Selective Interaction of AGS3 with G-proteins and the Influence
of AGS3 on the Activation State of G-proteins*
Michael L.
Bernard
,
Yuri K.
Peterson,
Peter
Chung,
Jane
Jourdan, and
Stephen M.
Lanier§
From the Department of Pharmacology, Medical University of South
Carolina, Charleston, South Carolina 29403
Received for publication, June 19, 2000, and in revised form, October 13, 2000
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ABSTRACT |
AGS3 (activator of
G-protein signaling 3) was isolated
in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role
of AGS3 in mammalian signal processing, we defined the AGS3 subdomains
involved in G-protein interaction, its selectivity for G-proteins, and
its influence on the activation state of G-protein. Immunoblot analysis
with AGS3 antisera indicated expression in rat brain, the neuronal-like
cell lines PC12 and NG108-15, as well as the smooth muscle cell line
DDT1-MF2. Immunofluorescence studies and confocal
imaging indicated that AGS3 was predominantly cytoplasmic and enriched
in microdomains of the cell. AGS3 coimmunoprecipitated with
G
i3 from cell and tissue lysates, indicating that
a subpopulation of AGS3 and G
i exist as a complex in the
cell. The coimmunoprecipitation of AGS3 and G
i was
dependent upon the conformation of G
i3 (GDP
GTP
S (guanosine 5'-3-O-(thio)triphosphate)). The regions
of AGS3 that bound G
i were localized to four amino acid
repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus
(Pro463-Ser650), each of which were capable of
binding G
i. AGS3-GPR domains selectively interacted with
G
i in tissue and cell lysates and with purified
G
i/G
t. Subsequent experiments with
purified G
i2 and G
i3 indicated that the
carboxyl-terminal region containing the four GPR motifs actually bound
more than one G
i subunit at the same time. The AGS3-GPR
domains effectively competed with G
for binding to
G
t(GDP) and blocked GTP
S binding to
G
i1. AGS3 and related proteins provide unexpected
mechanisms for coordination of G-protein signaling pathways.
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INTRODUCTION |
Signal processing via heterotrimeric G-protein proteins generally
involves an initial input sensed by a cell surface receptor with seven
membrane-spanning regions. Conformational changes in receptor
subdomains then transfer this signal to a G-protein, promoting exchange
of GTP for GDP and subunit dissociation with both the G
and G
subunits regulating effector molecules. These events are tightly
regulated to maximize signal efficiency, optimize signal specificity,
and integrate cellular responses to diverse stimuli. Regulatory
mechanisms include the segregation of specific signaling molecules in
cell microdomains, receptor phosphorylation and internalization,
cross-talk between signaling pathways, and proteins that regulate the
basal activation state of G-proteins independently of the receptor.
We partially purified a direct G-protein activator from NG108-15 cells
(1, 2) and subsequently used a functional screen to identify three
proteins (AGS1-3, for activator of G-protein signaling 1-3) that
activated heterotrimeric G-protein signaling in the absence of a cell
surface receptor (3-5). The identification of such proteins raises
many interesting and unexpected questions relative to signal processing
by heterotrimeric G-proteins. As an initial approach to address these
issues, we focused on the biochemical and functional characterization
of AGS1 proteins, and this
report deals specifically with AGS3 (AF107723, calculated
molecular weight 72,049). AGS3, isolated from a rat brain cDNA
library, contains seven tetratricopeptide repeats (TPRs) and four GPR
(G-protein-regulatory) motifs separated by a linker in the middle of
the protein (4)2 (Fig.
1).
AGS3 is one member of a larger protein family defined by a two-domain
structure (Fig. 1). In rodents and
humans, this family is defined by rat AGS3 and human LGN (U54999),
which was isolated in a yeast two-hybrid screen using
G
i2 as bait (8). A single AGS3-related protein is found
in Caenorhabditis elegans (AAA81387) and Drosophila
melanogaster (AF36967). Analysis of genome and expressed sequence
tag data bases indicated that in addition to human LGN cDNAs, there
are partial human cDNAs exhibiting higher homology to AGS3
versus LGN (e.g. AL117478 (360 amino acids), 95%
sequence similarity to AGS3 and 57% sequence similarity to LGN;
AI272212 (190 amino acids), 93% sequence similarity to AGS3 and 57%
sequence similarity to LGN). Likewise, analysis of mouse/rat genome and
expressed sequence tag data bases indicate that in addition to AGS3
cDNAs (e.g. L23316) there are mouse cDNAs
(e.g. AA543923, AA166402, and AW539573) exhibiting higher
sequence homology to human LGN versus AGS3. Thus, AGS3 and
LGN are distinct proteins, and perhaps there are additional related
proteins in the primate genome yet to be identified.

