRKBD (aa
375-770) fusion proteins (1 µg) were pulled down by GST-Piccolo, or
GST alone (2 µg), and analyzed by immunoblotting with His antibodies.
IB, immunoblotting. D, HEK293T cell lysates
transfected with FLAG-tagged full-length GITs (GIT1-FLAG, GIT2
long-FLAG, and GIT2 short-FLAG) or PAP /PAG3 (an unrelated ARF GAP)
were pulled down by GST-Piccolo or GST alone (5 µg) and analyzed by
immunoblotting with FLAG antibodies. PH, pleckstrin homology
domain; SH3, Src homology 3 domain. E, the
affinity and stoichiometry of the Piccolo-GIT1 interaction determined
by surface plasmon resonance measurements. H6-Piccolo was perfused over
the H6-GIT1 GAP-SHD immobilized on a CM5 sensor chip. Responses at the
steady state (Req) were plotted against the concentration of
H6-Piccolo and analyzed by non-linear curve fitting.
/PAG3, which lacks the SHD (32), as control (Fig.
2D). GST-Piccolo brought down GIT1, GIT2 long, and GIT2
short but not PAP
/PAG3, consistent with the yeast two-hybrid results
(Fig. 1A), indicating that Piccolo selectively interacts
with the GIT family proteins. The relatively inefficient pull-down of
GIT2 short by GST-Piccolo differs from the yeast two-hybrid results
(Fig. 1A). This could be due to differences in the assay,
but both assays (yeast two-hybrid and pull-down) clearly showed that
GIT2 short can bind Piccolo.
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Fig. 3.
Piccolo and GIT1 colocalize at synaptic sites
in cultured neurons. Cultured hippocampal neurons (DIV 21)
were labeled by double-immunofluorescence staining with GIT1 (1176) and
Piccolo (gp- -44a-GST-affi) antibodies. GIT1 (A1,
red) distributes to a number of small intracellular
structures scattered throughout the neurons, and several of the
GIT1-positive structures colocalize with presynaptic Piccolo puncta
(B1, green). C1 and C2 are
merged images. A2, B2, and
C2 represent enlargement of the boxes in
A1, B1, and C1, respectively.
Scale bar: 10 µm.
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Fig. 4.
Piccolo forms a complex with GIT1 in
heterologous cells and brain. A, HEK293T cell lysates
doubly transfected with EGFP-tagged Piccolo (EGFP-Piccolo, aa
2011-2350) plus GIT1-FLAG (full-length), or EGFP-Piccolo alone, were
immunoprecipitated with FLAG antibodies, and analyzed by immunoblotting
with EGFP and FLAG antibodies. IP, immunoprecipitation.
B, HEK293T cell lysates doubly transfected with EGFP-Piccolo
plus GIT1-FLAG, or GIT1-FLAG alone, were immunoprecipitated with
Piccolo (1203) antibodies and analyzed by immunoblotting with FLAG and
EGFP antibodies. C, detergent lysates of the crude
synaptosomal fraction of adult rat brain were immunoprecipitated with
Piccolo (1203) antibodies (untreated or boiled) and analyzed by
immunoblotting with GIT1, Piccolo, PIX, FAK, paxillin, liprin-
,
GRIP1, vinculin, and synaptophysin (SynPhy) antibodies.
200, 200-kDa molecular mass marker.
PIX (16, 18, 25), FAK
(25), paxillin (18), liprin-
,2 and the
liprin-
-associated GRIP1 (33) but not vinculin and synaptophysin
(negative control). The multiple bands in the
PIX immunoblot
represent the splice variants of
PIX expressed in the brain (34,
35). Immunoprecipitation with boiled Piccolo antibodies did not bring
down any of the above proteins. These results indicate that Piccolo
associates with GIT1 and GIT1-associated proteins in
vivo.
PIX, and FAK Bind to the SHD of GIT1 by Different
Mechanisms--
The GIT1 SHD contains two highly conserved repeats
that are ~30 aa residues in length (Fig.
5A, diagram in Fig.
5B). Using a yeast two-hybrid assay, we examined whether
both repeats were required for binding to Piccolo or
PIX. We found
that deletion of either repeat 1 or repeat 2 from the aa 1-374 GIT1
backbone eliminated its interaction with Piccolo and
PIX (Fig.
