Kalirin Inhibition of Inducible Nitric-oxide Synthase*
Edward A.
Ratovitski
,
M. Rashidul
Alam§,
Richard A.
Quick
,
Audrey
McMillan
,
Clare
Bao
,
Chaim
Kozlovsky
,
Tracey A.
Hand§,
Richard C.
Johnson§,
Richard E.
Mains§,
Betty A.
Eipper§, and
Charles J.
Lowenstein
¶*
From the Division of Cardiology,
Department of
Medicine, and § Department of Neurosciences, School of
Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
 |
ABSTRACT |
Nitric oxide (NO) acts as a neurotransmitter.
However, excess NO produced from neuronal NO synthase (nNOS) or
inducible NOS (iNOS) during inflammation of the central nervous system
can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide
biosynthetic enzyme. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the
formation of iNOS homodimers. Expression of exogenous Kalirin in
pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the
central nervous system by inhibiting iNOS activity.
 |
INTRODUCTION |
Nitric oxide (NO)1
mediates numerous physiological functions in the central nervous
system, including neurotransmission, synaptic plasticity, and hormone
secretion (1-5). In neurons that express nNOS, excitatory amino acids
can activate NMDA-type glutamate receptors, temporarily elevating
intracellular calcium concentrations, resulting in transient synthesis
of NO by nNOS. NO then acts as a neurotransmitter, diffusing into
adjacent neurons. However, excessive NO production is neurotoxic and
may be involved in diseases of the central nervous system. Various
experimental systems demonstrate the neurotoxicity of NO. For example,
NO derived from nNOS or NO donors can kill neurons in vitro
(6, 7). Large amounts of excitatory amino acids generated during
strokes can trigger neurons that contain nNOS to produce high
concentrations of NO (8). Conversely, studies with nNOS null mice show
that NMDA excitotoxicity is reduced, and cerebral infarct size is
smaller in the absence of NO generated from NOS (9-11). In humans, NO derived from nNOS may play a neurotoxic role in strokes and in diverse
neurodegenerative diseases, including Alzheimer's disease, Huntington's chorea, and amyotrophic lateral sclerosis. Neuronal NOS
is also found in a significant fraction of peripheral neurons, where it
is thought to function as a neurotransmitter (12).
Another source of high concentrations of NO in the central nervous
system is the iNOS isoform, which can be induced in neurons or
microglia during inflammation (13-16). In contrast to nNOS, iNOS
synthesizes large amounts of NO continuously, which can also be
neurotoxic to neurons in vitro (17). Furthermore, cerebral infarct size is also reduced in iNOS null mice (18). The iNOS isoform
is also expressed in experimental autoimmune encephalomyelitis, and NOS
inhibition reduces the severity of the disease (19-22). In humans, NO
derived from iNOS may play a role in the pathogenesis of inflammatory
disorders of the brain, such as AIDS dementia and multiple sclerosis
(23-25), and may also contribute to the pathogenesis of Alzheimer's
disease (26). NO may also play a more subtle role during brain
inflammation, interfering with physiological functions such as hormone
secretion. Experimental evidence from neuronal cultures and animal
models shows that NO can also inhibit specific secretory responses of
neurons. For example, some but not all modes of stimulating hormone
secretion from the pituitary are enhanced in the presence of NOS
inhibitors (27-36).
Because iNOS expression may lead to neurotoxicity in the brain,
disrupting hormone secretion or killing neurons, we tested the
hypothesis that proteins are expressed in the central nervous system
which regulate iNOS activity, minimizing its neurotoxic effects. A
yeast two-hybrid screen for hippocampal proteins that may bind to iNOS
revealed that Kalirin interacts with iNOS.
Kalirin is a cytosolic protein with nine spectrin-like repeats, a
Dbl-homology domain that acts as a GDP/GTP exchange factor for Rac1,
and pleckstrin homology and SH3 domains (see Fig. 1) (37, 38). Kalirin
is closely related to two other proteins, Trio and UNC-73, which also
possess spectrin-like motifs, Dbl homology domains, and pleckstrin
homology domains (39, 40). Kalirin was initially identified by its
ability to interact with the cytosolic domain of peptidylglycine
-amidating monooxygenase (PAM), an enzyme located in the
trans-Golgi network and large dense core vesicles. PAM is a
bifunctional type I integral membrane protein with both functional
domains in the lumen of the secretory pathway; PAM is responsible for
the
-amidation of neuropeptides and hormones, a modification
essential for the biological potency of more than half of all the known
bioactive peptides (41). Kalirin is expressed with PAM at high levels
in cerebral cortex, piriform cortex, amygdala, hippocampus, and
olfactory bulb. Although the precise role of Kalirin is unknown, it may
serve to transduce signals between large dense-core vesicles and the
actin cytoskeleton. Our data suggest a novel role for Kalirin in the
regulation of hormone secretion by its effects upon iNOS.
 |
EXPERIMENTAL PROCEDURES |
Antibodies, cDNAs, Vectors, and Reagents--
For
immunoblotting and immunoprecipitation, the following antibodies were
used: a monoclonal antibody raised against c-Myc (Santa Cruz
Biotechnology, Santa Cruz, CA) or prepared from hybridoma 9E10 cells
(42); a polyclonal antibody raised against a murine iNOS peptide
generated by us (43); a monoclonal antibody raised against murine iNOS
(Transduction Laboratories); polyclonal antibodies to eNOS and nNOS
(Transduction Laboratories), and a polyclonal antibody (JH2582) to
Kalirin (spectrin repeats 4-7) generated by us. Standard cloning
methods were used to join the Kalirin cDNA fragment identified
using the yeast two-hybrid screen (encoding Kalirin amino acids
447-1124) in reading frame 3' to the sequence of the c-Myc epitope and
a Gly5 linker (MEQKLISEEDLNGGGGG-Kalirin-(447-1124)). This
cDNA was cloned 3' to the CMV promoter in the mammalian expression vector pSCEP to create pSCEP.myc.Kalirin-(447-1124) (37, 38). The
murine iNOS cDNA was generated as described (44). The
(His)6-iNOS fusion protein was generated from a cDNA
based on the pET vector from Novagen (45) and expressed in bacteria and
then purified over a nickel resin (according to manufacturer
instructions). Lipopolysaccharide (LPS) was obtained from Sigma.
Interferon-
(IFN-
) was a generous gift from the American Cancer Society.
Cell Culture--
The interaction of Kalirin and iNOS was
examined in non-transfected AtT-20/D-16v cells, and in
AtT-20 cells stably expressing Kalirin-(447-1124). The vector
pSCEP.myc.Kalirin-(447-1124) was transfected into AtT-20 cells by
lipofection. AtT-20 cells stably expressing myc-Kalirin-(447-1124)
were first selected for hygromycin resistance (200 units/ml) and were
then screened for expression of the Kalirin transcript by Northern blot
analysis. Cells were maintained as described (37, 38). Induction of
iNOS in cells was performed using IFN-
(20 units/ml) and LPS (1 µg/ml) for 2-16 h (46, 47).
