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INTRODUCTION |
Neurogranin (RC3, BICKS) is a postnatal expressed brain-specific
protein that is localized postsynaptically in neuronal cell bodies and
dendrites of the cerebral cortex, hippocampus, and striatum (1-5). It
was first identified by subtractive hybridization in a screen for
mRNAs enriched in rat forebrain and was independently purified from
brain as a protein kinase C
(PKC)1 substrate (6-9).
Neurogranin possesses sequence similarity to neuromodulin (GAP-43),
another brain-specific PKC substrate located presynaptically in axons
and growth cones (10-12). Neurogranin and neuromodulin share high
sequence homology within 20 amino acids designated as the IQ motif (11,
13). This sequence, AAAAKIQASFRGHMARKKIK in neurogranin, contains a
binding domain for CaM and a PKC phosphorylation site (8, 14, 15). The interaction between CaM and the IQ domains of neurogranin and neuromodulin has been characterized extensively in vitro (8, 15-20). CaM binds to both proteins with equivalent or higher affinity in the absence of Ca2+ compared with the presence of
Ca2+. PKC phosphorylation within the IQ domain inhibits CaM
binding, and conversely, CaM binding inhibits PKC phosphorylation at
the serine within the IQ domain (21, 22). These observations led to the
general hypothesis that neuromodulin and neurogranin may bind and
concentrate CaM at specific sites in neurons and that PKC
phosphorylation or changes in Ca2+ may result in the
release of CaM (16, 23).
Although the physiological functions of neurogranin have not been
defined, its biochemical properties and postsynaptic localization have
implicated it in several signal transduction pathways. For example,
both neurogranin and neuromodulin have been shown to regulate
CaM-dependent nitric oxide synthase activity through sequestration of CaM (24, 25). Conversely, nitric oxide modifies neurogranin, reducing its ability to bind CaM or to be phosphorylated by PKC (26). Neurogranin and neuromodulin are also in vitro substrates for phosphorylase kinase and may interact with membrane phospholipids (27-29). It has also been hypothesized that neuromodulin and neurogranin may play pivotal roles in LTP by releasing CaM. Free
CaM could then activate enzymes, including CaM kinases or adenylyl
cyclases, that regulate synaptic plasticity (30, 31).
The hypothesis that neurogranin may regulate postsynaptic CaM levels is
based upon data showing that the two proteins interact in
vitro. However, it has not been demonstrated that neurogranin binds CaM in vivo, and the possibility that neurogranin may
interact with other proteins has not been investigated. Here, we
utilized yeast two-hybrid technology to detect neurogranin-binding
proteins and to characterize neurogranin/CaM interactions in
vivo.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes, T4 DNA ligase, and
-agarase were purchased from New England Biolabs. The TA cloning kit
was from Invitrogen. The ABI Prism sequencing kit was from
Perkin-Elmer. Centri-Sep spin columns were from Princeton Separations
Inc. The Quick-change site-directed mutagenesis kit was from
Stratagene. Yeast media was purchased from Difco. Sheared DNA and Lex A
antibody were from CLONTECH. X-Gal was purchased
from 5 Prime
3 Prime, Inc., Boulder, CO. Amino acids and other
chemicals were from Sigma.
Cell Culture--
HEK-293 cells were maintained in
Hepes-buffered Dulbecco's modified Eagle's medium plus 10% fetal
bovine serum and 1% penicillin and streptomycin in a humidified 95%
O2/5% CO2 incubator.
Yeast Media--
Yeast were grown on YPAD or YC solid or liquid
media containing specific amino acids (32).
Preparation of Neurogranin and Calmodulin Constructs--
The
coding sequence of rat neurogranin was polymerase chain
reaction-amplified with the addition of new BamHI and
EcoRI restriction sites using the neurogranin primers
5'-CCCCGAATTCATGGACTGCTGC-3' and 5'-CAATGGATCCTTAATCTCCGCTG-3'. The
polymerase chain reaction product was cloned into a TA vector and then
digested with BamHI and EcoRI. The gel-isolated
insert was ligated into the two-hybrid BTM116 Lex A DNA-binding domain
vector (33) or pcDNA 3.1 (Invitrogen). The coding sequence of a
synthetic mammalian CaM (34) was polymerase chain reaction-amplified
with the addition of two flanking BamHI sites using the CaM
primers 5'-CCCCGGATCCGGATGGCTGACCAACTCACC-3' and
5'-CCCCGGATCCTCACTTAGCCGTCATC-3'. The insert was cloned into a TA
vector, removed by digestion with BamHI, and was ligated into the VP16 activation domain vector (35). Each construct was
sequenced to ensure accuracy during cloning.