5B), suggesting both repeats of the SHD are required for
binding to SHD ligands. This is consistent with the 1:1 stoichiometry
of the Piccolo-GIT1 interaction obtained from the surface plasmon
resonance results (Fig. 2E).
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Fig. 5.
Piccolo, PIX, and
FAK bind to the SHD of GIT1 by different mechanisms. A,
multiple aa sequence alignment of the two repeats (~30 residues long)
in the SHD of GITs from various species and yeast Spa2p. Four invariant
residues are indicated by arrowheads and
highlighted as white characters in black
boxes. The residue numbers are from the repeat 1 of rat GIT1. The
sources of the aa sequences in the data base are as follows; rGIT1
(AF085693, rat), hGIT2 (NM_057169, human), Cat-2 (AF148693, mouse),
p95-APP1 (AF216970, chicken), p95-APP2 (AF134571, chicken), PKL
(AF112366, chicken), dGIT (NM_136755, Drosophila), cGIT
(NM_077360, C. elegans), aGIT (EAA03954,
Anopheles), and Spa2p (NP_013079, yeast). B, a
yeast-two hybrid assay was used to investigate the effects of deletions
and point mutants in the GIT1 SHD (pGAD10) on binding to Piccolo and
PIX (pBHA). R1, repeat 1; R2, repeat 2. C: panels a-c, HEK293T cell lysates doubly
transfected with GIT1-FLAG mutants plus either EGFP-Piccolo
(a), Myc-
PIX (b), or HA-FAK (c)
were immunoprecipitated with FLAG antibodies and analyzed by
immunoblotting with the indicated antibodies. Transf,
transfection; WT, wild-type.
PIX interactions, suggesting the effects of these mutations on
GIT1-Piccolo interactions are unlikely to be caused by nonspecific
changes in the SHD structure, and that Piccolo and
PIX bind to the
SHD of GIT1 by different mechanisms. Introduction of the same mutations
to full-length GIT1 gave similar results; the L288A mutation eliminated
the GIT1-Piccolo interaction. However, the inhibitory effects of the
other mutations on the GIT1-Piccolo interaction were slightly reduced.
This could be due to an enhanced affinity between GIT1 and Piccolo in
the context of full-length GIT1 (Figs. 1B and
2B).
PIX, and FAK) in
heterologous cells (Fig. 5C). The L288A mutation in the GIT1
SHD abolished the GIT1-Piccolo association, and the other mutations
also reduced GIT1-Piccolo interactions, with effects in the order of
R299A
F285A > R298A (Fig. 5C, panel
a), consistent with the yeast two-hybrid results (Fig.
5B). However, these mutations did not affect GIT1
coimmunoprecipitation with
PIX (Fig. 5C, panel
b), also consistent with the yeast two-hybrid results (Fig. 5B). Intriguingly, all four mutations of the GIT1 SHD
eliminated association of GIT1 with FAK, another protein that binds to
the GIT1 SHD (25), whereas wild-type GIT1 formed a complex with FAK
(Fig. 5C, panel c). Taken together, these results
suggest that Leu-288 of GIT1 plays an important role in binding
to Piccolo, and that Piccolo,
PIX, and FAK bind to the SHD of GIT1
by different mechanisms.
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Fig. 6.
GIT family proteins form homo- and
heteromultimers through the C-terminal GRKBD. A,
HEK293T cell lysates doubly transfected with EGFP-GIT1 and FLAG-tagged
GITs (GIT1 or GIT2 long) were immunoprecipitated with FLAG antibodies
and analyzed by immunoblotting with EGFP and FLAG antibodies.
B, GIT1 (full-length and deletions) and GIT2 long in pGAD10
were tested for binding to GIT1 and GIT2 long in pBHA in the yeast
two-hybrid assay. C, HEK293T cell lysates transfected with
EGFP-GIT1 (full-length and deletions) were pulled down by GST-GIT1
GRKBD (GST-GRKBD, aa 375-770, 4 µg), or GST alone, and
analyzed by immunoblotting with EGFP antibodies. D, H6-GIT1
GRKBD fusion proteins (1 µg) were pulled down by GST-GRKBD (2 µg),
or GST alone, and analyzed by immunoblotting with His antibodies.
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Fig. 7.
The entire GRKBD is involved in
multimerization. A, deletions of the GRKBD of GIT1 in
pGAD10 were tested for binding to full-length GIT1 and GIT2 in pBHA.