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Fig. 1.
Schematic representation of AGS3 and related
proteins. Full-length rat AGS3 (AAF08683) was aligned with the
human LGN protein (AAB40385), the D. melanogaster PINS
protein (AAF36967), and the C. elegans protein
(CE) (AAA81387) by PILEUP (University of Wisconsin GCG
program) and visual adjustment. Amino acid sequence similarity and
identity are indicated below the four sequences by + or residue,
respectively. The shaded and lined sequences
represent TPR I-VII and a repeated segment of amino acids (GPR I-IV).
The amino-terminal half of the AGS3 contains six TPRs, as defined by
SMART analysis (7), that exist as a cluster of two
(Ser43-Gln116,
Gly183-Ile336) and four motifs with a spacer
of ~60 amino acids between the two clusters. Visual inspection of the
spacer region between the two clusters of TPR motifs indicates the
likely existence of an additional TPR motif defined by the
I129GN and A162SEFYERNL sequence, in which the
helical structure of this TPR may be extended. The carboxyl-terminal
half of AGS3 contains the second functional domain consisting of four
~20 amino acid repeats. as discussed in the text. PINS contains three
GPRs with highest homology to GPRs I, III, and IV of AGS3, LGN, and
C. elegans protein.
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The first insight as to the functional role of LGN and AGS3 in signal
processing was their identification as a G
i-binding protein (8) and isolation as a receptor-independent G-protein activator
(4), respectively. Additional insight was provided by recent studies in
D. melanogaster, where the AGS3/LGN homolog PINS (Partner of
Inscuteable) is required for events involved in the asymmetric cell
division of neuroblasts in the early stages of development (9, 10).
PINS is part of a multiprotein complex that is translocated from the
cytosol to one pole of the dividing neuroblast. In this article, we
report the existence of an AGS3-G
i complex within the
cell, define the G
-interacting domains of AGS3, and determine the
selectivity of AGS3 for different G
subunits. AGS3, which
preferentially binds to G
GDP, can bind multiple G
subunits and hence may function as a scaffolding protein to provide spatially and temporally discrete signaling events. AGS3 and G
actually competed with each other for interaction with
G
t(GDP), and AGS3 inhibited guanine nucleotide exchange
on G
i1. The properties of the AGS3-G
interactions add
unexpected dimensions to signal processing by G-protein-regulated
signaling systems.
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EXPERIMENTAL PROCEDURES |
Materials--
[35S]GTP
S (1250 Ci/mmol) was
purchased from PerkinElmer Life Sciences. Tissue culture
supplies were obtained from JRH Bioscience (Lenexa, KS). Acrylamide,
bisacrylamide, Bio-Rad protein assay kits, and sodium dodecyl sulfate
were purchased from Bio-Rad. Ecoscint A was purchased from National
Diagnostics (Manville, NJ). Guanosine diphosphate, guanosine
triphosphate, and Thesit (polyoxyethylene-9-lauryl ether) were obtained
from Rche Molecular Biochemicals. Polyvinylidene difluoride membranes
were obtained from Pall Gelman Sciences (Ann Arbor, MI). Gammabind
G-Sepharose was obtained from Amersham Pharmacia Biotech, and
nitrocellulose BA85 filters were purchased from Schleicher & Schuell.
Poly-L-lysine. normal goat serum, biotinylated goat
anti-rabbit IgG, and Extravidin fluorescein isothiocyanate were
purchased from Sigma. Immuno Fluore mounting medium was purchased from
ICN Biomedicals. Purified bovine brain G-protein and antisera to the
COOH-terminal 10 amino acids of G
1, which recognizes G
1-4, were
kindly provided by Dr. John Hildebrandt (Department of Pharmacology,
Medical University of South Carolina, Charleston, SC) (11, 12).
G
i1-3 and G
o were purified from
Sf9 insect cells infected with recombinant virus as described
(13) and kindly provided by Dr. Stephen Graber (West Virginia
University School of Medicine, Morgantown, WV). G
s and
G
q, similarly expressed in Sf9 insect cells, were
kindly provided by Dr. Elliott Ross (University of Texas Southwestern Medical Center, Dallas, TX) (14). Purified G
t and
G

t (15) were kindly provided by Dr. Heidi Hamm
(Northwestern University Medical School, Chicago, IL). Polyclonal
G
i3 antisera generated against the COOH-terminal 10 amino acids was kindly provided by Dr. Thomas W. Gettys (Department of
Medicine, Medical University of South Carolina, Charleston, SC) (16).