Western Blot Analysis--
Cells were suspended in lysis buffer
(100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% of Triton X-100, 0.5% Brij-50, 1 mM PMSF, 4 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 mM Na3VO4, 50 mM NaF). Protein extracts were resolved by SDS-PAGE and
transferred onto polyvinylidene difluoride membranes. Membranes were
incubated for 1 h with primary antibodies diluted 1:1000-1:5000 in blocking solution containing 3% bovine serum albumin, 0.1% Tween
20 in Tris-buffered saline and after washing were incubated for 1 h with secondary antibodies coupled to horseradish peroxidase diluted
1:5000-1:10,000. Labeled bands were visualized by an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech). For determination of iNOS apparent molecular mass, cell lysates were loaded
onto a Superdex 200 gel filtration column (0.75 cm, inner diameter × 50 cm, length) in lysis buffer without detergents, and fractions
were collected, resolved by SDS-PAGE, and immunoblotted as above.
Immunoprecipitation--
Cells were suspended in lysis buffer
for 30 min on ice. Supernatants were recovered by centrifugation at
15,000 × g for 15 min, and 500 µl of supernatant was
incubated with 10 µl of normal rabbit serum for 30 min and then
incubated with 50 µl of protein A-Sepharose 4B for 30 min. Following
centrifugation, 500 µl of supernatant was incubated for 3 h with
primary antibodies and then with 40 µl of a 50% suspension of
protein A-Sepharose 4B (or goat anti-mouse agarose) for 4 h at
4 °C and then was washed three times with 1 ml of cold 20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1 mM EDTA, 0.2% Triton X-100, 0.2 mM PMSF. Samples were boiled with SDS and
-mercaptoethanol, fractionated by
SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Membranes were incubated for 1-2 h at room temperature with antibodies to c-Myc, or iNOS (1:5,000), washed, incubated for 1-2 h with goat
anti-mouse or goat anti-rabbit antibody (1:10,000) coupled to
horseradish peroxidase, and visualized with an ECL reagent (Amersham
Pharmacia Biotech). For the detection of iNOS and Kalirin complexes
in vivo, (C57Bl6, 129)F2 wild-type and iNOS null mice from
Jackson Laboratories were injected intraperitoneally with 500 µg of
LPS, and after 24 h, their brains were homogenized and analyzed by
immunoprecipitation as above.
NOS Assay--
The NOS assay was performed as described
previously (48). Cells were sonicated in 50 mM Tris-HCl, pH
7.4, 1 mM EDTA, 0.1 mM tetrahydrobiopterin, 2 mM dithiothreitol, 10% (v/v) glycerol, aprotinin (25 µg/ml), leupeptin (25 µg/ml), 100 µM PMSF, 10 µM FMN, and 10 µM FAD. A reaction mix was
made of cell lysate containing 50 µg of protein, 50 µl of
[U-14C]arginine (50,000 cpm), 5 µl of 5 mM
FAD, 5 µl of 100 µM H4B, 1 µl of 30 µM calmodulin, and the volume was brought up to 250 µl
with 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA. The reaction was initiated by addition of 50 µl
of 10 mM NADPH, and incubated for 60 min at 37 °C,
applied to a Dowex 50-W resin (H+ form) column, and the flow-through
was counted in a liquid scintillation counter.
Yeast Two-hybrid Screen--
We employed a yeast two-hybrid
system to screen for potential binding partners of iNOS (49). The pPC86
library contained cDNA prepared from rat hippocampus (generous gift
of Dr. A. Lanahan) (50). To prepare bait plasmids, murine iNOS cDNA
sequences 1-546 base pairs or 1-210 base pairs) were prepared by the
polymerase chain reaction and inserted 3' to the cDNA for the Gal4
binding domain of plasmid pPC97. The resultant pPC97-iNOS bait plasmids encoded a fusion protein comprised of the Gal4 binding domain and an
amino-terminal fragment of iNOS (amino acid residues 1-182 or 1-70).
YRG-2 yeast cells were co-transformed with pPC97-iNOS and the
pPC86-based library and grown on a selective medium lacking tryptophan,
leucine, and histidine, or combinations thereof. Colonies that contain
cDNA encoding target library proteins interacting with the bait
fusion protein were identified by transcription of the HIS3-
and lacZ-genes. A total of 2.6 × 106 yeast
transformants were placed under selection. Plasmid preparations from
-galactosidase positive yeast colonies were isolated and retransformed into competent Escherichia coli DH5
cells.
For quantitative liquid
-galactosidase assay, Saccharomyces
cerevisiae (strain SFY526) were co-transformed by the lithium acetate method with various combinations of bait plasmid (pPC97 expressing a fusion protein of the Gal4-BD (binding domain) and portions of iNOS) and target plasmids (pPC86 expressing a fusion protein of the Gal4-AD (activation domain) and portions of Kalirin). Transformants were plated for 3 days at 30 °C on selective medium lacking tryptophan, leucine, and histidine. After 3 days, cells were
assayed for
-galactosidase activity using ONPG as a substrate (nmol
ONPG cleaved/min/mg protein measured at
A420).
Size Exclusion Chromatography--
Lysates of cells were loaded
onto a Superdex 200 chromatography column, equilibrated, and eluted
with 20 mM Tris-HCl, pH 7.4, 125 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1 mM EDTA, 0.2% Triton X-100, 0.2 mM PMSF
buffer, and 1 ml fractions were collected. Molecular weight markers
were analyzed separately to determine approximate molecular weights of
substances in the eluted fractions.
ACTH Secretion Assay--
For secretion experiments using LPS
and IFN-
, identical wells of cells were treated for 12 h with
control medium or medium containing 50 ng/ml LPS and 10 units/ml
IFN-
; for treated cultures, LPS and IFN-
were added fresh
throughout the secretion experiment. Cells were transferred to basal
medium (Dulbecco's modified Eagle's medium-air with 2 mg/ml bovine
serum albumin, 0.1 mg/ml lima bean trypsin inhibitor, 1 µg/ml
insulin, 0.1 µg/ml transferrin) for the secretion experiments and
equilibrated in release medium for sequential 30-min periods.
Supernatants were harvested from cells exposed to basal media, 100 nM corticotrophin releasing hormone (Sigma), or to 1 mM BaCl2 (51-53). Cell extracts were prepared using 5 N acetic acid with protease inhibitors, lyophilized, and then dissolved in radioimmunoassay buffer with inhibitors. ACTH radioimmunoassays were performed using antiserum Kathy, which detects ACTH biosynthetic intermediate and ACTH but does not detect intact pro-opiomelanocortin (54).
 |
RESULTS |
We used the yeast two-hybrid system to search for proteins that
might associate with iNOS (49). Yeast expressing a fusion protein
consisting of the amino acids 1-182 of iNOS and the Gal4 DNA binding
domain (BD) were transformed with a library of plasmids encoding fusion
proteins consisting of rat hippocampal cDNAs and the Gal4
activation domain (AD) (50). The amino-terminal region of iNOS was
selected as a potential target of interacting proteins because it is
not homologous to the other NOS isoforms. Screening of 2.6 × 106 yeast transformants isolated a cDNA encoding the
protein Kalirin (Table I). Our yeast
two-hybrid screen also showed that Ki nuclear
autoantigen, nel-related protein, and homologues to a serine/threonine
kinase, and bicaudal D may also interact with the amino terminus of
iNOS. These interactions have not been confirmed by in
vivo testing, yet.