CC, coiled-coil; PBS, paxillin-binding subdomain;
SP, the spacer region between the CC and PBS domains.
B, HEK293T cell lysates transiently transfected with
EGFP-tagged GIT1 GRKBD were pulled down by GST-GRKBD (aa 375-770),
GST-CC (aa 405-485), GST-SP (aa 486-645), GST-PBS (aa 646-770), or
GST alone (4 µg each) and analyzed by immunoblotting with EGFP
antibodies.
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Fig. 8.
Sedimentation of GIT1 in a sucrose density
gradient suggests the formation of dimers. A, H6-GIT1
full-length or H6-GIT1 GAP-SHD was sedimented using 15-30% linear
sucrose density gradient centrifugation. The molecular mass standards
(bovine serum albumin (67 kDa), catalase (232 kDa), and thyroglobulin
(670 kDa)) were sedimented in a parallel gradient. Collected fractions
were analyzed by immunoblotting using His antibodies. Peak fractions of
the standards were determined by Coomassie Brilliant Blue staining.
B, quantitative analysis of the immunoblot results
(A). The band intensity in each fraction was normalized to
that of the peak fraction. Closed squares, H6-GIT1 GAP-SHD;
closed circles, H6-GIT1 full-length. C,
sedimentation of molecular weight standards. Molecular weights in log
scale were plotted against the number of fractions, followed by linear
fitting.
PIX--
Our results and
previous reports (16, 18, 25) indicate that Piccolo,
PIX, and FAK
bind to the same SHD of GIT1. One of the possible functions of GIT
multimerization is to enable GIT proteins to interact with multiple
SHD-binding proteins as depicted in Fig.
9A. To test this hypothesis,
we first attempted to identify smallest possible GIT1 deletions that
result in multimerization defects. We generated deletion variants of
full-length GIT1 that lacked the CC domain (GIT1
CC; deletion of aa
432-483) and the PBS domain (GIT1
PBS; deletion of aa 646-770) and
tested their multimerization by immunoprecipitation experiments. When
HEK293T cells doubly transfected with EGFP-GIT1 and GIT1-FLAG
(full-length,
CC or
PBS) were immunoprecipitated with FLAG
antibodies, coimmunoprecipitation of EGFP-GIT1 was greatly reduced in
GIT1
CC but not in GIT1 full-length or
PBS (Fig. 9B),
indicating that the CC domain is required for GIT multimerization. The
slightly smaller effect of CC domain deletion on GIT
multimerization in the yeast two-hybrid assay (Fig.
7A), compared with that in coimmunoprecipitation
experiments, could be due to the deletion of the different regions
containing the CC domain (aa 432-483 versus aa 375-485) or
the different size of the backbones for deletion (full-length
versus the GRKBD) in GIT1-FLAG
CC and pGAD10 GIT1 GRKBD
(aa 486-770), respectively.
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Fig. 9.
GIT multimerization is required for the
formation of a ternary complex between Piccolo, GIT1, and
PIX. A, a schematic diagram
illustrating the formation of a ternary complex between Piccolo, GIT,
and
PIX. B, HEK293T cell lysates doubly transfected with
EGFP-GIT1 and GIT1-FLAG (full-length,
CC, or
PBS) were
immunoprecipitated with FLAG antibodies and analyzed by immunoblotting
with EGFP and FLAG antibodies. C, HEK293T cell lysates
triply transfected with EGFP-Piccolo, Myc-
PIX, and GIT1-FLAG
(full-length or
CC) were immunoprecipitated with Myc antibodies and
analyzed by immunoblotting with the antibodies indicated. D
and E, HEK293T cell lysates doubly transfected with
EGFP-Piccolo plus GIT1-FLAG (full-length or
CC), or Myc-
PIX plus
GIT1-FLAG (full-length or
CC), were immunoprecipitated with FLAG
antibodies and analyzed by immunoblotting with the antibodies
indicated. F, lysates from HEK293T cells transfected with
EGFP-
PIX GBD were subjected to pull-down assays using GST-GIT1
GAP-SHD (50 pmol) and increasing amounts of H6-Piccolo. Complexes were
analyzed by immunoblotting with EGFP and His antibodies. GST fusion
proteins on the membrane were visualized by Ponceau S staining. The
asterisk indicates H6-Piccolo. GBD, GIT-binding
domain. G, schematic diagram illustrating the formation of a
complex containing GST-GIT1 GRKBD, H6-GIT1 full-length, and EGFP-
PIX
GBD. H, lysates from HEK293T cells transfected with
EGFP-
PIX GBD were subjected to pull-down assays using GST-GIT1
GRKBD, or GST alone (6 µg), in the presence or absence of H6-GIT1
full-length (15 µg). Complexes were analyzed by immunoblotting with
EGFP and His antibodies. GST fusion proteins on the membrane were
visualized by Ponceau S staining.