Purified GA antibody, which selectively recognizes
Gi/G
o, was kindly provided by Drs. Paul
Goldsmith, Andrew Shenkar, and Allen Spiegel (17). All other materials
were obtained as described elsewhere (4, 18).
Generation of AGS3 Subdomains--
AGS3 subdomains were
generated as glutathione fusion proteins by polymerase chain reaction
using the full-length cDNA of AGS3 as a template. Primers were
designed to add BamHI and EcoRI sites to the 5'
and 3' ends, respectively, of AGS3 subdomains to fuse the AGS3 open
reading frame with the reading frame of glutathione S-transferase contained in the pGEX4T1 vector. The
polymerase chain reactions were generally performed using 250 nM primers and 125 pM template DNA in a total
volume of 50 µl. Cycles were 1 × 3 min at 94 °C; 30 × 1.5 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C; and
1 × 10 min at 72 °C. Primers used to generate specific
constructs were as follows.
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Preparation of Cell/Tissue Lysates--
NG108-15, PC12, and
DDT1-MF2 cells were grown as described previously (1, 2).
Caco-2 cells were obtained from the American Type Culture Collection
and cultured in Eagle's minimum essential medium supplemented with 1%
minimum essential medium nonessential amino acids. CHO cells were grown
on Falcon tissue culture dishes at 37 °C (5% CO2) in
Ham's F-12 medium supplemented with 10% fetal bovine serum plus
penicillin (100 units/ml), streptomycin (100 µg/ml), and fungizone
(0.25 µg/ml) (19). Rat brain was homogenized in 3 ml of buffer/g of
tissue of lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40).
Confluent 100-mm dishes of cells were washed with cell washing solution
(137 mM NaCl, 2.6 mM KCl, 1.8 mM
KH2PO4, 10 mM
Na2HPO4) and resuspended in 1 ml of lysis
buffer/dish by homogenization. Following a 1-h incubation on ice at
4 °C, the cell homogenate was centrifuged at 27,000 × g for 30 min. Supernatants were collected and spun at
100,000 × g for 1 h to generate a
detergent-soluble fraction. The supernatant was immediately processed
for immunoblotting or immunoprecipitation. In some experiments, cells
and tissue were also fractionated to generate a crude membrane pellet
and a 100,000 × g supernatant containing cytosol.
Tissues were homogenized in 5 mM Tris, 5 mM
EDTA, 5 mM EGTA, pH 7.4, and centrifuged at 100,000 × g for 30 min at 4 °C and washed at least three times by
homogenization in membrane buffer. For preparation of cell homogenates,
12 confluent 100-mm dishes were lysed in 3 ml of 5 mM Tris,
5 mM EDTA, 5 mM EGTA, pH 7.4, and centrifuged
at 100,000 × g for 30 min at 4 °C. Cell membrane
pellets were washed three times with intervening homogenization and
pelleting at 100,000 × g. The washed membrane pellets
were resuspended in 250 µl of membrane buffer (50 mM
Tris, 0.6 mM EDTA, 5 mM MgCl2, pH
7.4) by homogenization. Protein concentrations were determined by a
Bio-Rad protein assay.
Protein Interaction Assays--
The interaction of AGS3 with
G-proteins was assessed by both coimmunoprecipitation and protein
interaction experiments using tissue/cell lysates or purified
G-proteins. Protein concentrations in the lysates were determined by a
Bio-Rad protein assay. For immunoprecipitation from mammalian cells,
cell/tissue lysates (1-3 mg of protein in 0.5-1 ml) were pre-cleared
by rotating incubation with Gammabind G-Sepharose (12.5 µl of packed
resin equilibrated in lysis buffer) for 30 min at 4 °C. Following
centrifugation, G
i3 antisera (1:250 dilution) was added
to pre-cleared lysates and incubation continued overnight at 4 °C.
Protein complexes were captured by adding Gammabind G-Sepharose (12.5 µl packed volume) and continuing the incubation for 30 min at
4 °C. The mixture was then microcentrifuged at 4 °C and the
pellets washed (3× 500 µl of incubation buffer) and resuspended in
2× Laemmli buffer. Resuspended samples were placed in a boiling water
bath for 5 min and microcentrifuged for 10 min prior to loading on denaturing 10% polyacrylamide gels. Proteins were transferred to
polyvinylidene difluoride membranes for immunoblotting.