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Table I
Specific interaction between iNOS and Kalirin in the yeast two-hybrid
assay
S. cerevisiae (strain SFY526) were co-transformed by the
lithium acetate method with various combinations of bait plasmid (pPC97
expressing a fusion protein of the Gal4 binding domain and portions of
iNOS) and target plasmids (pPC86 expressing a fusion protein of the
Gal4 activation domain and portions of Kalirin). Transformants were
plated for 3 days at 30 °C on selective medium lacking tryptophan,
leucine, and histidine. After 3 days, cells were assayed for
-galactosidase activity by liquid culture assay method using ONPG as
a substrate (nmol ONPG cleaved/min/mg of protein measured at
A420) (5). Assays were performed in triplicate.
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|
Kalirin Interacts with iNOS in Vitro--
To define more precisely
the regions of iNOS and Kalirin that interact, yeast expressing a
Kalirin fusion protein (consisting of amino acid residues 570-753 of
Kalirin fused to the Gal4-AD) were transformed with various plasmids
representing different domains of iNOS. Analysis of transfected yeast
revealed that the first 70 amino acid residues of iNOS are capable of
interacting with Kalirin in yeast (Table I, Fig.
1). The region of iNOS between 70-220
amino acids does not interact with Kalirin in yeast.

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Fig. 1.
Domains of iNOS and Kalirin. For iNOS,
amino-terminal Hook Domain (dimerization segment), heme binding domain
(HEME), calmodulin binding domain (CAL), flavin
adenine mononucleotide binding domain (FMN), flavin adenine
dinucleotide binding domain (FAD), nicotinamide adenine
dinucleotide phosphate binding domain (NADPH) are shown. The
Kalirin interaction domain of iNOS identified in this study is also
indicated. For Kalirin, spectrin motifs (ovals),
Dbl-homology domain (DH), pleckstrin homology domain
(PH), and SH3 domain are shown.
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|
We next examined the ability of iNOS and Kalirin to interact in
vitro. Bacterial expression vectors were constructed which express
a (His)6-iNOS fusion protein (45) or a glutathione
S-transferase-Kalirin-(amino acid residues 447-1124) fusion
protein. Purified (His)6-iNOS fusion protein interacts with
purified glutathione S-transferase-Kalirin-(447-1124) fusion protein in vitro (Fig.
2). This interaction was demonstrated by
co-immunoprecipitation of GST-Kalirin with an iNOS antibody and by the
binding of iNOS to GST-Kalirin immobilized on glutathione-agarose resin.

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Fig. 2.
Complexes of iNOS and Kalirin in
vitro. (His)6-iNOS, GST-Kalirin-(447-1124), and
an irrelevant target, GST-midkine (93), were expressed and purified
from bacteria. Various combinations of pure proteins were incubated
together as indicated. A, immunoprecipitation with antibody
to iNOS and immunoblot with antibody to GST. B, purification
with glutathione-agarose and immunoblot with antibody to iNOS. Only
iNOS and Kalirin fusion proteins are capable of interaction. (The
experiment was repeated twice with similar results.)
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Kalirin Interacts with iNOS in Cells and in Mice--
We next
examined the ability of iNOS and Kalirin to interact in mammalian
cells. The mouse pituitary tumor cell line AtT-20 (in which Kalirin is
not normally expressed) was stably transfected with a plasmid
expressing a fragment of Kalirin (amino acids 447-1124); this fragment
of Kalirin contains spectrin motif repeat domains, the region of
Kalirin interacting with iNOS. Cells were then treated with LPS and
IFN-
to induce expression of iNOS. Kalirin is expressed only in
AtT-20 cells that are transfected, and iNOS is expressed only in cells
that are treated with LPS and IFN-
, as detected by immunoblotting
(Fig. 3A). Kalirin and iNOS
also associate in these cells, as shown by co-immunoprecipitation (Fig.
3B). Most of the Kalirin-(447-1124) and most of the iNOS
appear to be associated with each other.

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Fig. 3.
Complexes of iNOS and Kalirin in cells.
A, treated AtT-20 cells express iNOS, and transfected AtT-20
cells express Kalirin. Wild-type AtT-20 cells or AtT-20 cells
expressing Kalirin-(447-1124) tagged with c-Myc were incubated with
media alone ( ) or with IFN- and LPS (+). Cell lysates were
immunoblotted with an antibody to iNOS or antibody to c-Myc (myc
antibody). B, iNOS and Kalirin interact in cells.
AtT-20 cells expressing c-Myc-tagged Kalirin-(447-1124) were incubated
with media alone or with IFN- and LPS. Cell lysates were
immunoprecipitated with antibody to iNOS (iNOS ab) followed
by immunoblotting with antibody to c-Myc (myc ab) (to detect
c-Myc-tagged Kalirin), or were immunoprecipitated with antibody to Myc
followed by immunoblotting with antibody to iNOS. (The experiment was
repeated twice with similar results.)
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|
We then examined the interaction of iNOS and Kalirin in mice. The
availability of mice lacking iNOS provides a control for the
specificity of this interaction. Kalirin is expressed in the brains of
wild-type mice and iNOS null mice (Fig.
4A). (Native Kalirin-(1-1899)
has a molecular mass of approximately 210 kDa when expressed in mouse
brains or in AtT-20 cells. The recombinant Kalirin-(447-1124) fragment
expressed in AtT-20 cells and used in the other figures has a molecular
mass of approximately 73 kDa.) As expected, LPS injected
intraperitoneally into wild-type and iNOS null mice induces iNOS
expression only in wild-type mice (Fig. 4A). Complexes of
endogenous Kalirin and endogenous iNOS are detected by
immunoprecipitation in brain extracts of LPS-treated wild-type mice,
but not in brain extracts of LPS-treated mice lacking iNOS (Fig.
4B) (55). Thus iNOS and Kalirin interact in a variety of
experimental systems.

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Fig. 4.
Complexes of iNOS and Kalirin in mice.
Wild-type mice (+/+) or iNOS null mice ( / ) were injected with 500 µg LPS intraperitoneally, and after 24 h, their brains were
harvested. A, Kalirin and iNOS expression in murine brains.
Brain lysates were resolved by SDS-PAGE and analyzed by immunoblotting
with antibody to Kalirin or to iNOS. B, physiological
complexes of iNOS and Kalirin in mice. Brain lysates were prepared from
mice as above, and either immunoprecipitated with antibody to iNOS
followed by immunoblotting with antibody to Kalirin, or
immunoprecipitated with antibody to Kalirin followed by immunoblotting
with antibody to iNOS. C, Kalirin does not interact with
nNOS in vivo. Lysates from wild-type mice (lanes
1 and 3) or from iNOS null mice (lanes 2 and
4) were fractionated by SDS-PAGE (lanes 1 and
2) or immunoprecipitated with antibody to kalirin,
fractionated by SDS-PAGE (lanes 3 and 4), and
then probed with an antibody to nNOS. D, Kalirin does not
interact with eNOS in vivo. Samples were analyzed as in
panel C using an eNOS antiserum. The experiment was repeated
in three wild-type and three iNOS null mice with similar results.
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Specificity of Kalirin Interaction with NOS Isoforms--
To test
the specificity of the Kalirin interaction with NOS isoforms, we
analyzed mouse brain extracts for the interaction of Kalirin and nNOS
or eNOS. Lysates from brains of wild-type and iNOS null mice treated
with LPS as above were fractionated by SDS-PAGE and probed with
antibody to nNOS, showing that nNOS is expressed in mouse brain (Fig.