CC) to determine whether GIT1
multimerization is required for the formation of a ternary complex
between Piccolo, GIT1, and
PIX. When HEK293T cells triply transfected with EGFP-Piccolo, GIT1-FLAG (full-length or
CC), and
Myc-
PIX were immunoprecipitated with Myc antibodies, Piccolo was
brought down only in the presence of full-length GIT1, but not in the
presence of GIT1
CC (Fig. 9C). In control experiments, GIT1
CC and full-length GIT1 showed a similar coimmunoprecipitation with Piccolo (Fig. 9D) or
PIX (Fig. 9E),
indicating that the lack of the ternary complex in GIT1
CC (Fig.
9C) did not arise from a reduced binding of GIT1
CC to
Piccolo or
PIX. In additional control experiments to support our
model, we found that the addition of increasing amounts of H6-Piccolo
reduced the pull down of EGFP-
PIX GBD (GIT-binding domain) by
GST-GIT1 GAP-SHD (Fig. 9F). This indicates that Piccolo and
PIX compete for binding to the GIT1 SHD and suggests that the GIT1
SHD cannot bind two different ligands simultaneously. As an independent
way of demonstrating the function of GIT multimerization, we performed
the pull-down assay depicted in Fig. 9G. GST-GIT1 GRKBD,
which cannot pull down EGFP-
PIX by itself, was able to pull down
PIX only in the presence of H6-GIT1 full-length fusion proteins
(Fig. 9H). Taken together, these results suggest that GIT
multimerization is required for the formation of the
Piccolo·GIT1·
PIX ternary complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
PIX, FAK, and paxillin. Intriguingly, Piccolo,
PIX, and FAK bind to the SHD of GIT1 by different mechanisms. In addition, GIT proteins form homo- and heteromultimers through the GRKBD, and this
multimerization enables GIT proteins to simultaneously interact with
multiple SHD-binding proteins, including Piccolo and
PIX. These
results suggest that GIT proteins, through multimerization and
interaction with Piccolo, are involved in organizing the CAZ.
(41, 42). Mutations in
Dliprin and syd-2, Drosophila, and
Caenorhabditis elegans homologs of liprin-
cause
morphological defects in presynaptic active zones (43, 44). In support
of the genetic evidence for its presynaptic function, liprin-
associates with the receptor tyrosine phosphatase LAR, a regulator of
axon guidance and synaptic target recognition (45-47), and with the
active zone scaffolding protein RIM, which regulates neurotransmitter
release (8, 48). In addition, liprin-
associates with GRIP/ABP, a
family of multi-PDZ domain proteins that interacts with various
membrane and signaling proteins, including
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
glutamate receptors, the RasGEF GRASP-1, and ephrin ligands and their Eph receptors (28, 49-54). Immuno-EM studies indicate that
GIT1, liprin-
, and GRIP are localized at the presynaptic nerve
terminal in addition to postsynaptic sites (28, 33, 38,
39).2 Taken together, these results suggest that Piccolo
and GIT proteins, in concert with liprin-
and various
liprin-
-associated proteins, including LAR, RIM, and GRIP,
participate in the organization of the CAZ.
PIX through GIT multimers (Fig. 9), suggesting that GIT multimerization may mediate the association of Piccolo with other SHD-binding proteins, including
PIX and FAK. In support of the synaptic function of
PIX, genetic deletion of dPix, a
Drosophila homolog of
PIX, causes defects in synaptic
structure and protein targeting (55). FAK is widely expressed in brain
and implicated in the recruitment of various signaling and adaptor
proteins, including the Src family of tyrosine kinases
(56). Thus
PIX and FAK that are brought to the vicinity of Piccolo
through GIT multimers and may participate in the organization of
presynaptic active zones. Intriguingly, point mutations in the GIT1 SHD
had different effects on the interaction of GIT1 with Piccolo,
PIX, and FAK (Fig. 5), suggesting that Piccolo,
PIX, and FAK bind to the
SHD of GIT1 by different mechanisms. Although determination of the
exact nature of these differences requires further study, these point
mutations may be useful for testing the in vivo functional significance of these interactions in future studies.