Site-directed Mutagenesis--
Specific amino acids were mutated
within the IQ domain of the BTM116-neurogranin wild-type construct.
Oligonucleotides were designed to introduce the following
changes: Ile-33
Gln,
5'-CCGCTGCAGCCAAACAACAGGCGAGTTTTCGGGGCCATATGGCGAGG-3' and
5'-CCTCGCCATATGGCCCCGAAAACTCGCCTGTTGTTTGGCTGCAGCGG-3'; Arg-38
Gln,
5'-GCCAAAATCCAGGCGAGTTTTCAGGGCCATATGGCGAGG-3' and
5'-CCTCGCCATATGGCCCTGAAAACTCGCCTGGATTTTGGC-3'; Ser-36
Asp,
5'-GCCAAAATCCAGGCGGATTTTCGGGGCCATATGGCGAGG-3' and 5'-CCTCGCCATATGGCCCCGAAAATCCGCCTGGATTTTGGC-3'; Ser-36
Ala,
5'-GCCAAAATCCAGGCGGCTTTTCGGGGCCATATGGCGAGG-3' and
5'-CCTCGCCATATGGCCCCGAAAAGCCGCCTGGATTTTGGC-3'. Point mutants were
made using the quick-change site-directed mutagenesis kit following
manufacturer's directions. Mutations were confirmed by restriction
digest and sequencing.
Yeast Transformations--
Small and large scale yeast
transformations and two-hybrid screens were performed essentially as
described (32). Briefly, for small scale transformations,
BTM116-neurogranin wild-type and mutant constructs were transformed
into the L40 strain of yeast in combination with VP16-CaM. Positive
transformants were grown for 2 days on YC media lacking leucine and
tryptophan (
Leu/
Trp) and were then streaked to YC media lacking
histidine (
His) or again to
Leu/
Trp. Growth was assayed on the
His plates after 2 days. Filter lifts and
-galactosidase assays
were performed on the yeast re-streaked to the
Leu/
Trp media. For
large scale transformations and two-hybrid screens, BTM116-neurogranin
wild-type and mutant constructs were transformed singly into L40 and
were grown on YC media lacking tryptophan (
Trp) for 2 days. A single positive transformant colony was grown in 10 ml of liquid
Trp YC
media overnight. The culture was diluted into 100 ml of
Trp YC media
for another 24 h and was then diluted to an OD of 0.4 in YPAD
media. Large scale transformations were performed using either a rat
brain library (CLONTECH) or a mouse brain library (36). Library cDNAs were isolated from colonies that grew in the
absence of histidine and had
-galactosidase activity. To check for
specificity, each cDNA was retested in a mating assay against
BTM116 lamin (37) and against the original bait construct. cDNAs
that retested as positive were sequenced and identified.
Western Blotting--
Western blotting was utilized to
demonstrate appropriate expression of wild-type and mutant neurogranin
constructs in yeast. The constructs were transformed singly into the
L40 strain of yeast, and positive transformants were grown in YC media
(
Trp) for 2 days. The cultures were pelleted at 1200 × g and were rinsed once in 10 mM Hepes, pH 7.5. Cultures were re-pelleted and resuspended in an ice-cold lysis buffer
composed of 10 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM dithiothreitol,
2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin,
and 20 µg/ml leupeptin. Acid-washed beads (Sigma) were added, and
cultures were vortexed 5 × 30 s each. Tubes were returned to
ice following each vortex. The yeast lysate was separated from the
beads by centrifugation at 100 × g. Lysates were
re-centrifuged, and the supernatant was saved. The Pierce BCA kit was
used to assay protein content in each sample, and SDS-polyacrylamide
gel electrophoresis sample buffer was added. Fifteen µg of protein
were loaded per well and were electrophoresed on 12.5%
SDS-polyacrylamide gel electrophoresis. The proteins were transferred
to nitrocellulose, and membranes were blocked in 5% non-fat milk.
Western blotting was carried out using a Lex A primary antibody
(CLONTECH) at 20 ng/ml for 2 h. Membranes were
washed extensively in phosphate-buffered saline plus 0.2% Tween 20. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody
was added at a 1:7,500 dilution of a 1 mg/ml stock for 1 h.