4C). Lysates from these brains were then immunoprecipitated
with antibody to Kalirin, fractionated by SDS-PAGE, and probed with
antibody to nNOS. No nNOS was co-immunoprecipitated with Kalirin (Fig.
4C). We then repeated this experiment with antibody to eNOS.
No eNOS was co-immunoprecipitated with Kalirin (Fig. 4D).
Thus, Kalirin interacts only with iNOS and not with the other NOS isoforms.
Kalirin Inhibits iNOS Activity by Blocking iNOS
Homodimerization--
We then measured the effect of Kalirin on iNOS
enzymatic activity. NOS activity was measured in AtT-20 cells and in
AtT-20 cells expressing Kalirin-(447-1124); cells were either resting or treated with IFN-
and LPS. As expected, treatment of AtT-20 cells
with LPS and IFN-
induces iNOS activity (Fig.
5A). Expression of
Kalirin-(447-1124) reduces iNOS activity by more than 90% (Fig. 5A). Because expression of Kalirin-(447-1124) does not
reduce the amount of iNOS protein expressed in response to IFN-
/LPS treatment (Fig. 5B), we explored other mechanisms by which
Kalirin could inhibit iNOS activity.

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Fig. 5.
Kalirin inhibits iNOS activity.
A, AtT-20 cells and AtT-20 cells expressing
Kalirin-(447-1124) were incubated with media alone or with media
containing IFN- and LPS, and 50 µg of cell lysate was assayed for
the conversion of arginine to citrulline. Control samples had
negligible amounts of NOS activity and are not visible on the chart.
(n = 3 ± S.D.; the experiment was repeated three
times.) B, Kalirin expression does not change the amount of
iNOS in cells. AtT-20 cells (lanes 1 and 2) and
AtT-20 cells expressing Kalirin-(447-1124) (lanes 3 and
4) were incubated with media alone (lanes 1 and
3) or with media containing IFN- and LPS (lanes
2 and 4); cell lysates were fractionated by SDS-PAGE
and probed with an antibody to iNOS.
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We hypothesized that Kalirin inhibits iNOS activity by affecting
iNOS homodimerization, which is required for iNOS activity (56, 57). To
test this hypothesis, cell lysates were prepared from either LPS- and
IFN-
-treated AtT-20 cells or from LPS- and IFN-
-treated AtT-20
cells expressing Kalirin-(447-1124), and these lysates were
fractionated on a gel filtration column. Aliquots were then
electrophoresed on a denaturing gel. Immunoblotting of gel filtration
fractions with antibody to iNOS suggests that most of the iNOS exists
as a homodimer (Mr 260,000) in cells without Kalirin (eluted in fractions 11-20 at approximately 300 kDa) (Fig. 6). The less abundant iNOS monomer eluted
in fractions 27-30 at approximately 110 kDa. In contrast, in cells
expressing Kalirin-(447-1124), most of the iNOS exists as an
intermediate-sized form (fractions 18-23), probably representing
iNOS/Kalirin heterodimers; some dimer was detected, but very little
iNOS monomer was present in these cells (Fig. 6). Kalirin-(447-1124)
eluted with iNOS in fractions 18-23, and trailed into later fractions.
Thus Kalirin-(447-1124) inhibits iNOS activity and iNOS
homodimerization.

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Fig. 6.
Kalirin inhibits iNOS homodimerization.
AtT-20 cells were treated with IFN- and LPS for 16 h, and then
cell lysates were fractionated on a Superdex 200 gel filtration column.
Fractions of 1 ml were collected, resolved by SDS-PAGE, and
immunoblotted with antibody to iNOS (A) or antibody to
Kalirin (B). AtT-20 cells expressing Kalirin-(447-1124)
were treated with IFN- and LPS for 16 h and then fractionated
and analyzed in the same manner as above with antibody to iNOS
(C) or with antibody to kalirin (D). The column
was calibrated with the following standards: thyroglobulin (relative
molecular weight Mr 670,000) in fractions 8-12,
gamma globulin (Mr 158,000) in fractions 18-22,
ovalbumin (Mr 44,000) in fractions 32-40,
myoglobin (Mr 17,000), and cyanocobalamin
(Mr 1350). The experiment was repeated twice
with similar results.
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Kalirin Associates with iNOS Monomers--
In theory, Kalirin
could associate with iNOS monomers, preventing iNOS homodimerization
and eliminating NOS activity; or Kalirin could associate with iNOS
homodimers, converting iNOS homodimers into monomers and thereby
inhibiting NOS activity. To distinguish between these two
possibilities, we performed experiments mixing cell lysates. Lysates
were prepared from non-transfected resting AtT-20 cells,
non-transfected AtT-20 cells treated with LPS and
IFN, resting
AtT-20 cells expressing Kalirin-(447-1124), or AtT-20 cells expressing
Kalirin-(447-1124) treated with LPS and IFN-
. Pairs of these
lysates were mixed together and either analyzed by immunoblotting or by
immunoprecipitation with antibody to Kalirin followed by immunoblotting
with the antibody to iNOS (Fig. 7). Kalirin can interact with iNOS if both proteins are produced in the
same cell or if each protein is produced in different cells. However,
because iNOS exists not only as homodimers but also as monomers in
LPS/IFN-
-treated AtT-20 cells, this experiment does not prove that
Kalirin only interacts with iNOS monomers.

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Fig. 7.
Physical interaction of Kalirin and iNOS in
mixed lysates. Non-transfected AtT-20 cells and AtT-20 cells
expressing Kalirin-(447-1124) were treated with LPS and IFN- or not
for 16 h. Pairs of cell lysates were mixed together as indicated.
Top, immunoblot with antibody to iNOS; middle,
immunoblot with antibody to Kalirin; bottom,
immunoprecipitation with antibody to Kalirin followed by immunoblot
with antibody to iNOS.
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If Kalirin-(447-1124) only associates with iNOS monomers, then we
would predict that addition of Kalirin-containing extracts to
iNOS-containing extracts would not affect iNOS activity. This is indeed
the case. Lysates were prepared from AtT-20 cells that were treated or
not with LPS/IFN-
, and mixed with lysates prepared from AtT-20 cells
expressing Kalirin-(447-1124) that were treated or not with
LPS/IFN-
, and the NOS activity in the mixtures was assayed as above.
Resting non-transfected AtT-20 cells have little NOS activity (113 ± 54 cpm/mg); resting AtT-20 cells expressing Kalirin-(447-1124) also
have little NOS activity (527 ± 92). LPS/IFN-
treatment
resulted in increased NOS activity in non-transfected AtT-20 cells
(4222 ± 125). Although iNOS activity was inhibited when iNOS and
Kalirin were expressed in the same cells (481 ± 70), iNOS
activity was not inhibited when Kalirin and iNOS were made in separate
cells and the lysates were mixed (4661 ± 292).
To confirm that Kalirin-(447-1124) only associates with iNOS monomers,
we mixed Kalirin-containing cell extracts with iNOS monomers or iNOS
homodimers, which had been isolated by column chromatography of
LPS-treated AtT-20 cell lysates, as in Fig. 6A. Mixtures of
Kalirin-(447-1124) and iNOS dimers and mixtures of Kalirin-(447-1124)
and iNOS monomers both contain Kalirin-(447-1124) and iNOS, as
verified by immunoblotting. Co-immunoprecipitation of iNOS with
antiserum to Kalirin was used to determine whether the proteins could
interact; iNOS was detected in the Kalirin immunoprecipitate only when
Kalirin-containing extracts were mixed with iNOS monomers (Fig.