-arrestin- and dynamin-sensitive manner (15, 24).
Importantly, the inhibition of agonist-induced internalization of
2-adrenergic receptors by GIT1 requires an intact ARF
GAP domain (15). ARF6, a substrate of GIT1 (57), is unique among various ARFs in that it regulates actin cytoskeleton rearrangement in
various cell types (21, 58-63). ARF6 proteins are expressed in
developing hippocampus, and the ARF-specific GEF ARF nucleotide-binding site opener suppresses dendritic branching in cultured hippocampal neurons through ARF6 and Rac1 pathways (64). Consistently,
wild-type and truncated GIT1/p95-APP1, through ARF6 and Rac1, induces
actin-rich protrusions in fibroblasts (17). Another ARF-specific GEF
msec7-1/cytohesin-1 increases neurotransmitter release at the
Xenopus neuromuscular junction (65). Taken together, these
results suggest that GIT proteins, through their ARF modulation, may be
involved in the regulation of receptor trafficking, actin cytoskeleton
rearrangement, and neurotransmitter release at the active zone.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Dongeun Park at Seoul
National University for the gift of PIX expression construct and
antibody, Dr. Cheol O. Joe at the Korea Advanced Institute of Science
and Technology for FAK expression construct, and Dr. Pann-Ghill Suh at
Pohang University of Science and Technology for the GRIP1 (43-8) antibody.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Korean Ministry of Science and Technology, the Korea Science and Engineering Foundation, the Korea Research Foundation (to E. K.), the National Research Laboratory (Grant M1-0104-00-0140) (to B. K. K.), and the Deutsche Forschungsgemeinschaft (SFB426/A1) (to E. D. G.) and by National Institutes of Health Grants RO1-NS39471 and PO1-AG06569 (to C. C. G.).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.: 42-869-2633;
Fax: 42-869-2610; E-mail: kime@mail.kaist.ac.kr.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M212287200
2 J. Ko, S. Kim, J. G. Valtschanoff, H. Shin, J. R. Lee, M. Sheng, R. T. Premont, R. Weinberg, and E. Kim. (2003) J. Neurosci. In press
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ABBREVIATIONS |
---|
The abbreviations used are: CAZ, cytoskeletal matrix assembled at active zones; GRKBD, G-protein-coupled receptor kinase-binding domain; GAP, GTPase-activating protein; ARF, ADP-ribosylation factor; SHD, Spa2 homology domain; GEF, guanine nucleotide exchange factor; FAK, focal adhesion kinase; EGFP, enhanced green fluorescent protein; aa, amino acid(s); CMV, cytomegalovirus; GST, glutathione S-transferase; PBS, paxillin-binding subdomain; GBD, GIT-binding domain; RU, resonance units; DIV, days in vitro; EM, electron microscopy; hemagglutinin.
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REFERENCES |
---|
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---|
1. | Landis, D. M. (1988) J. Electron Microsc. Tech. 10, 129-151[Medline] [Order article via Infotrieve] |
2. | Garner, C. C., Kindler, S., and Gundelfinger, E. D. (2000) Curr. Opin. Neurobiol. 10, 321-327[CrossRef][Medline] [Order article via Infotrieve] |
3. | Dresbach, T., Qualmann, B., Kessels, M. M., Garner, C. C., and Gundelfinger, E. D. (2001) Cell. Mol. Life Sci. 58, 94-116[Medline] [Order article via Infotrieve] |
4. | Cases-Langhoff, C., Voss, B., Garner, A. M., Appeltauer, U., Takei, K., Kindler, S., Veh, R. W., De, Camilli, P., Gundelfinger, E. D., and Garner, C. C. (1996) Eur. J. Cell Biol. 69, 214-223[Medline] [Order article via Infotrieve] |
5. | Fenster, S. D., Chung, W. J., Zhai, R., Cases-Langhoff, C., Voss, B., Garner, A. M., Kaempf, U., Kindler, S., Gundelfinger, E. D., and Garner, C. C. (2000) Neuron 25, 203-214[Medline] [Order article via Infotrieve] |
6. |
Wang, X.,
Kibschull, M.,
Laue, M. M.,
Lichte, B.,
Petrasch-Parwez, E.,
and Kilimann, M. W.