Membranes were washed again, and proteins were visualized using an ECL
kit (Amersham Pharmacia Biotech).
Quantitative
-Galactosidase Assay--
-Galactosidase
activity was measured using a fluorometric assay (38, 39). Yeast were
transformed with wild-type and mutant neurogranin constructs; lysates
were prepared as described above for Western blotting, and protein
levels were determined. A 15 mg/ml stock solution of the substrate
4-methylumbelliferyl
-D-galactosidase was diluted 1:500
into 0.8× Z buffer (32). One hundred µl of substrate and 10 µl of
yeast extract were added to a microtiter plate and were incubated at
37 oC for 15 min. The amount of yeast lysate added was
adjusted to yield activities within the linear range. The plate was
read on a microfluor microfluorimeter (Dynatech Industries, Inc.).
Values were divided by protein content to yield
-galactosidase
activities. Activity was determined in triplicate for each colony, and
four separate colonies were assayed for each condition.
cAMP Accumulation Assay--
Adenylyl cyclase activity was
measured as changes in intracellular cAMP ± stimulus (40).
Briefly, HEK-293 cells were plated on 12-well polylysine-coated plates
at a density of 2 × 105 cells per well. Transfections
were carried out for 5-6 h using LipofectAMINE (Life Technologies,
Inc.) following the manufacturer's instructions. Each well was
transfected with either pCEP-AC1 (0.125 µg) or pCEP-AC8 (0.125 µg)
in combination with green fluorescent protein (control-0.7 µg) or
pcDNA3-neurogranin (0.7 µg). A
-galactosidase construct (0.08 µg) was transfected and used to normalize transfection efficiency.
Cells were loaded with [3H]adenine in Dulbecco's
modified Eagle's medium plus 10% fetal bovine serum, 12 h prior
to assay. Cells were assayed 48-72 h following transfection.
Radioactive media was aspirated and replaced with Dulbecco's modified
Eagle's medium. Each well was pretreated with 1 mM
isobutylmethylxanthine for 20 min. Cells were then stimulated with 5 µM calcium ionophore, A23187, for 15 min. Reactions were stopped with the addition of 5% trichloroacetic acid plus 1 µM cAMP. Proteins were precipitated at 4 °C for 1-4
h, and acid-soluble nucleotides were separated by sequential Dowex
AG-50W-X4 and neutral alumina chromatography (41). Changes in
intracellular cAMP are reported in triplicate as the ratio of cAMP to
the complete adenine pool (cAMP + ATP + ADP + AMP). Activity was
normalized to
-galactosidase activity.
-Galactosidase Activity in HEK-293
Cells--
-Galactosidase activity was measured using a
galacto-light assay. 293 cells transfected with a pcDNA3
-galactosidase construct were lysed in 4 mM ATP, 100 mM NaPO4, pH 7.8, 6 mM
MgSO4, 1 mM dithiothreitol, and 0.1% Triton
X-100 for 10 min at room temperature. 100 µl of reaction buffer (100 mM Na2HPO4, 2 mM
MgCl2, 10 µl/ml galactone (Tropix), 20 mg/ml
D-galactose) was added to 5-25 µl of cell extract.
Samples were incubated in the dark for 30-60 min. Activity was
measured in a Berthold luminometer using Emerald luminescence
amplifying reagent (Tropix). Each condition was performed in triplicate.
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RESULTS |
Interaction between Neurogranin and CaM in Vivo--
To determine
if neurogranin interacts with CaM in vivo, we utilized yeast
two-hybrid technology. In this system, the interaction of two proteins,
expressed as fusion constructs, is monitored within the confines of a
yeast cellular environment (42). Interactions are measured by read-out
from two transcriptional reporters. Activation of one reporter results
in the production of histidine, allowing yeast to grow in the absence
of this amino acid. Activation of the other reporter construct results
in
-galactosidase activity which is measured by hydrolysis of an
X-Gal substrate that causes yeast to turn blue.
An L40 strain of yeast was transformed with a wild-type neurogranin
DNA-binding domain construct and a CaM activation domain construct as
described under "Experimental Procedures." Positive transformants
were streaked to media lacking histidine and were assayed for growth
(Fig. 1). This assay showed that
neurogranin interacts strongly with CaM in vivo but not with
the activation domain sequence (VP-16) alone. We also monitored
neurogranin/CaM interactions using
-galactosidase activity as a
reporter. Positive double transformants were streaked to media lacking
leucine and tryptophan. The yeast were transferred to nitrocellulose
filters, and
-galactosidase activity was assayed. This assay also
revealed strong neurogranin/CaM interactions that were detectable
within 30 min (data not shown).