For analysis of the interaction of AGS3-GPR with multiple G-protein
subunits, G
i2 (200 nM) was incubated with
G
i3 (50 nM) in the presence or absence of
the AGS3-GPR GST fusion protein (250 nM) in 250 µl of
buffer A (20 mM Tris, pH 7.5, 70 mM NaCl, 1 mM dithiothreitol, 0.6 mM EDTA, 0.01% Thesit)
for 1 h at 4 °C. G
i3 antisera (1: 500) was
added, and the incubation was continued for 3 h at 4 °C.
Protein complexes were isolated and evaluated by immunoblotting as
described above.
Protein interaction assays using purified G-protein subunits were
conducted as described previously (4, 18). All purified G-proteins used
in these studies were isolated in the GDP-bound form. Unless indicated
otherwise, all G-protein interaction assays contained 10 µM GDP. The AGS3-GST fusion proteins were expressed in
and purified from bacteria using a glutathione affinity matrix. The
AGS3-GST fusion proteins were eluted from the matrix with glutathione
and desalted by centrifugation (Centricon YM-3; Millipore, Bedford,
MA). For interaction assays with cell/tissue lysates, the AGS3-GST
fusion protein (100-300 nM) was incubated with purified G-protein (50-100 nM) or cell/tissue lysate (~4 mg of
protein/ml) for 1 h at 24 °C in a total volume of 250 µl.
12.5 µl of packed glutathione-Sepharose slurry was added and the
mixture rotated at 4 °C for 20 min, after which the affinity matrix
was pelleted and washed three times with 500 µl of incubation buffer.
Proteins retained on the matrix were solubilized in 2× Laemmli loading buffer and separated by electrophoresis on denaturing 10%
polyacrylamide gels. Proteins were transferred to polyvinylidene
difluoride membranes for immunoblotting. Each blot was checked by Amido
Black staining to verify equal loading of fusion proteins.
Immunofluorescence--
DDT1-MF2 control cells and
DDT1-MF2 cells stably transfected with AGS3 were plated
onto coverslips (18-mm round no. 1) precoated with 0.01% polylysine
and allowed to grow to 60% confluence. Coverslips were then rinsed
with 3 × 2 ml of cell washing solution (CWS) (137 mM
NaCl, 2.6 mM KCl, 1.8 mM
KH2PO4, 10 mM
Na2HPO4) and fixed in 4% paraformaldehyde for
10 min, followed by two 5-min incubations in CWS containing 0.1 M glycine (3 ml/coverslip). Coverslips were then incubated
in 0.01% Triton X-100 for 10 min, followed by three 5-min incubations
(3 ml/coverslip) with CWS. Fixed cells were then incubated in 10% goat
serum for 1 h and washed once with CWS. AGS3 antibody was diluted
into CWS containing 2% goat serum and 1% fetal bovine serum and then
centrifuged at 10,000 × g for 10 min prior to use.
Coverslips were incubated with 75 µl of AGS3 antibody (0.01 mg/ml)
for 1 h by placing the coverslips (cell side down) on parafilm in
a humidified chamber. Following incubation with AGS3 antibody,
coverslips were washed three times (3 ml/coverslip) with CWS and then
incubated with goat anti-rabbit biotin conjugate (1:800) for 40 min.
The fixed cells were washed three times in CWS and incubated in
Extravidin fluorescein isothiocyanate (1:500) for 40 min, followed by
three 10 min incubations with CWS. Washed coverslips were mounted in
Immuno Fluore, sealed with nail polish, and stored at 4 °C until
evaluated by fluorescent microscopy. All incubations were carried out
at 24 °C. Mounted slips were evaluated on a Leica DMLB fluorescent
microscope and by confocal microscopy using a Bio-Rad MRC-100 laser
scanning confocal imaging system. The cell nucleus was identified by
propidium iodide staining. Multiple series of experiments were
performed to determine the optimal conditions for signal detection and
to verify the specificity of observed signals. These experiments
included different methods of fixation and permeabilization as well as
a matrix with serial dilutions of primary antibodies and secondary
conjugates. We chose to generate stable transfectants to minimize any
artifacts introduced by transient transfection. Only very weak
immunofluorescence signals were detected in nontransfected
DDT1-MF2 cells as expected from the relative strengths of
the signals for control and AGS3-transfected cells observed by
immunoblotting. No immunofluorescent signal was detected in control or
AGS3 transfectants in the absence of any primary antibody.