(1999)
J. Cell Biol.
147,
151-162 |
7. |
tom Dieck, S.,
Sanmarti-Vila, L.,
Langnaese, K.,
Richter, K.,
Kindler, S.,
Soyke, A.,
Wex, H.,
Smalla, K. H.,
Kampf, U.,
Franzer, J. T.,
Stumm, M.,
Garner, C. C.,
and Gundelfinger, E. D.
(1998)
J. Cell Biol.
142,
499-509 |
8. | Wang, Y., Okamoto, M., Schmitz, F., Hofmann, K., and Sudhof, T. C. (1997) Nature 388, 593-598[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Brose, N.,
Hofmann, K.,
Hata, Y.,
and Sudhof, T. C.
(1995)
J. Biol. Chem.
270,
25273-25280 |
10. |
Ohtsuka, T.,
Takao-Rikitsu, E.,
Inoue, E.,
Inoue, M.,
Takeuchi, M.,
Matsubara, K.,
Deguchi-Tawarada, M.,
Satoh, K.,
Morimoto, K.,
Nakanishi, H.,
and Takai, Y.
(2002)
J. Cell Biol.
158,
577-590 |
11. |
Wang, Y.,
Liu, X.,
Biederer, T.,
and Sudhof, T. C.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
14464-14469 |
12. |
Martincic, I.,
Peralta, M. E.,
and Ngsee, J. K.
(1997)
J. Biol. Chem.
272,
26991-26998 |
13. |
Fujimoto, K.,
Shibasaki, T.,
Yokoi, N.,
Kashima, Y.,
Matsumoto, M.,
Sasaki, T.,
Tajima, N.,
Iwanaga, T.,
and Seino, S.
(2002)
J. Biol. Chem.
277,
50497-50502 |
14. | Zhai, R. G., Vardinon-Friedman, H., Cases-Langhoff, C., Becker, B., Gundelfinger, E. D., Ziv, N. E., and Garner, C. C. (2001) Neuron 29, 131-143[Medline] [Order article via Infotrieve] |
15. |
Premont, R. T.,
Claing, A.,
Vitale, N.,
Freeman, J. L.,
Pitcher, J. A.,
Patton, W. A.,
Moss, J.,
Vaughan, M.,
and Lefkowitz, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14082-14087 |
16. |
Bagrodia, S.,
Bailey, D.,
Lenard, Z.,
Hart, M.,
Guan, J. L.,
Premont, R. T.,
Taylor, S. J.,
and Cerione, R. A.
(1999)
J. Biol. Chem.
274,
22393-22400 |
17. | Di Cesare, A., Paris, S., Albertinazzi, C., Dariozzi, S., Andersen, J., Mann, M., Longhi, R., and de Curtis, I. (2000) Nat. Cell Biol. 2, 521-530[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Turner, C. E.,
Brown, M. C.,
Perrotta, J. A.,
Riedy, M. C.,
Nikolopoulos, S. N.,
McDonald, A. R.,
Bagrodia, S.,
Thomas, S.,
and Leventhal, P. S.
(1999)
J. Cell Biol.
145,
851-863 |
19. |
Premont, R. T.,
Claing, A.,
Vitale, N.,
Perry, S. J.,
and Lefkowitz, R. J.
(2000)
J. Biol. Chem.
275,
22373-22380 |
20. | Turner, C. E., West, K. A., and Brown, M. C. (2001) Curr. Opin. Cell Biol. 13, 593-599[CrossRef][Medline] [Order article via Infotrieve] |
21. | Donaldson, J. G., and Jackson, C. L. (2000) Curr. Opin. Cell Biol. 12, 475-482[CrossRef][Medline] [Order article via Infotrieve] |
22. | Jackson, T. R., Kearns, B. G., and Theibert, A. B. (2000) Trends Biochem. Sci. 25, 489-495[CrossRef][Medline] [Order article via Infotrieve] |
23. | Chavrier, P., and Goud, B. (1999) Curr. Opin. Cell Biol. 11, 466-475[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Claing, A.,
Perry, S. J.,
Achiriloaie, M.,
Walker, J. K.,
Albanesi, J. P.,
Lefkowitz, R. J.,
and Premont, R. T.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1119-1124 |
25. |
Zhao, Z. S.,
Manser, E.,
Loo, T. H.,
and Lim, L.