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Fig. 1.
Interactions between neurogranin and
CaM in vivo. L40 was transformed with BTM116
neurogranin and VP16 CaM, and positive transformants were streaked to
media lacking histidine. The plates were incubated for 2 days at
30 °C. BTM116 neurogranin (NG) was expressed with
VP16-CaM or VP16 alone. Two independent colonies are shown for each
condition. Results shown are representative of three independent
experiments.
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Interaction between CaM and Neurogranin IQ Domain
Mutants--
Site-specific point mutants were created to determine if
amino acids within the IQ domain of neurogranin are important for its
interaction with CaM in vivo. These point mutants included Ile-33
Gln, Arg-38
Gln, Ser-36
Ala, and Ser-36
Asp. To determine whether the mutants were adequately expressed in yeast, Western analysis was performed on yeast lysates as described under "Experimental Procedures." An antibody that recognizes the Lex A
DNA-binding domain was used to detect the fusion proteins. All of the
neurogranin mutants were expressed and electrophoresed with an apparent
mass of 30 kDa, the expected size for the fusion proteins
(Fig. 2).

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Fig. 2.
Expression of wild-type neurogranin and
neurogranin IQ domain mutants. L40 was transformed singly with
each BTM116-neurogranin construct or with BTM116 alone. Positive
transformants were grown at 30 °C for 36 h in liquid media
lacking tryptophan. Protein lysates were prepared as described under
"Experimental Procedures," and 15 µg of protein was loaded per
lane. Molecular mass markers are in kDa on the left. Lex A
protein is expressed from yeast transformed with the BTM116 vector.
Expression of each of the neurogranin (NG) constructs
results in a fusion protein of the appropriate size, approximately 30.5 kDa. From left to right the constructs are:
BTM116, wild-type BTM116 neurogranin; Ile-33 Gln, Arg-38
Gln, Ser-36 Asp, and Ser-36 Ala.
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Each of the neurogranin mutants was co-expressed with CaM, and the
interaction between the expressed proteins was examined. A range of
interaction strengths was observed by growth on media lacking histidine
(Fig. 3, A and B).
Each mutant was compared with wild-type neurogranin on the same plate.
The best indication of strong interactions is the ability of the yeast
to form single colonies at the interior of each yeast streak. The
Ile-33
Gln fusion protein did not interact with CaM, indicating
that the isoleucine within the IQ domain is particularly important for neurogranin/CaM interactions. The Arg-38
Gln mutant interacted with
CaM, but to a lesser degree than wild-type neurogranin (Fig. 3A).

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Fig. 3.
Interaction of neurogranin IQ domain mutants
with CaM. L40 was transformed with each neurogranin mutant and
VP16 CaM, and positive transformants were streaked to media lacking
histidine. Plates were incubated for 2 days at 30 °C. Each plate is
separated into three regions. Two separate colonies are shown for each
condition. A, the BTM116-wild-type neurogranin and VP16-CaM
interaction is shown as a control. Neurogranin (NG) Ile-33
Gln is on the left, and Arg-38 Gln is shown on the
right. B, the BTM116-wild-type neurogranin and
VP16-CaM interaction is shown as a control. The neurogranin Ser-36 Asp mutant is on the left, and neurogranin Ser-36 Ala is
on the right. Results shown are representative of five
separate experiments.
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In vitro data indicate that the serine within the IQ domain
is a PKC phosphorylation site in neurogranin and other IQ domain proteins (8, 15, 19, 22). When phosphorylated at this site, neurogranin
can no longer interact with CaM in vitro. The two-hybrid
point mutants at Ser-36 were examined to define the importance of this
amino acid for CaM binding and to determine if introduction of negative
charge at this site inhibits CaM binding in vivo. The Ser-36
Asp mutant was created to mimic neurogranin phosphorylated at this
site by introduction of a negative charge. Mutation of the serine to an
alanine (Ser-36
Ala) removes the putative PKC phosphorylation site.
The change from serine to alanine did not affect the neurogranin/CaM
interaction; yeast growth was comparable to that seen with wild-type
neurogranin and CaM (Fig. 3B). Conversion of serine to an
aspartic acid reduced but did not completely inhibit the
neurogranin/CaM interaction. This is consistent with the hypothesis
that introduction of negative charge by phosphorylation at Ser-36 may
reduce CaM binding.