Nucleotide Binding Assays--
Nucleotide binding assays were
conducted by a modification of a described previously techniques (2,
20). G-proteins (100 nM) were preincubated with varying
amounts of AGS3 subdomain proteins or GST controls for 15 min at
24 °C (binding buffer = 50 mM Hepes-HCl, pH 7.5, 1 mM EDTA, 2 mM MgCl2, 1 mM dithiothreitol, 50 µM adenosine triphosphate, 10 µg/ml bovine serum albumin) prior to addition of
0.5-1 µM GTP
S (4.0 × 104 dpm/pmol);
the final incubation volume was 50 µl. Samples were incubated with
GTP
S at 24 °C for 30 min. Incubated reactions were terminated by
rapid filtration through nitrocellulose filters (Schleicher & Schuell
BA85) with four 4-ml washes of stop buffer (50 mM Tris-HCl,
5 mM MgCl2, 1 mM EDTA, pH 7.4, 4 °C). Radioactivity bound to the filters was determined by liquid
scintillation counting.
Additional Methods--
DDT1-MF2 cells were stably
transfected with pcDNA3.AGS3 by DNA/calcium phosphate
coprecipitation (21). For antipeptide antisera, AGS3 peptides (P-32
Thr306-Ile436 and P-22
Asp528-Gly550) were synthesized and conjugated
for generation of rabbit polyclonal antisera using the Peptide
Synthesis and Antibody Production Facility at the Medical University of
South Carolina. Each of the three antisera specifically recognized
GST-AGS3 at reasonable dilutions of serum and were affinity-purified.
Denaturing gel electrophoresis and immunoblotting were performed as
described previously (18). For reprobing of membrane
transfers, the membrane transfers were washed with buffer A
containing 20 mM Tris-HCl, pH 7.6, 140 mM NaCl,
0.2% Tween each and then incubated with pre-heated stripping buffer
(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol) for 20 min in a 55 °C water bath with gentle
shaking. The membrane was then washed with buffer A and processed for
immunoblotting. For Coomassie Blue staining of proteins, gels were
incubated in 100 ml of staining buffer (0.25% Coomassie Blue in 45%
methanol, 45% H2O, 10% glacial acetic acid) for 30 min at
room temperature. Stained gels were then washed in 100 ml of destain
solution (45% methanol, 45% H2O, 10% glacial acetic
acid) and incubated for 30 min. Gels were then washed in fresh destain
solution every 30 min until protein bands were visible.
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RESULTS |
Expression Profile of AGS3 and Coimmunoprecipitation of AGS3 and
G-protein Subunits--
Immunoblots with AGS3 antipeptide antibodies
indicated expression of AGS3 (Mr ~ 74,000) in
rat brain, the neuroblastoma-glioma cell hybrid NG108-15 (rat/murine)
the rat pheochromocytoma cell line PC12, and the DDT1-MF2
smooth muscle cell line derived from hamster vas deferens (Fig.
2). Immunoreactive species with an apparent Mr of ~74,000 were not detected in
rat liver, rat kidney, Caco-2 cells, CHO cells, HEK cells, or NIH-3T3
fibroblasts (Fig. 2).3 The
same immunoreactive Mr ~74,000 species was
observed with two different antibodies (P-32, P-22) generated against
peptides derived from different regions of the protein (Fig. 2).
Fractionation of tissues/cells expressing AGS3 indicated that AGS3 is
enriched in the 100,000 × g supernatant consistent
with a major distribution of AGS3 in the cytosol (Fig.
3A). A similar fractionation
of AGS3 was observed in DDT1-MF2 cells stably transfected
with AGS3 (Fig. 3A). The subcellular localization of AGS3
was also addressed by immunofluorescence analysis following stable
expression of AGS3 in the DDT1-MF2 cell line (Fig.
3B). Confocal microscopy was used to generate an image
approximately through the middle plane of the cell. The
immunofluorescent image indicates that AGS3 is predominantly cytosolic
(Fig. 3B), as suggested by immunoblot analysis of the 100,000 × g supernatant from cell lysates illustrated
in Fig. 3A. Within the cell, the AGS3 signal is often
punctate and occasionally enriched in microdomains of the cell.

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Fig. 2.
Expression of AGS3 in different tissues and
cells. Tissues and cells were homogenized in Nonidet P-40 lysis
buffer and processed as described under "Experimental Procedures."
Membrane transfers were first immunoblotted with AGS3 antisera P-32
(upper panel). The blot was then stripped and
reprobed with another AGS3 antisera P-22 (lower
panel). The immunoblots are representative of results
obtained in five different experiments. The lines to the
right of the blot indicated the migration of size standards
(low molecular weight; Bio-Rad) × 10 3.
The arrows to the left of the immunoblot indicate
the migration of AGS3. Each lane contains 100 µg of protein.
CHO, Chinese hamster ovary K1 cell line; DDT,
DDT1-MF2 cells.