(2000)
Mol. Cell. Biol.
20,
6354-6363 |
26. | Kim, E., Niethammer, M., Rothschild, A., Jan, Y. N., and Sheng, M. (1995) Nature 378, 85-88[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Kim, S.,
Lee, S. H.,
and Park, D.
(2001)
J. Biol. Chem.
276,
10581-10584 |
28. |
Wyszynski, M.,
Valtschanoff, J. G.,
Naisbitt, S.,
Dunah, A. W.,
Kim, E.,
Standaert, D. G.,
Weinberg, R.,
and Sheng, M.
(1999)
J. Neurosci.
19,
6528-6537 |
29. |
Choi, J., Ko, J.,
Park, E.,
Lee, J. R.,
Yoon, J.,
Lim, S.,
and Kim, E.
(2002)
J. Biol. Chem.
277,
12359-12363 |
30. | Dick, O., Hack, I., Altrock, W. D., Garner, C. C., Gundelfinger, E. D., and Brandstatter, J. H. (2001) J. Comp. Neurol. 439, 224-234[CrossRef][Medline] [Order article via Infotrieve] |
31. | Goslin, K., and Banker, G. (1991) Culturing Nerve Cells , The MIT Press, Cambridge, MA |
32. |
Andreev, J.,
Simon, J. P.,
Sabatini, D. D.,
Kam, J.,
Plowman, G.,
Randazzo, P. A.,
and Schlessinger, J.
(1999)
Mol. Cell. Biol.
19,
2338-2350 |
33. | Wyszynski, M., Kim, E., Dunah, A. W., Passafaro, M., Valtschanoff, J. G., Serra-Pages, C., Streuli, M., Weinberg, R. J., and Sheng, M. (2002) Neuron 34, 39-52[Medline] [Order article via Infotrieve] |
34. | Kim, S., Kim, T., Lee, D., Park, S. H., Kim, H., and Park, D. (2000) Biochem. Biophys. Res. Commun. 272, 721-725[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Koh, C. G.,
Manser, E.,
Zhao, Z. S., Ng, C. P.,
and Lim, L.
(2001)
J. Cell Sci.
114,
4239-4251 |
36. | Kim, E., Cho, K. O., Rothschild, A., and Sheng, M. (1996) Neuron 17, 103-113[Medline] [Order article via Infotrieve] |
37. | Naisbitt, S., Kim, E., Tu, J. C., Xiao, B., Sala, C., Valtschanoff, J., Weinberg, R. J., Worley, P. F., and Sheng, M. (1999) Neuron 23, 569-582[Medline] [Order article via Infotrieve] |
38. |
Dong, H.,
Zhang, P.,
Song, I.,
Petralia, R. S.,
Liao, D.,
and Huganir, R. L.
(1999)
J. Neurosci.
19,
6930-6941 |
39. |
Srivastava, S.,
and Ziff, E. B.
(1999)
Ann. N. Y. Acad. Sci.
868,
561-564 |
40. |
Manabe Ri, R.,
Kovalenko, M.,
Webb, D. J.,
and Horwitz, A. R.
(2002)
J. Cell Sci.
115,
1497-1510 |
41. | Serra-Pages, C., Kedersha, N. L., Fazikas, L., Medley, Q., Debant, A., and Streuli, M. (1995) EMBO J. 14, 2827-2838[Abstract] |
42. |
Serra-Pages, C.,
Medley, Q. G.,
Tang, M.,
Hart, A.,
and Streuli, M.