8). These sets of experiments imply that Kalirin can only interact with iNOS monomers, and Kalirin cannot interact with iNOS homodimers.

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|
Fig. 8.
Kalirin interacts with iNOS monomers but not
iNOS homodimers. Lysates from AtT-20 cells treated with IFN-
and LPS were fractionated by column chromatography as in Fig. 6.
Fractions containing iNOS monomers (M) or iNOS homodimers
(D) were then mixed with lysates from non-treated AtT-20
cells expressing Kalirin-(447-1124). Entire lysate mixtures were then
fractionated by SDS-PAGE and probed with antibody to Kalirin or iNOS.
Lysate mixtures were also immunoprecipitated with antibody to iNOS,
fractionated by SDS-PAGE, and probed with antibody to Kalirin
(IP).
|
|
Kalirin Reduces the Inhibitory Effect of iNOS upon ACTH
Secretion--
Other investigators have shown that NO inhibits the
secretion of adrenocorticotropic hormone (ACTH) from the pituitary
during systemic or central nervous system inflammation (9-11, 13,
19-25). To study the functional significance of the interaction
between Kalirin and iNOS, we measured the ability of iNOS and Kalirin to alter ACTH secretion in AtT-20 cells. AtT-20 cells produce ACTH and
-endorphin from pro-opiomelanocortin, storing the peptide products
in large dense core vesicles (58), and release of ACTH can be
stimulated by secretagogues such as corticotropin-releasing hormone
(CRH) and BaCl2 (53, 59, 60).
We measured the ability of iNOS and Kalirin to alter ACTH secretion
from non-transfected AtT-20 cells and from stably transfected AtT-20
cells expressing Kalirin-(447-1124). LPS and IFN-
treatment decreases the basal release of ACTH from the non-transfected cells to
40%, compared with untreated non-transfected cells (Fig.
9). NO mediates this inhibition of ACTH
secretion by LPS and IFN-
, because pretreatment for 3 h with
the NOS inhibitor nitro-arginine methyl ester (NAME) largely negates
the effects of LPS and IFN-
(data not shown). Similar to the effects
of NAME, expression of Kalirin also blunts the effect of LPS and
IFN-
upon basal ACTH secretion (Fig. 9), presumably by inhibiting NO
synthesis from iNOS. Stimulated secretion of ACTH was also reduced to
half by LPS and IFN-
treatment in the non-transfected cells. Kalirin expression also blocked this inhibitory effect of LPS and IFN-
treatment on stimulated ACTH release (Fig. 9). Thus Kalirin reduces the
inhibition of basal and stimulated secretion of ACTH in cells exposed
to LPS and IFN-
.

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[in a new window]
|
Fig. 9.
Kalirin inhibits iNOS-induced changes in ACTH
secretion. Pairs of identical cultures of non-transfected AtT-20
cells and AtT-20 cells expressing Kalirin-(447-1124) were treated with
growth medium alone or with IFN- and LPS for 12 h, and ACTH
secretion was then measured after three 30-min wash periods. Media were
collected for four sequential 30-min periods with BaCl2 or
corticotropin-releasing hormone present only during the third time
period; the drop in ACTH concentration in the fourth collection period
is normal. Samples from two separate experiments were each assayed in
triplicate, and the results of the two experiments were averaged. The
medium blank (0.28 nM) was subtracted from all data. Data
are plotted as mean ± S.D. Similar effects were also seen with an
independent clone of AtT-20 cells expressing Kalirin-(447-1124). After
10 h of treatment with LPS and IFN- , some cells were treated
with 1 mM nitro-arginine methyl ester for 2 h prior to
stimulation with BaCl2, and most of the inhibition of
secretion by LPS and IFN- was blocked by pretreatment with
nitro-arginine methyl ester (not shown).
|
|
 |
DISCUSSION |
Calmodulin was the first protein shown to interact with NOS (48);
it is necessary for enzymatic activity of all NOS isoforms. Subsequently a variety of other proteins have been shown to interact with the constitutive NOS isoforms; syntrophin, post-synaptic density
protein (PSD) 93, and PSD95 can each interact with the amino-terminal
region of nNOS via a PDZ domain, localizing nNOS to specific
subcellular regions of myocytes or neurons (5, 61, 62). The mammalian
homologue of dynein light chain (PIN) can interact with nNOS,
maintaining it in a monomeric form (63); and caveolin 1 and caveolin 3 can interact with eNOS, localizing eNOS to the caveolae of endothelial
cells (64-72). Our report of the interaction between iNOS and Kalirin
suggests a novel mechanism for regulation of iNOS, which was previously
thought to be regulated primarily at the level of transcription
(73).
Dimerization of NOS activates NO synthesis by permitting electron
transfer between the reductase and oxygenase domains (74-76). Homodimerization of iNOS depends upon arginine, tetrahydrobiopterin and
heme (77-84). Biochemical and crystallographic studies show that
dimerization of iNOS involves the oxygenase domains of each iNOS
monomer (83, 85, 86). A portion of the oxygenase domain of iNOS, amino
acid residues 66-114, is particularly important for homodimerization
(78). Crystallography shows that in this region, Cys-109 forms an
interchain disulfide bond across the dimer interface (83). In our
experiments using the yeast two-hybrid system, we found that Kalirin
interacts with the initial 70 amino acids of the amino-terminal of
iNOS. Because this region partially overlaps the iNOS dimerization
domain, perhaps Kalirin prevents iNOS homodimerization by physically
interfering with the amino-terminal iNOS dimerization region.
The fact that LPS and interferon treatment inhibits basal and
stimulated ACTH secretion from AtT-20 cells is consistent with other
studies which show that NO inhibits ACTH secretion (27-36). The effect
of LPS upon the pituitary is complex: in the whole animal LPS is often
reported to stimulate ACTH secretion (87). Some of the confusion about
LPS and IFN-
action could be because of time-dependent
effects, because short term stimulation of ACTH secretion by IFN-
is
followed by prolonged inhibition of ACTH secretion in the longer
treatment periods used in many studies and in this work (88). In the
central nervous system, LPS induces iNOS which produces NO, which in
turn can interact with specific protein components of the 20 S
v-SNARE/t-SNARE complex, inhibiting exocytosis of synaptic vesicles
(89). Here we present data showing that expression of
Kalirin-(447-1124) inhibits the catalytic activity of iNOS, and thus
reduces the ability of LPS and interferon to inhibit secretion of ACTH.
