From the Department of Physiology and Program in Neuroscience, University of California at San Francisco, San Francisco, California 94143-0444
Received for publication, October 4, 2002, and in revised form, December 6, 2002
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ABSTRACT |
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Membrane-associated guanylate kinases (MAGUKs)
regulate cellular adhesion and signal transduction at sites of
cell-cell contact. MAGUKs are composed of modular protein-protein
interaction motifs including L27, PDZ, Src homology (SH) 3, and
guanylate kinase domains that aggregate adhesion molecules and
receptors. Genetic analyses reveal that lethal mutations of MAGUKs
often occur in the guanylate kinase domain, indicating a critical role
for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the
MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid
residues in the GMP binding pocket that differ between MAGUKs and
authentic guanylate kinase explain this lack of binding, as swapping
these residues largely prevent GMP binding to yeast guanylate kinase.
Conversely, these mutations restore GMP binding but not catalytic
activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase
domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear
not to interact with the canonical GMP binding pocket, and GMP binding
does not influence the intramolecular SH3/guanylate kinase (GK)
interaction within PSD-95. These studies indicate that MAGUK proteins
have lost affinity for GMP but may have retained the guanylate kinase
structure to accommodate a related regulatory ligand.
Tissue development, differentiation, and physiology require
specialized cellular adhesion and signal transduction at sites of
cell-cell contact. Scaffolding proteins that tether adhesion molecules,
receptors, and intracellular signaling enzymes organize macromolecular
protein complexes at cellular junctions to integrate these functions.
One family of such scaffolding proteins is the large group of membrane
associated guanylate kinases
(MAGUKs)1 (1-4). Genetic
studies have highlighted the critical roles for MAGUK proteins in the
development and physiology of numerous tissues from a variety of
metazoan organisms. Mutation of Drosophila discs large
(dlg) disrupts epithelial septate junctions and causes
overgrowth of the imaginal discs (5). Similarly, mutation of
lin-2, a related MAGUK in Caenorhabditits
elegans, blocks vulval development (2), and mutation of the
postsynaptic density protein PSD-95 impairs synaptic plasticity in
mammalian brain (6).
MAGUK proteins are composed of a set of protein-protein interaction
domains that explain their important roles in organizing protein
complexes at sites of cell-cell contact. MAGUKs contain L27
heterodimerization domains, one or three PDZ domains, an SH3 domain and
a C-terminal domain homologous to guanylate kinase (GK) (7, 8). PDZ
domains are small modular motifs that contain a single peptide binding
groove that associates with specific sequences often found at the
extreme C termini of interacting ion channels (9, 10), cell adhesion
molecules (11, 12), and other membrane-associated proteins (13-16).
Multiple PDZ domains in PSD-95 organize and accelerate signal
transduction at synapses by linking receptors to downstream signaling
enzymes (17, 18).
Whereas the functional roles for PDZ domains in MAGUKs are well
established, functions for the SH3 and GK domains are less certain. SH3
domains classically bind to proline-rich motifs (19-23); however, the
structure of the PSD-95 SH3 domain suggests that such interactions are
unlikely because a conserved helix in MAGUK SH3 domains occludes the
canonical polyproline binding site (24, 25). A variety of high affinity
protein ligands have been identified for GK domains from several MAGUKs
(26-30), but it is not yet clear how these interactions regulate MAGUK
functions. In addition to binding exogenous ligands, protein fragments
containing the proposed SH3 and GK regions of MAGUK proteins interact
with each other (31-34). The crystal structure of the SH3GK region of
PSD-95 reveals that this interaction is the assembly of the SH3 fold
from discontinuous structural components (24, 25). This SH3/GK
interaction, may oligomerize MAGUK scaffolds, but factors that regulate
intermolecular SH3 assembly remain uncertain. Despite our limited
understanding of the biochemical roles for the SH3 and GK domains,
these regions are clearly critical as most genetically identified
mutations of MAGUKs occur in the SH3 and GK domains (2, 5).