(1998)
J. Biol. Chem.
273,
15611-15620 |
43. | Zhen, M., and Jin, Y. (1999) Nature 401, 371-375[CrossRef][Medline] [Order article via Infotrieve] |
44. | Kaufmann, N., DeProto, J., Ranjan, R., Wan, H., and Van Vactor, D. (2002) Neuron 34, 27-38[Medline] [Order article via Infotrieve] |
45. | Krueger, N. X., Van Vactor, D., Wan, H. I., Gelbart, W. M., Goodman, C. S., and Saito, H. (1996) Cell 84, 611-622[Medline] [Order article via Infotrieve] |
46. | Clandinin, T. R., Lee, C. H., Herman, T., Lee, R. C., Yang, A. Y., Ovasapyan, S., and Zipursky, S. L. (2001) Neuron 32, 237-248[Medline] [Order article via Infotrieve] |
47. | Maurel-Zaffran, C., Suzuki, T., Gahmon, G., Treisman, J. E., and Dickson, B. J. (2001) Neuron 32, 225-235[Medline] [Order article via Infotrieve] |
48. | Schoch, S., Castillo, P. E., Jo, T., Mukherjee, K., Geppert, M., Wang, Y., Schmitz, F., Malenka, R. C., and Sudhof, T. C. (2002) Nature 415, 321-326[CrossRef][Medline] [Order article via Infotrieve] |
49. | Bruckner, K., Pablo Labrador, J., Scheiffele, P., Herb, A., Seeburg, P. H., and Klein, R. (1999) Neuron 22, 511-524[Medline] [Order article via Infotrieve] |
50. |
Lin, D.,
Gish, G. D.,
Songyang, Z.,
and Pawson, T.
(1999)
J. Biol. Chem.
274,
3726-3733 |
51. | Torres, R., Firestein, B. L., Dong, H., Staudinger, J., Olson, E. N., Huganir, R. L., Bredt, D. S., Gale, N. W., and Yancopoulos, G. D. (1998) Neuron 21, 1453-1463[Medline] [Order article via Infotrieve] |
52. | Ye, B., Liao, D., Zhang, X., Zhang, P., Dong, H., and Huganir, R. L. (2000) Neuron 26, 603-617[Medline] [Order article via Infotrieve] |
53. | Dong, H., O'Brien, R. J., Fung, E. T., Lanahan, A. A., Worley, P. F., and Huganir, R. L. (1997) Nature 386, 279-284[CrossRef][Medline] [Order article via Infotrieve] |
54. | Srivastava, S., Osten, P., Vilim, F. S., Khatri, L., Inman, G., States, B., Daly, C., DeSouza, S., Abagyan, R., Valtschanoff, J. G., Weinberg, R. J., and Ziff, E. B. (1998) Neuron 21, 581-591[Medline] [Order article via Infotrieve] |
55. | Parnas, D., Haghighi, A. P., Fetter, R. D., Kim, S. W., and Goodman, C. S. (2001) Neuron 32, 415-424[Medline] [Order article via Infotrieve] |
56. | Girault, J. A., Costa, A., Derkinderen, P., Studler, J. M., and Toutant, M. (1999) Trends Neurosci. 22, 257-263[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Vitale, N.,
Patton, W. A.,
Moss, J.,
Vaughan, M.,
Lefkowitz, R. J.,
and Premont, R. T.
(2000)
J. Biol. Chem.
275,
13901-13906 |
58. | Radhakrishna, H., Klausner, R. D., and Donaldson, J. G. (1996) J. Cell Biol. 134, 935-947[Abstract] |
59. |
Song, J.,
Khachikian, Z.,
Radhakrishna, H.,
and Donaldson, J. G.
(1998)
J. Cell Sci.
111,
2257-2267 |
60. |
Radhakrishna, H.,
and Donaldson, J. G.
(1997)
J. Cell Biol.
139,
49-61 |
61. |
Zhang, Q.,
Cox, D.,
Tseng, C. C.,
Donaldson, J. G.,
and Greenberg, S.
(1998)
J. Biol. Chem.
273,
19977-19981 |
62. |
Al-Awar, O.,
Radhakrishna, H.,
Powell, N. N.,
and Donaldson, J. G.
(2000)
Mol. Cell. Biol.
20,
5998-6007 |
63. |
Frank, S. R.,
Hatfield, J. C.,
and Casanova, J. E.
(1998)
Mol. Biol. Cell
9,
3133-3146 |
64. | Hernandez-Deviez, D. J., Casanova, J. E., and Wilson, J. M. (2002) Nat. Neurosci. 5, 623-624[Medline] [Order article via Infotrieve] |
65. |
Ashery, U.,
Koch, H.,
Scheuss, V.,
Brose, N.,
and Rettig, J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1094-1099 |