Kalirin interacts with other molecules in addition to iNOS that may be
involved in the secretory process. Notably, Huntingtin associated
protein-1 was also found to interact with the spectrin-like domains of
Kalirin (90), as well as with p150glued (91). We
have also shown that Kalirin interacts directly with the protein kinase
P-CIP2, which was originally identified as another interactor with the
carboxyl domain of PAM (38) and as an interactor with stathmin, a
regulator of microtubule depolymerization (92)
Expression of iNOS in the central nervous system is associated with a
variety of inflammatory states, which can lead to neurotoxicity, apoptosis, and aberrant regulation of hormone release. Expression of
iNOS and production of NO inhibit ACTH secretion by the pituitary. Our
data show that Kalirin can inhibit iNOS activity in AtT-20 cells; in
the central nervous system, Kalirin would preserve neuropeptide secretion during inflammation. In this manner, Kalirin may play a
neuroprotective role, reducing neurotoxicity and restoring hormone and
neuropeptide secretion from large dense core vesicles.
 |
FOOTNOTES |
*
This work was supported in part by American Heart
Association-Maryland Affiliate (to E. A. R.), National Institutes of
Health Grants R01 DK32948 (to R. E. M.), DA00266 (to B. A. E.), P50
HL52315 (to C. J. L.), and R01 HL5361 (to C. J. L.), the Ciccarone
Center for the Prevention of Heart Disease (to C. J. L.), the Cora
and John H. Davis Foundation (to E. A. R. and C. J. L.), and the
Bernard Bernard Foundation (to C. J. L.).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 and reprint requests should be
addressed: Division of Cardiology, Dept. of Medicine, Johns Hopkins University School of Medicine, 950 Ross Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-1530; Fax: 410-614-5129; E-mail: clowenst{at}welchlink.welch.jhu.edu.
The abbreviations used are:
NO, nitric oxide; NOS, nitric-oxide synthase; nNOS, NO synthase; iNOS, inducible NOS; PAM, peptidylglycine
-amidating monooxygenase; ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; LPS, lipopolysaccharide; IFN-
, interferon-gamma; NAME, nitro-arginine
methyl ester; ONGP, o-nitrophenyl-
-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl
fluoride; GST, glutathione S-transferase; NMDA, N-methyl-D-aspartate.
 |
REFERENCES |
-
Nelson, R. J.,
Demas, G. E.,
Huang, P. L.,
Fishman, M. C.,
Dawson, V. L.,
Dawson, T. M.,
and Snyder, S. H.
(1995)
Nature
378,
383-386[CrossRef][Medline]
[Order article via Infotrieve]
-
Brenman, J. E.,
and Bredt, D. S.
(1997)
Curr. Opin. Neurobiol.
7,
374-378[CrossRef][Medline]
[Order article via Infotrieve]
-
Szabo, C.
(1996)
Brain Res. Bull.
41,
131-141[CrossRef][Medline]
[Order article via Infotrieve]
-
Zhang, J.,
and Snyder, S. H.
(1995)
Annu. Rev. Pharmacol. Toxicol.
35,
213-233[CrossRef][Medline]
[Order article via Infotrieve]
-
Bredt, D. S.
(1996)
Proc. Soc. Exp. Biol. Med.
211,
41-48[Abstract]
-
Dawson, V. L.,
Dawson, T. M.,
London, E. D.,
Bredt, D. B.,
and Snyder, S. H.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
6368-6371[Abstract]
-
Dawson, V. L.,
Dawson, T. M.,
Bartley, D. A.,
Uhl, G. R.,
and Snyder, S. H.
(1993)
J. Neurosci.
13,
2651-2661[Abstract]
-
Dawson, T. M.,
Dawson, V. L.,
and Snyder, S. H.
(1992)
Ann. Neurol.
32,
297-311[Medline]
[Order article via Infotrieve]
-
Ayata, C.,
Ayata, G.,
Hara, H.,
Matthews, R. T.,
Beal, M. F.,
Ferrante, R. J.,
Endres, M.,
Kim, A.,
Christie, R. H.,
Waeber, C.,
Huang, P. L.,
Hyman, B. T.,
and Moskowitz, M. A.
(1997)
J. Neurosci.
17,
6908-6917[Abstract/Free Full Text]
-
Huang, Z.,
Huang, P. L.,
Panahian, N.,
Dalkara, T.,
Fishman, M. C.,
and Moskowitz, M. A.
(1994)
Science
265,
1883-1885[Medline]
[Order article via Infotrieve]
-
Hara, H.,
Huang, P. L.,
Panahian, N.,
Fishman, M. C.,
and Moskowitz, M. A.
(1996)
J. Cerebr. Blood Flow Metab.
16,
605-611[Medline]
[Order article via Infotrieve]
-
Zhou, Y.,
and Ling, E. A.
(1998)
J. Comp. Neurol.
394,
496-505[CrossRef][Medline]
[Order article via Infotrieve]
-
Wong, M. L.,
Rettori, V.,
al-Shekhlee, A.,
Bongiorno, P. B.,
Canteros, G.,
McCann, S. M.,
Gold, P. W.,
and Licinio, J.
(1996)
Nat. Med.
2,
581-584[Medline]
[Order article via Infotrieve]
-
Eissa, N. T.,
Strauss, A. J.,
Haggerty, C. M.,
Choo, E. K.,
Chu, S. C.,
and Moss, J.
(1996)
J. Biol. Chem.
271,
27184-27187[Abstract/Free Full Text]
-
Mitrovic, B.,
St. Pierre, B. A.,
Mackenzie-Graham, A. J.,
and Merrill, J. E.
(1994)
Ann. N. Y. Acad. Sci.
738,
436-446[Medline]
[Order article via Infotrieve]
-
Merrill, J. E.,
Ignarro, L. J.,
Sherman, M. P.,
Melinek, J.,
and Lane, T. E.
(1993)
J. Immunol.
151,
2132-2141[Abstract/Free Full Text]
-
Mitrovic, B.,
Ignarro, L. J.,
Vinters, H. V.,
Akers, M. A.,
Schmid, I.,
Uittenbogaart, C.,
and Merrill, J. E.
(1995)
Neuroscience
65,
531-539[CrossRef][Medline]
[Order article via Infotrieve]
-
Iadecola, C.,
Zhang, F.,
Casey, R.,
Nagayama, M.,
and Ross, M. E.
(1997)
J. Neurosci.
17,
9157-9164[Abstract/Free Full Text]
-
Brenner, T.,
Brocke, S.,
Szafer, F.,
Sobel, R. A.,
Parkinson, J. F.,
Perez, D. H.,
and Steinman, L.
(1997)
J. Immunol.
158,
2940-2946[Abstract]
-
Okuda, Y.,
Nakatsuji, Y.,
Fujimura, H.,
Esumi, H.,
Ogura, T.,
Yanagihara, T.,
and Sakoda, S.
(1995)
J. Neuroimmunol.
62,
103-112[CrossRef][Medline]
[Order article via Infotrieve]
-
Cross, A. H.,
Misko, T. P.,
Lin, R. F.,
Hickey, W. F.,
Trotter, J. L.,
and Tilton, R. G.
(1994)
J. Clin. Invest.
93,
2684-2690[Medline]
[Order article via Infotrieve]
-
Lin, R. F.,
Lin, T. S.,
Tilton, R. G.,
and Cross, A. H.
(1993)
J. Exp. Med.
178,
643-648[Abstract]
-
Mitrovic, B.,
Ignarro, L. J.,
Montestruque, S.,
Smoll, A.,
and Merrill, J. E.
(1994)
Neuroscience
61,
575-585[CrossRef][Medline]
[Order article via Infotrieve]
-
Bo, L.,
Dawson, T. M.,
Wesselingh, S.,
Mork, S.,
Choi, S.,
Kong, P. A.,
Hanley, D.,
and Trapp, B. D.
(1994)
Ann. Neurol.
36,
778-786[Medline]
[Order article via Infotrieve]
-
Adamson, D. C.,
Wildemann, B.,
Sasaki, M.,
Glass, J. D.,
McArthur, J. C.,
Christov, V. I.,
Dawson, T. M.,
and Dawson, V. L.