The GK domains of MAGUKs share 40% sequence homology with authentic GK
enzymes, which phosphorylate GMP to form GDP. The connection of MAGUK
GK domains to guanine nucleotide metabolism is uncertain. Most MAGUKs
lack enzymatic activity, although p55, a MAGUK in red blood cells,
apparently shows modest GK catalysis (35). Rather than having robust GK
activity MAGUKs are suggested to bind potently to GMP (36, 37), which
may then regulate GK domain interactions.
To explore for a regulatory role of GMP on MAGUK function, we have now
quantitated GMP binding using a straightforward and sensitive assay,
equilibrium dialysis. Surprisingly, we found that MAGUK proteins PSD-95
and CASK fail to interact detectably with GMP, whereas yeast GK (YGK)
binds with an affinity of 30 µM. This lack of binding by
MAGUKs is explained in part by two residues in the GMP binding pocket
of YGK that are not conserved in MAGUK proteins. Mutating these
residues in YGK to those in PSD-95 drastically reduces GMP binding by
YGK. Conversely, replacing these two residues in PSD-95 with those
occupying the identical positions in YGK facilitates GMP binding,
albeit of low affinity (~1 mM), but does not confer GK
activity on the double mutant PSD-95 GK domain. This double mutant
PSD-95 construct can still properly assemble the SH3 domain as well as
bind the exogenous GK ligands GKAP and MAP1A, and these protein binding
activities of the double mutant PSD-95 are not competitive with GMP.
These studies demonstrate that MAGUK proteins lack detectable GMP
binding activity and suggest that other related ligands may associate with the canonical GMP binding pocket in MAGUK proteins.
Protein Expression and Purification--
DNA sequences encoding
YGK (residues 2-187), rat PSD-95 (SH3GK, residues 417-724; GK,
residues 532-711), and rat CASK (SH3GK, residues 591-909) were
amplified by PCR and cloned in-frame into a His6-tagged
expression vector (16). Mutations were introduced by site-directed
PCR-based mutagenesis. Escherichia coli strain BL21 (DE3)
(Stratagene) expressing PSD-95 and YGK constructs were grown in LB to
an OD600 = 0.8, induced with 100 µM
isopropyl-1-thio- Equilibrium Dialysis--
Dialysis was performed at room
temperature in 100 mM Tris (pH 7.8), 100 mM
KCl, 5 mM dithiothreitol, and different
concentrations of GMP ranging from 10 nM to 10 mM. Tracer amounts of [8,3'-3H]GMP (Amersham
Biosciences) were included to determine bound and free
concentrations of GMP. Samples were collected after 24 h of
dialysis, and the content of radioactivity was determined using liquid
scintillation. Data were analyzed by nonlinear least square fit to the
following equation,
Guanylate Kinase Assay--
GK activity was measured as
described previously by Agarwal et al. (38). Briefly, 100 µl of reaction buffer consisted of 100 mM Tris (pH 7.6),
100 mM KCl, 10 mM MgCl2, 1.5 mM sodium phospho(enol)pyruvate, 0.25 unit of pyruvate
kinase, 0.33 unit of lactate dehydrogenase, 150 µM
Cell Culture and Transfections--
COS cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Cells were transfected with LipofectAMINE Plus (Invitrogen) according
to the manufacture's protocols. 48 h post-transfection cells were
harvested and solubilized in TEEN (100 mM Tris (pH 7.8),
300 mM NaCl, 2 mM EDTA, 2 mM EGTA)
containing 1% Triton X-100. The soluble fraction was collected
following a 20,000 × g spin for 15 min at 4 °C and
used for in vitro binding assays.