Since the results described above using growth on media lacking
histidine are qualitative, the
-galactosidase reporter was used to
provide a more quantitative evaluation of these interactions (Fig.
4). The data obtained with
-galactosidase expression are consistent with those reported in Fig.
3. The Ile-33
Gln, Arg-38
Gln, and Ser-36
Asp mutants did
not show any interaction with CaM over background (BTM116 alone). Both
wild-type neurogranin and Ser-36
Ala constructs interacted strongly
with CaM. Because of variation between individual colonies and the
sensitivity of the assay, it cannot be concluded that the Ser-36
Ala mutation has a higher affinity for CaM than native neurogranin.
However, the slightly stronger interaction seen with the Ser-36
Ala
mutant may reflect basal phosphorylation of native neurogranin that
lowers the signal somewhat compared with the alanine mutant.

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Fig. 4.
Interaction of wild-type neurogranin and
neurogranin IQ domain mutants with CaM measured by
-galactosidase activity. L40 was transformed
with BTM116 neurogranin (NG) constructs and VP16-CaM.
Positive transformants were grown in liquid media lacking leucine and
tryptophan for 36 h at 30 °C. Yeast lysates were prepared as
described under "Experimental Procedures." The interaction between
each neurogranin construct and CaM was measured by conversion of
4-methylumbelliferyl -D-galactosidase, indicating
-galactosidase activity. Each bar represents the average
of four separate yeast colonies, each measured in triplicate. Errors
are ± S.D.
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Does Neurogranin Interact with Other Proteins in
Vivo?--
Although the data described above indicate that neurogranin
interacts with CaM in vivo, this does not preclude
interactions between neurogranin and other proteins. Therefore, it was
of interest to search for other neurogranin-binding proteins using the
yeast two-hybrid screen. To address this question, we performed large scale two-hybrid screens using wild-type neurogranin as bait and two
different activation domain brain cDNA libraries. A summary of all
large scale neurogranin two-hybrid screens performed is reported in
Table I. Yeast were sequentially
transformed with neurogranin and either a rat brain or a mouse brain
cDNA library. Positive transformants were plated to media lacking
histidine, and histidine-positive colonies were retested for
-galactosidase activity. In the rat brain transformation, 100 histidine-positive colonies were isolated. Of these, all were positive
for
-galactosidase activity, and the color change occurred within 30 min. For the mouse brain transformation, 500 histidine-positive
colonies were retested for
-galactosidase activity. All colonies
retested as positive, with approximately half of the colonies turning
blue within 30 min. cDNAs were isolated from all of the rat brain
positives and from a mixture of 150 strong and weaker positive colonies from the mouse brain transformation.
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Table I
Summary of wild type and mutant neurogranin two-hybrid screens
Steps of each two-hybrid screen are outlined to demonstrate screen
efficiency. Each bait was screened against a rat and/or mouse brain
library. The transformation efficiency is shown as number of
transformants. Each library expresses between 1 × 106 and
3 × 106 independent clones. Histidine-positive
transformants grew on media lacking histidine 48 h following
transformation. These colonies were retested for -galactosidase
( -Gal) activity. Strong activity indicates conversion of X-Gal,
demonstrated by the color blue, in 30-60 min. Any activity seen from
4-24 h was identified as weak. Library and bait plasmids were
segregated. L40 containing the library plasmid was then mated to the
AMR70 strain transformed with the bait plasmid or lamin. Mated colonies
that regrew on media lacking histidine and had -galactosidase
activity in the presence of the bait plasmid, but not lamin, were
identified as positive after mating. The percent of colonies that
retested as positive is shown in the mating column. Proteins found to
interact with the neurogranin baits are indicated as isolated clones.
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The neurogranin bait construct was segregated from the library cDNA
in each L40 colony. Mating assays were used to test the specificity of
interaction between the neurogranin and the isolated library cDNAs.
The AMR70 strain of yeast was transformed with the wild-type BTM116
neurogranin bait, or with a BTM116-lamin fusion protein. Each L40
colony containing an isolated cDNA was mated to each of the AMR70
bait colonies. Interactions were scored by growth on media lacking
histidine and by
-galactosidase activity. cDNAs were isolated
only from colonies that showed positive growth on (
His) media and
-galactosidase activity during the mating. Any cDNA that also
interacted with the BTM116-lamin fusion protein was identified as a
nonspecific neurogranin binding partner and was discarded.