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Fig. 3.
Subcellular distribution of AGS3.
A, tissues or confluent dishes of cell lines were
fractionated into a 100,000 × g supernatant
(S) and 100,000 × g pellet (P)
as described under "Experimental Procedures." Membrane transfers
were immunoblotted with AGS3 antisera P-32. The immunoblot is
representative of results obtained in three different experiments. The
lines to the right of the blot indicated the
migration of size standards (low molecular weight; Bio-Rad) × 10 3. The arrows to the
left of the immunoblot indicate the migration of AGS3. Each
lane contains 100 µg of protein. DDT, DDT1-MF2
cells; DDT-AGS3, DDT1-MF2 cells stably
transfected with rat AGS3. B, immunofluorescent analysis of
AGS3 distribution in DDT1-MF2 cells stably transfected with
AGS3. Cells were fixed and processed as described under "Experimental
Procedures." Confocal microscopy was used to evaluate images through
different planes of the cells, and the micrograph shown is the image
taken from approximately the middle plane of the cells. The large
rounded area in the middle of the cell devoid of signal corresponds to
the cell nucleus as defined by propidium iodide staining. This image
was generated with a Bio-Rad MRC-100 laser scanning confocal imaging
system (magnification, ×63; 30% laser power; gain, 1250; IRS, 0.7).
This figure is representative of five to seven images obtained by
different fixation methods and using different antibody concentrations.
Only very weak immunofluorescence signals were detected in
nontransfected DDT1-MF2 cells as expected from the relative
strengths of the signals for control and AGS3-transfected cells
observed by immunoblotting in A. No immunofluorescent signal
was detected in control or AGS3 transfectants in the absence of any
primary antibody.
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We previously reported that the carboxyl-terminal 74 amino acids of
AGS3 were active in the yeast functional screen and that this peptide
fragment directly bound to G
i (4). We thus asked if
full-length AGS3 was complexed with G
i3 in lysates of
rat brain or DDT1-MF2 cells stably transfected with AGS3.
As AGS3 preferentially regulated G
i2 and
G
i3 in the yeast functional assay (4), we first
approached this issue by immunoprecipitation of G
i3.
Approximately 30% of brain lysate G
i3 was
immunoprecipated with a G
i3 carboxyl terminus antibody.
Immunoblots of membrane transfers containing G
i3
immunoprecipitates indicated that AGS3 coimmunoprecipitated with
G
i in a nucleotide-dependent manner (Fig.
4). The absence of G
in the
GTP
S-treated samples provided internal controls for G-protein
activation and subunit dissociation by added GTP
S/Mg2+.
Immunoprecipitation experiments were also conducted with the AGS3
antisera P-32. Although AGS3 was effectively immunoprecipitated by the
P-32 antisera in each cell/tissue extract, coimmunoprecipitation of
G
i3 was variable, which may reflect lower
immunoprecipitation efficiency for P-32 and/or a masking of the P-32
epitope in the AGS3-G
complex (data not shown).3
Nevertheless, these data indicated that a subpopulation of
G
i3 and AGS3 exists as a complex in the cell and that
this interaction is regulated by nucleotide binding to G
.

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Fig. 4.
Coimmunoprecipitation of AGS3 and
G i3. Rat brain (2.5 mg;
A) and DDT-AGS3 cell (1.25 mg; B) lysates were
preincubated with 30 µM GDP or 30 µM
GTP S/25 mM MgCl2 at 24 °C for 30 min.
Lysates were then precleared and G i3 immunoprecipitated
as described under "Experimental Procedures." Membrane transfers
were first blotted with AGS3 P-32 antisera and then stripped and
sequentially reprobed with G i and G antisera. Input
lanes contained 5 µl (A) or 20 µl (B) of the
Nonidet P-40 lysate used for immunoprecipitation. The aberrant
migration of G i and G in the input lane in
B is due to the larger amount of 1% Nonidet P-40 in the
samples. The data are representative of two to four experiments. The
input lane contains one-tenth of the lysate volume used for
immunoprecipitation. IP, immunoprecipitation; IB,
immunoblot. Antipeptide antisera recognizing AGS3 were generated as
described under "Experimental Procedures." P-32 antisera was
purified on a peptide affinity matrix and used for immunoblotting at a
concentration of 1.0 µg/ml. DDT-AGS3, DDT1-MF2
cells stably transfected with rat AGS3.