GST Binding Assays--
DNA sequences encoding the wild type and
mutant PSD-95 GK domains and MAP1A (residues 1861-1930) were amplified
by PCR and cloned into pGEX 4T-1 (Amersham Biosciences). The SH3
(residues 417-532) and E-GK-F (residues 523-724) constructs were used
as described previously (24), and the GFP-GKAP construct was subcloned in-frame into the pEGFP-C1 plasmid from a cDNA product kindly provided by M. Sheng (MIT). E. coli strain DH5 We assessed possible binding of [3H]GMP to the SH3GK
domain of PSD-95 by equilibrium dialysis. For a typical experiment, we dialyzed SH3GK or YGK at 100 µM protein concentration in
a volume of 100 µl against a 1-liter volume of buffer containing
[3H]GMP at varying concentrations. When only tracer
concentrations of [3H]GMP were present in the buffer (100 nM), the YGK dialysis compartment accumulated
[3H]GMP levels 3-fold those in the dialysis buffer. On
the other hand, dialysate containing either the SH3GK region of
PSD-95 or of CASK showed no increased accumulation of
[3H]GMP above that in the dialysis buffer.
Previous studies have suggested that the SH3GK domain of PSD-95 binds
GMP with an affinity of 300 nM (36). Such an affinity would
have yielded counts in the dialysis bag ~250-fold above those in the
dialysis buffer in our experiments containing only tracer levels of
[3H]GMP. An affinity as low as 1 mM would
have yielded counts in the dialysis bag 10% above those in the buffer,
and this would have been detectable. We therefore conclude that the
SH3GK domains of PSD-95 and CASK do not bind GMP with affinities <1
mM. Our protein appeared stable during the dialysis, as it
was not degraded when evaluated by SDS-PAGE (Fig.
1A), and dialyzed PSD-95 SH3GK was able to specifically interact with GST-MAP1A (data not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 3 h, harvested by centrifugation, and sonicated in Buffer A (50 mM NaPO4 (pH 8.0), 300 mM NaCl,
10% glycerol). His6-tagged proteins were purified from
bacterial lysates using nickel-nitrilotriacetic acid resin (Qiagen).
Following incubation with bacterial lysates, resin was extensively
washed with 50 mM NaPO4 (pH 6.5), 300 mM NaCl, 10% glycerol, and subsequently with Buffer A
containing 5 and 15 mM imidazole. Proteins were eluted with
Buffer A containing 200 mM imidazole and concentrated with
15 ml Centriprep centrifugal filters (Millipore). Purity was >95% as
determined by Coomassie-stained SDS-PAGE gels.
where Lb and Lf are the
bound and free GMP concentrations, respectively; Pt
is the protein concentration, and Kd is the
dissociation constant.
(Eq. 1)
-nicotinamide adenine dinucleotide, and 4 mM ATP. To
initiate the GK reaction, different amounts of GMP were added to the
reaction buffer containing protein, and the rate of
A340 decay was recorded. Background decay rate
of A340 absorbance was determined by addition of
protein to reaction buffer in the absence of GMP and subtracted from
total reaction rate.
expressing
the various constructs were grown in LB to OD600 = 0.8, induced with 100 µM
isopropyl-1-thio-
-D-galactopyranoside for 3 h, harvested by centrifugation, and sonicated in MTPBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM
KH2PO4, 2 mM EDTA). Approximately 20 µg of GST fusion protein was coupled to glutathione-Sepharose (Amersham Biosciences) beads and washed extensively with MTPBS. Prior to the interaction assays, His6-tagged proteins, COS
cell lysate, and coupled GST proteins were incubated in the presence or
absence of 5 mM GMP for 1 h at 4 °C. Coupled GST
fusion proteins were then incubated with 20 µg of
His6-tagged fusion proteins or 80 µl of COS cell lysate
for 40 min ± 5 mM GMP at 4 °C and extensively
washed with either Buffer A or TEEN ± 5 mM GMP.
Retained proteins were eluted with SDS protein loading buffer,
separated by PAGE, and analyzed by immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
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Fig. 1.