(1996)
Science
274,
1917-1921[Abstract/Free Full Text]
-
Vodovotz, Y.,
Lucia, M. S.,
Flanders, K. C.,
Chesler, L.,
Xie, Q. W.,
Smith, T. W.,
Weidner, J.,
Mumford, R.,
Webber, R.,
Nathan, C.,
Roberts, A. B.,
Lippa, C. F.,
and Sporn, M. B.
(1996)
J. Exp. Med.
184,
1425-1433[Abstract]
-
Rivier, C.
(1995)
Endocrinology
136,
3597-3603[Abstract]
-
Rivier, C.,
and Shen, G. H.
(1994)
J. Neurosci.
14,
1985-1993[Abstract]
-
Lee, S.,
and Rivier, C.
(1994)
Alcohol Clin. Exp. Res.
18,
1242-1247[Medline]
[Order article via Infotrieve]
-
Brunetti, L.,
Preziosi, P.,
Ragazzoni, E.,
and Vacca, M.
(1993)
Life Sci.
53,
PL219-PL222[Medline]
[Order article via Infotrieve]
-
Turnbull, A. V.,
and Rivier, C.
(1996)
Endocrinology
137,
455-463[Abstract]
-
Duvilanski, B. H.,
Zambruno, C.,
Seilicovich, A.,
Pisera, D.,
Lasaga, M.,
Diaz, M. C.,
Belova, N.,
Rettori, V.,
and McCann, S. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
170-174[Abstract]
-
Seilicovich, A.,
Duvilanski, B. H.,
Pisera, D.,
Theas, S.,
Gimeno, M.,
Rettori, V.,
and McCann, S. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3421-3424[Abstract]
-
Duvilanski, B. H.,
Zambruno, C.,
Lasaga, M.,
Pisera, D.,
and Seilicovich, A.
(1996)
J. Neuroendocrinol.
8,
909-913[Medline]
[Order article via Infotrieve]
-
Seilicovich, A.,
Lasaga, M.,
Befumo, M.,
Duvilanski, B. H.,
del Carmen Diaz, M.,
Rettori, V.,
and McCann, S. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
11299-11302[Abstract]
-
Volpi, R.,
Chiodera, P.,
Caffarri, G.,
Vescovi, P. P.,
Capretti, L.,
Gatti, C.,
and Coiro, V.
(1996)
Neuropeptides
30,
528-532[CrossRef][Medline]
[Order article via Infotrieve]
-
Alam, M. R.,
Johnson, R. C.,
Darlington, D. N.,
Hand, T. A.,
Mains, R. E.,
and Eipper, B. A.
(1997)
J. Biol. Chem.
272,
12667-12675[Abstract/Free Full Text]
-
Alam, M. R.,
Caldwell, B. D.,
Johnson, R. C.,
Darlington, D. N.,
Mains, R. E.,
and Eipper, B. A.
(1996)
J. Biol. Chem.
271,
28636-28640[Abstract/Free Full Text]
-
Debant, A.,
Serra-Pages, C.,
Seipel, K.,
O'Brien, S.,
Tang, M.,
and Park, S. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5466-5471[Abstract/Free Full Text]
-
Steven, R.,
Kubiseki, T. J.,
Zheng, H.,
Kulkarni, S.,
Mancillas, J.,
Morales, A. R.,
Hogue, C. W. V.,
Pawson, T.,
and Culotti, J.
(1998)
Cell
92,
785-796[Medline]
[Order article via Infotrieve]
-
Eipper, B. A.,
Milgram, S. L.,
Husten, E. J.,
Yun, H. Y.,
and Mains, R. E.
(1993)
Protein Sci.
2,
489-497[Abstract/Free Full Text]
-
Evan, G.,
Lewis, G.,
Ramsay, G.,
and Bishop, J. M.
(1985)
Mol. Cell. Biol.
5,
3610-3616[Medline]
[Order article via Infotrieve]
-
Lowenstein, C. J.,
Hill, S. L.,
Lafond-Walker, A.,
Wu, J.,
Allen, G.,
Landavere, M.,
Rose, N. R.,
and Herskowitz, A.
(1996)
J. Clin. Invest.
97,
1837-1843[Abstract/Free Full Text]
-
Lowenstein, C. J.,
Glatt, C. S.,
Bredt, D. S.,
and Snyder, S. H.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6711-6715[Abstract]
-
Ghosh, S.,
and Lowenstein, J. M.
(1996)
Gene (Amst.)
176,
249-255[CrossRef][Medline]
[Order article via Infotrieve]
-
Stuehr, D. J.,
and Nathan, C. F.
(1989)
J. Exp. Med.
169,
1543-1555[Abstract]
-
Stuehr, D. J.,
and Marletta, M. A.
(1987)
J. Immunol.
139,
518-525[Abstract/Free Full Text]
-
Bredt, D. S.,
and Snyder, S. H.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
682-685[Abstract]
-
Fields, S.,
and Song, O.
(1989)
Nature
340,
245-246[CrossRef][Medline]
[Order article via Infotrieve]
-
Li, X. J.,
Li, S. H.,
Sharp, A. H.,
Nucifora, F. C., Jr.,
Schilling, G.,
Lanahan, A.,
Worley, P.,
Snyder, S. H.,
and Ross, C. A.
(1995)
Nature
378,
398-402[CrossRef][Medline]
[Order article via Infotrieve]
-
Mains, R. E.,
and Eipper, B. A.
(1981)
J. Biol. Chem.
256,
5683-5688[Abstract/Free Full Text]
-
Mains, R. E.,
and Eipper, B. A.
(1980)
Ann. N. Y. Acad. Sci.
343,
94-110[Medline]
[Order article via Infotrieve]
-
Milgram, S. L.,
Mains, R. E.,
and Eipper, B. A.
(1993)
J. Cell Biol.
121,
23-36[Abstract]
-
Schnabel, E.,
Mains, R. E.,
and Farquhar, M. G.
(1989)
Mol. Endocrinol.
3,
1223-1235[Abstract]
-
MacMicking, J. D.,
Nathan, C.,
Hom, G.,
Chartrain, N.,
Fletcher, D. S.,
Traumbauer, M.,
Stevens, K.,
Xie, Q. W.,
Sokol, K.,
Hutchinson, N.,
Chen, H.,
and Mudgett, J. S.
(1995)
Cell
81,
641-650[Medline]
[Order article via Infotrieve]
-
Stuehr, D. J.,
Cho, H. J.,
Kwon, N. S.,
Weise, M. F.,
and Nathan, C. F.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
7773-7777[Abstract]
-
Hevel, J. M.,
White, K. A.,
and Marletta, M. A.
(1991)
J. Biol. Chem.
266,
22789-22791[Abstract/Free Full Text]
-
Mains, R. E.,
and Eipper, B. A.
(1990)
Trends Endocrinol. Metab.
1,
388-394
-
Mains, R. E.,
and Eipper, B. A.
(1981)
J. Cell Biol.
89,
21-28[Abstract]
-
Mains, R. E.,
and Eipper, B. A.
(1984)
Endocrinology
115,
1683-1690[Abstract]
-
Brenman, J. E.,
Chao, D. S.,
Gee, S. H.,
McGee, A. W.,
Craven, S. E.,
Santillano, D. R.,
Wu, Z.,
Huang, F.,
Xia, H.,
Peters, M. F.,
Froehner, S. C.,
and Bredt, D. S.