Following the mating assays, we isolated 98 positive cDNAs from the
rat brain transformation and 90 from the mouse brain transformation. Each of these DNAs was digested with Sau3A1, and the
restriction patterns were compared. All of the digested DNAs had the
same restriction pattern, suggesting that they were identical. Twenty cDNAs from each transformation were sequenced. Each of these
cDNAs were identified as either rat or mouse CaM, indicating that
CaM is the only neurogranin-binding protein detectable in yeast
two-hybrid screens from two different rodent cDNA libraries.
Although the data described above suggests that CaM may be the only
protein that interacts with neurogranin in vivo, there may
be other neurogranin-binding proteins whose interactions are inhibited
by CaM. The high levels of CaM in brain (10-20 µM) may have obscured the detection of other neurogranin-binding proteins. Furthermore, phosphorylated neurogranin may bind to other proteins, which would not have been detected in the original yeast two-hybrid screen. For these reasons, we re-screened the brain cDNA libraries using the Ile-33
Gln and Ser-36
Asp neurogranin mutants,
neither of which interact strongly with CaM in vivo.
The Ile-33
Gln mutant was screened against both libraries. Results
are shown in Table I. In both screens, none of the isolated cDNAs
retested as positive in the mating assay. In addition, each of the
positive cDNAs were tested for interactions against wild-type neurogranin and the other neurogranin mutants in mating assays. None of
the cDNAs showed positive interactions with any of the baits
tested. A subset of these cDNAs were sequenced, and none of the
sequences correlated to "in frame" sequences. Similarly, in the
Ser-36
Asp neurogranin mouse brain screen, none of the isolated
cDNAs retested as positive in mating assays or when retransformed in yeast with each of the neurogranin constructs. Sequencing of 12 of
the putative positives did not reveal in frame sequences.
Obvious differences were seen between the wild-type and mutant
neurogranin two-hybrid screens. All transformations were equally efficient, but many fewer positives were found in the mutant screens. The
-galactosidase activity in the wild-type screens appeared within
minutes, whereas positives detected in the mutant screens appeared in
hours. At each step in the screening process, the number of mutant
positives decreased substantially, while very few of the positives in
the wild-type screens were lost during successive screens. We were
unable to detect any neurogranin-binding proteins by screening the
mutant neurogranins suggesting that CaM is the major protein that
interacts with neurogranin in vivo.
Neurogranin Regulates Calmodulin-sensitive Targets in
Vivo--
Previous studies have shown that neurogranin and
neuromodulin can regulate calcium/CaM-sensitive enzymes in
vitro (24, 25). For example, nitric oxide synthase activity
decreases in the presence of increasing concentrations of neuromodulin
and neurogranin. We used the CaM-stimulated adenylyl cyclases, AC1 and
AC8, to determine whether neurogranin could regulate CaM-sensitive
targets in vivo. AC1 and AC8 are both stimulated by
Ca2+ and CaM, in vivo. In these experiments, 293 cells were transiently co-transfected with neurogranin and either AC1
or AC8. Enzyme activity was measured in response to the calcium
ionophore, A23187. Ca2+ stimulation of AC1 or AC8 in
vivo was markedly reduced when neurogranin was co-expressed (Fig.
5). These data indicate that neurogranin has the potential to regulate the activity of CaM-regulated enzymes in vivo by complexing and lowering the effective
concentration of free CaM.

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Fig. 5.
Neurogranin inhibits adenylyl cyclase
activity in vivo. 293 cells were transiently
transfected with pcDNA3-neurogranin and either AC1 (A)
or AC8 (B). As a control, when not co-expressed with
neurogranin, adenylyl cyclases were expressed with equivalent amounts
of a green fluorescent protein. Adenylyl cyclase activity was measured
as the change in intracellular cAMP levels ± 5 µM
A23187 as described under "Experimental Procedures." Enzyme
activity is shown as the ratio of cAMP to a total pool of adenine
nucleotides. Activities were measured in triplicate and were
normalized to -galactosidase expression. Error shown is ± S.D.
Data shown are representative of three separate experiments.
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DISCUSSION |
Neurogranin and the related IQ domain protein, neuromodulin, are
hypothesized to regulate neuronal CaM levels in response to PKC
phosphorylation and increases in intracellular Ca2+ (3, 16,
23). Therefore, it has been important to determine whether these
proteins bind CaM and are phosphorylated by PKC in vivo.