PSD-95 and CASK do not bind GMP or have GK
activity. A, YGK (closed circles) binds GMP
with an affinity of ~29 µM as determined by equilibrium
dialysis. In contrast, GMP binding was not observed for the SH3GK
domains of PSD-95 (open circles) and CASK (closed
triangles). Little to no degradation of protein occurred during
dialysis as shown by the Coomassie-stained gel. B, among the
proteins tested, only YGK was catalytically active
(Km = 41 µM).
By varying the concentrations of [3H]GMP in the dialysis buffer, we determined the affinity for YGK to be ~30 µM (Fig. 1A and Table I), which is similar to that reported by others (39). We also evaluated GK activity and found that YGK was active with a Km of ~40 µM, but that neither PSD-95-SH3GK nor CASK-SH3GK showed catalytic activity (Fig. 1B and Table I).
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The SH3 domain structurally flanks the GK domain in MAGUK proteins
potentially constraining the GK domain from binding GMP. We therefore
expressed the GK domain from PSD-95 alone. Again, we found that the
isolated GK domain failed to bind [3H]GMP (Fig.
2). A previously published crystal
structure of the SH3GK domain of PSD-95 showed that GMP co-crystallized
with PSD-95 only in the presence of 100 mM added guanidine
(25). However, we found that addition of guanidine did not influence
[3H]GMP binding to PSD-95 (Fig. 2).
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The GK domain of PSD-95 shares 40% sequence identity with YGK (Fig.
3A), and the crystal
structures of these domains are similar (24). The crystal structure of
YGK bound to GMP showed that nine residues from the enzyme make
specific contact with the GMP molecule (40). Seven of these nine
residues are conserved in the GK domains of PSD-95 and CASK (Fig. 3,
A and B). To test whether these amino acid
differences explained the failure of PSD-95 or CASK GK domains to bind
GMP, we first mutated one or both of these residues in YGK and
evaluated the effects on GMP binding and GK activity. Strikingly, we
found that mutation of serine 35 in YGK to proline, which resembles the
residue in PSD-95 and is conserved within all PSD-95 members, reduced
the affinity for GMP by 40-fold (Table I). This mutation also
drastically reduced the guanylate kinase activity nearly 1000-fold
(Fig. 4 and Table I). A double mutation
of YGK, changing both serine 35 to proline and aspartic acid 101 to
serine to mimic that in PSD-95, yielded an enzyme with compromised
binding properties (Table I) and GK catalytic activity similar to the
single mutation (Fig. 4).
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These changes suggested that differences at these crucial residues may explain our failure to detect [3H]GMP binding to PSD-95. Therefore, we mutated these residues in the SH3GK domain of PSD-95 to resemble those in YGK. This double mutant of PSD-95 showed very weak binding to GMP (Kd = 1.3 mM; Table I). This mutant, however, lacked guanylate kinase activity (Fig. 4).
Rather than binding to GMP, PSD-95 interacts with several synaptic
proteins, including GKAP/SAPAP (synapse-associated protein-associated protein) (29, 30) and MAP1A (26). We therefore asked whether the
canonical GMP binding pocket in PSD-95 might be the site for interaction with these proteins. To evaluate this possibility, we
examined binding of GKAP to PSD-95 GK constructs containing the
mutations in the GMP binding pocket that permit low affinity GMP
binding. As previously reported, we found that the isolated GK domain
of PSD-95 expressed as a GST fusion protein binds to GKAP protein
expressed in heterologous cells in a "pull-down" assay. The double
mutation of the GK domain of PSD-95 that permits binding to GMP does
not abolish binding to GKAP (Fig.
5A). Furthermore, addition of
high concentrations of GMP, which bind to this mutant PSD-95 construct,
do not compete with GKAP binding, suggesting that GMP and these protein
ligands interact noncompetitively (Fig. 5A). We did similar
experiments to evaluate binding of the GK domain of PSD-95 to a GST
fusion of MAP1A. Again, we found that mutations that enable PSD-95 to
bind GMP do not impair binding to MAP1A and that addition of 5 mM GMP does not disrupt binding of wild type or mutant
PSD-95 to MAP1A (Fig. 5B).