(1996)
Cell
84,
757-767[Medline]
[Order article via Infotrieve]
-
Brenman, J. E.,
Chao, D. S.,
Xia, H.,
Aldape, K.,
and Bredt, D. S.
(1995)
Cell
82,
743-752[Medline]
[Order article via Infotrieve]
-
Jaffrey, S. R.,
and Snyder, S. H.
(1996)
Science
274,
774-777[Abstract/Free Full Text]
-
Michel, J. B.,
Feron, O.,
Sase, K.,
Prabhakar, P.,
and Michel, T.
(1997)
J. Biol. Chem.
272,
25907-25912[Abstract/Free Full Text]
-
Garcia-Cardena, G.,
Martasek, P.,
Masters, B. S. S.,
Skidd, P. M.,
Couet, J.,
Li, S.,
Lisanti, M. P.,
and Sessa, W. C.
(1997)
J. Biol. Chem.
272,
25437-25440[Abstract/Free Full Text]
-
Ju, H.,
Zou, R.,
Venema, V. J.,
and Venema, R. C.
(1997)
J. Biol. Chem.
272,
18522-18525[Abstract/Free Full Text]
-
Song, K. S.,
Sargiacomo, M.,
Galbiati, F.,
Parenti, M.,
and Lisanti, M. P.
(1997)
Cell. Mol. Biol. Res.
43,
293-303
-
Feron, O.,
Smith, T. W.,
Michel, T.,
and Kelly, R. A.
(1997)
J. Biol. Chem.
272,
17744-17748[Abstract/Free Full Text]
-
Venema, V. J.,
Zou, R.,
Ju, H.,
Marrero, M. B.,
and Venema, R. C.
(1997)
Biochem. Biophys. Res. Commun.
236,
155-161[CrossRef][Medline]
[Order article via Infotrieve]
-
Michel, J. B.,
Feron, O.,
Sacks, D.,
and Michel, T.
(1997)
J. Biol. Chem.
272,
15583-15586[Abstract/Free Full Text]
-
Garcia-Cardena, G.,
Fan, R.,
Stern, D. F.,
Liu, J.,
and Sessa, W. C.
(1996)
J. Biol. Chem.
271,
27237-27240[Abstract/Free Full Text]
-
Feron, O.,
Belhassen, L.,
Kobzik, L.,
Smith, T. W.,
Kelly, R. A.,
and Michel, T.
(1996)
J. Biol. Chem.
271,
22810-22814[Abstract/Free Full Text]
-
Nathan, C.,
and Xie, Q. W.
(1994)
Cell
78,
915-918[Medline]
[Order article via Infotrieve]
-
Siddhanta, U.,
Wu, C.,
Abu-Soud, H. M.,
Zhang, J.,
Ghosh, D. K.,
and Stuehr, D. J.
(1996)
J. Biol. Chem.
271,
7309-7312[Abstract/Free Full Text]
-
Sheta, E. A.,
McMillan, K.,
and Masters, B. S. S.
(1994)
J. Biol Chem.
269,
15147-15153[Abstract/Free Full Text]
-
Stevens-Truss, R.,
Beckingham, K.,
and Marletta, M. A.
(1997)
Biochemistry
36,
12337-12345[CrossRef][Medline]
[Order article via Infotrieve]
-
Baek, K. J.,
Thiel, B. A.,
Lucas, S.,
and Stuehr, D. J.
(1993)
J. Biol. Chem.
268,
21120-21129[Abstract/Free Full Text]
-
Ghosh, D. K.,
Wu, C.,
Pitters, E.,
Moloney, M.,
Werner, E. R.,
Mayer, B.,
and Stuehr, D. J.
(1997)
Biochemistry
36,
10609-10619[CrossRef][Medline]
[Order article via Infotrieve]
-
Mayer, B.,
Wu, C.,
Gorren, A. C.,
Pfeiffer, S.,
Schmidt, K.,
Clark, P.,
Stuehr, D. J.,
and Werner, E. R.
(1997)
Biochemistry
36,
8422-8427[CrossRef][Medline]
[Order article via Infotrieve]
-
Hellermann, G. R.,
and Solomonson, L. P.
(1997)
J. Biol. Chem.
272,
12030-12034[Abstract/Free Full Text]
-
Klatt, P.,
Schmidt, K.,
Lehner, D.,
Glatter, O.,
Bachinger, H. P.,
and Mayer, B.
(1995)
EMBO J.
14,
3687-3695[Abstract]
-
Tzeng, E.,
Billiar, T. R.,
Robbins, P. D.,
Loftus, M.,
and Stuehr, D. J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
11771-11775[Abstract]
-
Crane, B. R.,
Arvai, A. S.,
Ghosh, D. K.,
Wu, C.,
Getzoff, E. D.,
Stuehr, D. J.,
and Traintor, J. A.
(1998)
Science
279,
2121-2125[Abstract/Free Full Text]
-
Presta, A.,
Siddhanta, U.,
Wu, C.,
Sennequier, N.,
Huang, L.,
Abu-Soud, H. M.,
Erzurum, S.,
and Stuehr, D. J.
(1998)
Biochemistry
37,
298-310[CrossRef][Medline]
[Order article via Infotrieve]
-
Crane, B. R.,
Arvai, A. S.,
Gachhui, R.,
Wu, C.,
Ghosh, D. K.,
Getzoff, E. D.,
Stuehr, D. J.,
and Tainer, J. A.
(1997)
Science
278,
425-431[Abstract/Free Full Text]
-
Venema, R. C.,
Ju, H.,
Zou, R.,
Ryan, J. W.,
and Venema, V. J.
(1997)
J. Biol. Chem.
272,
1276-1282[Abstract/Free Full Text]
-
Aurenhammer, C. J.,
Chesnokova, V.,
and Melmed, S.
(1998)
Endocrinology
139,
2201-2208[Abstract/Free Full Text]
-
Katahira, M.,
Iwasaki, Y.,
Aoki, Y.,
Oiso, Y.,
and Saito, H.
(1998)
Endocrinology
139,
2414-2422[Abstract/Free Full Text]
-
Meffert, M. K.,
Calakos, N. C.,
Scheller, R. H.,
and Schulman, H.
(1996)
Neuron
16,
1229-1236[Medline]
[Order article via Infotrieve]
-
Colomer, V.,
Engelender, S.,
Sharp, A. H.,
Duan, K.,
Cooper, J. K.,
Lanahan, A.,
Lyford, G.,
Worley, P.,
and Ross, C. A.
(1998)
Hum. Mol. Genet.
6,
1519-1525[Abstract/Free Full Text]
-
Li, S. H.,
Gutekunst, C. A.,
Hersch, S. M.,
and Li, X. J.
(1998)
J. Neurosci.
18,
1261-1269[Abstract/Free Full Text]
-
Curmi, P. A.,
Anderson, S. S. L.,
Lachkar, S.,
Gavet, O.,
Karsenti, E.,
Knossow, M.,
and Sobel, A.
(1997)
J. Biol. Chem.
272,
25029-25036[Abstract/Free Full Text]
-
Ratovitski, E. A.,
Kotzbauer, P. T.,
Milbrandt, J.,
Lowenstein, C. J.,
and Burrow, C. R.
(1998)
J. Biol. Chem.
273,
3654-3660[Abstract/Free Full Text]
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