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AGS3 Domains That Interact with G-proteins--
The interaction
between AGS3 and G-proteins was further explored in in vitro
binding assays to define the regions of AGS3 actually involved in
binding to G
. We generated the amino-terminal half of AGS3
(AGS3-TPR, Met1-Ile462) and the COOH-terminal
half of AGS3 (AGS3-GPR, Pro463-Ser650) as GST
fusion proteins (Fig. 5). The AGS3-TPR,
AGS3-GPR, and the 74 amino acid carboxyl terminus (AGS3-CT,
Met577-Ser650) isolated in the original yeast
functional screen were incubated with DDT1-MF2 cell lysates
and proteins bound to the AGS3 subdomains identified by immunoblotting
of gel transfers. The G
i1/2 binding domains of AGS3 were
found in the COOH-terminal half of the protein (Fig. 5B,
left panel). The TPR domains of AGS3 did not
interact with G
i1/2 or G
(Fig. 5B,
left panel).

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Fig. 5.
AGS3 domains interacting with
G-proteins. Subdomains of AGS3 (A) were generated as
GST fusion proteins and purified following expression in bacteria for
protein interaction studies as described under "Experimental
Procedures." The left panels in B
and C are Coomassie Blue-stained gels indicating the
migration of GST and each of the fusion proteins (1 µg) used for
protein interaction studies. Right panels of
B and C, lysates were prepared from
DDT1-MF2 cells and 1 mg of lysate protein was incubated
with 300 nM GST-AGS3 fusion proteins as described
previously (4). Membrane transfers of bound proteins were probed with
G-protein subunit antisera. B, TPR,
Met1-Ile462; GPR,
Pro463-Ser650; CT,
Met577-Ser650. The Roman
numbers in C correspond to the GPR domains in
A: GPR-I, Pro463-Glu501;
GPR-II, Ser516-Leu555;
GPR-III, Gly563-Thr602;
GPR-IV, Thr602-Ser650. Similar
results were obtained in 3-5 individual experiments using different
batches of lysate. The input lane contains one-tenth of the lysate
volume used in each individual interaction assay.
|
|
Within the COOH-terminal region of AGS3 that binds to G
, there are
four repeats (~20 residues each) termed GPR for G-protein regulatory
motifs (4). Previous data indicated that the 74-amino acid domain at
the carboxyl terminus of AGS3 was functional in the yeast functional
screen and the interaction of this peptide with G
i was
disrupted by targeted mutations in GPR IV highlighting the importance
of the GPR motifs. We then asked if each GPR domain was indeed capable
of binding G
. Each GPR motif was generated as a GST fusion protein
(Fig. 5) and evaluated in protein interaction assays using
DDT1-MF2 lysates (Fig. 5B, right
panel). Each GPR motif bound G
i1/2, although
GPR I, at least in this context, bound less G
than did GPR II-IV
(Fig. 5B, right panel). These data
suggest that interaction of AGS3 with G
i3 observed by
coimmunoprecipitation experiments (Fig. 4) reflects interaction of
G
i3 with the GPR domains in AGS3.
The preceding data also suggested that AGS3 is capable of binding
multiple G
i subunits. To address this issue, we asked if a GST-AGS3 fusion protein containing GPRs I-IV indeed bound more than
one G
i at the same time. A GST-AGS3 fusion protein
containing GPRs I-IV was incubated with a mixture of G
i3
and G
i2. Samples were then immunoprecipitated with
antisera directed against the carboxyl terminus of G
i3.
In the presence of AGS3, G
i2 was also found in the
G
i3 immunoprecipitate (Fig.
6). G
i2 was not found in
the G
i3 immunoprecipitate in the absence of AGS3 (Fig.
6). These data clearly indicate that AGS3 is capable of binding more than one G
i subunit consistent with a putative role of
AGS3 as a scaffolding protein within a larger signal transduction
complex.

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Fig. 6.
Interaction of AGS3 with multiple
G i subunits.
G i2 (200 nM) was incubated with
G i3 (50 nM) in the presence or absence of
the AGS3-GPR GST-fusion protein (250 nM). Samples were
immunoprecipitated with G i3 antisera and processed for
SDS-PAGE as described under "Experimental Procedures." Membrane
transfers were immunoblotted with the indicated antisera. The
arrows to the left indicate the migration of the
indicated proteins. Similar amounts of G i3 antisera were
pelleted in each lane as indicated by detection with secondary
antibody. Input lanes represent one-tenth of the the sample processed
for immunoprecipitation. This experiment is representative of three
such experiments.
|
|
Selectivity of AGS3 for G-proteins--
The preceding data clearly
established the interaction of AGS3 with G
within the cell and
defined the regions of AGS3 involved in G-protein binding. We then
asked if the interaction of AGS3 with G
was selective for different
G-protein families. We approached this question using crude tissue/cell
lysates and purified G
subunits. The AGS3-GPR GST fusion protein was
incubated with rat brain lysate and bound proteins identified by
immunoblotting with G
specific antisera. AGS3-GPR effectively bound
G
i1-3, but not G
s, G
o,
G
q, or G
(Fig.