Both neurogranin and neuromodulin are phosphorylated by PKC in
vivo (8, 43, 44), and recently it was shown that neuromodulin
binds CaM in vivo (45, 46). The objectives of this study
were to determine if neurogranin binds CaM in vivo through
its IQ domain and to determine if other neurogranin-binding proteins
can be detected by yeast two-hybrid technology. The discovery of other
neurogranin-binding proteins would provide mechanistic insights
concerning the role of neurogranin in signal transduction and would
suggest alternative biochemical mechanisms for neurogranin in neurons.
Our data indicate that neurogranin and CaM are also in vivo
binding partners and that the interaction occurs primarily through the
IQ domain of neurogranin. The Ile-33
Gln mutation completely inhibited the CaM/neurogranin interaction, illustrating the importance of hydrophobic interactions for CaM binding to neurogranin. Reduced binding of CaM to the Ser-36
Asp mutant of neurogranin is
consistent with the hypothesis that introduction of negative charge at
Ser-36 by PKC phosphorylation lowers CaM binding affinity.
CaM was the only neurogranin-interacting protein isolated from the rat
and mouse brain two-hybrid screens. In addition, neurogranin-binding proteins were not detected using neurogranin mutant proteins that do
not bind CaM. It is interesting that other two-hybrid screens performed
on IQ domain proteins have yielded similar results. After extensive
testing, CaM was the only protein found to interact with neuromodulin
(45), and it was also the only protein isolated using Igloo,
a Drosophila neuromodulin homologue, as bait (47). In a
screen of IQGAP1, a human putative Ras GTPase-activating protein that
contains four IQ domains, CaM was the most frequent interacting protein
identified (48). Yeast two-hybrid screens do not necessarily detect all
significant interactions. For example, although neurogranin and
neuromodulin are PKC substrates, yeast two-hybrid screens did not
identify PKC as an interacting protein with either neuromodulin or
neurogranin. Nevertheless, our data indicate that CaM is the only
neurogranin-binding protein detectable by an extensive yeast two-hybrid
screen; no other neurogranin-interacting proteins other than PKC have
been identified in vitro or in vivo.
If neuromodulin and neurogranin regulate free CaM levels, these
proteins should regulate the activity of CaM-stimulated enzymes. In
support of this hypothesis, both neurogranin and neuromodulin have been
shown to regulate the activity of CaM-dependent enzymes (24, 25). Addition of neurogranin or neuromodulin to
Ca2+/CaM-activated nitric oxide synthase reduces NO
synthase activity. This inhibition is not seen when neurogranin or
neuromodulin are phosphorylated, or at high Ca2+
concentrations, indicating that sequestration of CaM by neurogranin or
neuromodulin regulates the activity of CaM-stimulated enzymes. Our
results show that neurogranin may also regulate calmodulin-stimulated targets in vivo. When co-expressed with neurogranin, the
calcium/calmodulin-stimulated activities of adenylyl cyclases AC1 and
AC8 were inhibited. As both AC1 and AC8 are expressed in brain, they
may represent physiological targets for neurogranin.
The existence of presynaptic and postsynaptic CaM-binding proteins that
may regulate free CaM in neurons has led to the interesting hypothesis
that these proteins may play a pivotal role during synaptic plasticity,
e.g. long term potentiation (LTP) (30, 31). For example,
neurogranin and neuromodulin are both phosphorylated by PKC during LTP
(49-53). There are several forms of mechanistically distinct LTP
expressed in the hippocampus and other areas of brain, including forms
that are predominantly presynaptic and others that are hypothesized to
be postsynaptic (54). In either case, initial increases in
intracellular Ca2+ arising because of activation of
voltage-sensitive Ca2+ channels or glutamate receptors are
thought to initiate signal transduction cascades leading to enhanced
synaptic efficacy (55-57). CaM-stimulated enzymes including CaM
kinases, NO synthase (58), and adenylyl cyclases (59, 60) are thought
to play major roles in the initiation, maintenance, and propagation of
LTP. Consequently, neuromodulin and neurogranin may be critical
components of the molecular machinery used for modulation of synaptic
plasticity and the development of learning and memory in vertebrates.
Our demonstration that neurogranin can regulate brain adenylyl cyclases through its interactions with calmodulin is consistent with this theory.