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We also asked whether the GMP binding site might regulate assembly of
the SH3 domain, which mediates the previously observed interaction of
the SH3 and GK domains (24). As previously reported (24),
intermolecular interaction of SH3 and GK domains in vitro is
only observed when the SH3 is split and subdomains are expressed in
separate molecules (Fig. 5C). The disrupted SH3 domain does not interact with a complete SH3GK module, because intramolecular assembly is favored (Fig. 5C). Similarly, an SH3GK construct
containing the double mutations in the GMP binding site also
preferentially assembled in an intramolecular manner as it failed to
bind to the partial SH3 domain. Addition of GMP failed to promote an
intermolecular SH3/GK interactions (Fig. 5C), suggesting
that the GMP binding site is not involved in assembly of SH3GK modules.
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DISCUSSION |
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This study shows that the GK domains of MAGUK proteins PSD-95 and CASK do not bind to GMP with detectable affinity. This lack of binding is explained in part by two residues that differ between MAGUK proteins and authentic GK enzymes. However, even a PSD-95 protein with "back mutations" to contain these residues shows only weak binding, which suggests that other differences between MAGUK proteins and authentic GK also contribute to the lack of binding. Our study also suggests that the residues involved in GMP binding likely do not contribute to interactions of MAGUK proteins with certain neuronal protein ligands, including GKAP and MAP1A.
Previous studies suggested that MAGUK proteins bind to GMP with very high affinity (36, 37). One study showed that full-length PSD-95 as a GST fusion protein bound to a Cibacron blue dye column and could be eluted from such a column with 1 µM GMP (36). We repeated those experiments and indeed found that PSD-95 either expressed in bacteria or from brain extracts does bind to a Cibacron blue dye column. However, we were unable to elute PSD-95 with concentrations of GMP up to 1 mM (data not shown). Reasons for this discrepancy are unclear; it is conceivable that differences in the Cibacron blue preparation might explain the disparate results. Another study used equilibrium dialysis and found that the GK region of CASK binds to [3H]GMP with an affinity of 300 nM (37). This high affinity, which is 100-fold greater than that of YGK for GMP, is very difficult to reconcile with our results. Unfortunately, the binding reported in that study (37) was presented as "data not shown," so it is impossible for us to compare our conditions with theirs.
Conceivably, the GK constructs used in our study could have failed to bind GMP, because they were inactivated during purification. We feel this is unlikely for three reasons. First, our proteins migrated as single bands by SDS-PAGE and were not degraded during the dialysis experiments. Second, we used very mild conditions for purification of these proteins from bacteria. Third, our GK constructs retained binding to other ligands such as GKAP and MAP1A. Another possibility is that GMP may have been tightly bound to our constructs purified from bacteria; therefore, the site would have been unavailable for binding to exogenous GMP. This is also unlikely because the SH3GK protein used here is identical to that which we previously crystallized and found no GMP in the canonical binding site (24).
Crystal structures of GK domains from PSD-95 and CASK have yielded ambiguous results concerning GMP binding (24, 25, 37). For all three published structures, GMP did not co-purify with the GK domain from bacteria and was not found in the native structure. Attempts to co-crystallize CASK with added GMP were unsuccessful (37). For PSD-95, co-crystallization with GMP was achieved only in the presence of 100 mM guanidine (25), an additive screened to obtain high quality crystals. Indeed, a guanidine bound near the GMP and formed one of the crucial hydrogen bonds with the phosphate group of GMP and with an Asp residue from a crystallographically related GK molecule (25). Based on these results and our new findings, GMP binding in the crystal likely occurred as a result of the crystallization conditions and likely has no biological relevance.
Rather than binding GMP, the GK domain of MAGUK proteins associate with specific protein ligands such as GKAP (29, 30), MAP1A (26), BEGAIN (41), GAKIN (28), and SPAR (42). As no crystal structure has yet been solved for a MAGUK bound to a protein ligand, the binding site(s) for these protein ligands remain(s) uncertain. One reasonable location would be the crevice into which GMP sits in the structure of YGK. Our experiments suggest that this is not the site for binding to MAP1A or GKAP. That is, mutation of two residues in this binding pocket that produces weak binding to GMP do not abolish binding to MAP1A or GKAP. Furthermore, adding high concentrations of GMP to these mutants, which should occupy the nucleotide binding site, does not prevent additional contemporaneous binding of MAP1A or GKAP. This does not exclude the possibility that MAP1A and GKAP interactions are not disrupted, because the GMP binding affinity is too low to efficiently compete. Future structural studies of MAGUK proteins complexed with ligands would seem essential to determine decisively binding sites for these protein partners.
Why might the GK structure be so tightly conserved in MAGUK proteins? This conservation includes all four residues that coordinate the phosphate of GMP and three of five residues that bind the guanine base. This may suggest that MAGUK proteins interact with a distinct phosphorylated nucleotide or even a phosphorylated residue from a polypeptide. Our results here showing that back mutations of the residues in PSD-95 that differ from GK restore weak binding suggests that MAGUK proteins may have lost their ability to bind GMP in favor of a distinct ligand.
In addition to binding exogenous ligands, the SH3 and GK domains of
MAGUK proteins mediate a unique intramolecular association (31-34).
This SH3/GK interaction actually reflects assembly of the unique MAGUK
SH3 domain, which has a peculiar pair of -strands that surround the
GK domain (24, 25). Interestingly, this assembly of MAGUK SH3 domains
can occur in both an intramolecular as well as an intermolecular
fashion and can thereby mediate a regulated oligomerization of MAGUK
proteins (32, 33, 43). It is unlikely that residues of the NBD are
directly involved in intermolecular assembly, as the GK double mutant
retains the ability to interact with the SH3 domain (data not shown).
Instead, this swapping between monomer and MAGUK oligomer may be
regulated by protein interactions or increased rigidity of the HOOK
insert within the SH3 domain (24, 32). It is conceivable that
conformational changes associated with binding sites in the GK domain
of MAGUKs could also induce oligomerization.
Although biochemical functions for the SH3GK region of MAGUK proteins
remain uncertain, genetic studies indicate that this region is critical
for MAGUK function (2, 5). A single point mutation in the SH3 domain of
Drosophila discs large yields a lethal phenotype,
as do small truncations in the GK domain (5). Furthermore, truncations
of the GK domain of lin-2 in C. elegans cause a
vulvaless phenotype (2). These mutations all disrupt the previously
identified SH3/GK interaction, suggesting a critical role for that mode
of protein assembly (31). Whether the largely conserved GMP binding
pocket also is essential for MAGUK protein function has not yet been
investigated at the genetic level. Future biochemical and genetic
studies of this region are warranted. Also critical will be to
determine whether another nucleotide or other phosphorylated molecule
occupies this pocket and how this binding controls MAGUK protein function.
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ACKNOWLEDGEMENTS |
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We thank A. McGee, S. Dakoji, and K. Moore for discussions and technical help.
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FOOTNOTES |
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* This work was supported by grants (to D. S. B. and O. O.) from the National Institutes of Health and from the Christopher Reeves Paralysis Foundation (to D. S. B.) and by the Human Frontier Research Program.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.
Established investigator for the American Heart Association. To
whom all correspondence should be addressed: University of California
at San Francisco School of Medicine, 513 Parnassus Ave., San Francisco,
CA 94143-0444. Tel.: 415-476-6310; Fax: 415-476-4929; E-mail:
bredt@itsa.ucsf.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210165200
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ABBREVIATIONS |
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The abbreviations used are: MAGUK, membrane-associated guanylate kinase; PSD-95, postsynaptic density-95; GK, guanylate kinase; YGK, yeast GK; GKAP, guanylate kinase-associated protein; MAP1A, microtubule-associated protein 1A; GFP, green fluorescent protein; GST, glutathione S-transferase; SH, Src homology.
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