7A). Based upon the comparison of the signal intensity in the input versus sample lane, it
is estimated that AGS3-GPR binds ~20-% of the total
G
i protein in the lysate sample. Similar results were
obtained in DDT1-MF2 cell lysates. Each of the protein
interaction experiments in the tissue/cell lysates were done in the
presence of GDP which would stabilize heterotrimeric G

;
however, immunoblotting with G-protein
subunit antisera indicated
that AGS3 was complexed with G
i in the absence of G
(Fig. 7A). Thus, either AGS3 effectively promoted subunit
dissociation or there is a population of G
i that exists free of G
. The selectivity of AGS3 for different G-proteins was
also observed using purified G
subunits. AGS3 bound to
G
i1-3 and purified G
t, but it did not
interact with G
s and weakly bound G
q and
G
o (Fig. 7B). A similar profile of AGS3
selectivity for G
subunits was also observed in a yeast functional
assay (4). Comparison of the relative intensities of the bound G
versus input G
for G
o/G
q
and G
i (Fig. 7B) indicated a higher apparent
affinity of AGS3 for Gi versus
Go/Gq, which may account for the inability of
AGS3 to interact with G

q and
G

o in brain lysates (Fig. 7A).

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Fig. 7.
Selective interaction of AGS3 with G-proteins
from brain lysates and with purified G-proteins. One milligram of
lysate protein from rat brain was incubated with 300 nM
GST-AGS3 fusion (TPR Met1-Ile462, GPR
Pro463-Ser650, CT
Met577-Ser650) as described under
"Experimental Procedures". Membrane transfers of bound proteins
were probed with the indicated antisera. B) Recombinant G (100 nM) or purified G t (100 nM) were
incubated with 300 nM GST or AGS3-GPR. All interactions
were done in the presence of 10 µM GDP. The input lanes
represent one-tenth of the G-protein used in each interaction assay.
Membrane transfers of bound proteins were probed with the indicated
antisera with intervening stripping of the blot as described under
"Experimental Procedures." The G i3 antibody exhibits
some cross-reactivity with G o that likely accounts for
the broad immunoreactive band observed in the input lane in
panel A. Similar results were obtained in two to
three separate experiments.
|
|
AGS3 and G-protein Activation--
As both AGS3 and G
interact with the GDP-bound conformation of G
, the two proteins may
actually compete with each other for interaction with G
and thus
AGS3 would essentially promote subunit dissociation in the absence of
nucleotide exchange. This issue was addressed by determining the
influence of G
on the interaction of AGS3 with G
t.
We first compared the ability of AGS3 to interact with purified
G
t versus heterotrimeric Gt (Fig. 8A). At equimolar
concentrations of purified G
t and heterotrimeric Gt, AGS3 bound equivalent amounts of G
t. As
observed with the AGS3-G
i complex isolated from
tissue/cell lysates, G
was not present in the
AGS3-G
t complex isolated from purified heterotrimeric Gt, indicating that AGS3 effectively dissociated
Gt from G
. We thus asked if G
would interfere
with formation of the AGS3-G
t complex. In these
experiments, G
t was first incubated with equimolar or
excess G
to generate heterotrimeric Gt prior to
exposure of the complex to AGS3. The interaction of AGS3 with G
was
not altered by G
at concentrations equivalent to G
, as
observed in the experiments using heterotrimeric Gt (Fig.
8B), but it was completely blocked by 10-fold higher
concentrations of G
(Fig. 8B), indicating that AGS3
and G
are effectively competing with each other for binding to
G
GDP.

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Fig. 8.
Influence of
G on the interaction of AGS3
with G t. A,
AGS3-GPR (300 nM) was incubated with purified
G t or G  t (100 nM) and
processed for protein interaction studies as described under
"Experimental Procedures." The data presented are representative of
five individual experiments with G t and two experiments
with G  t using different batches of fusion
proteins. B, AGS3-GPR (100 nM) was added to
tubes containing G t (50 nM) that had been
preincubated with G (50 or 500 nM) and samples
processed for protein interaction assays. Similar data were obtained in
two experiments. The blot in A was first probed with the
G antisera and then stripped for reprobing with the G antisera.
The input lane in A and B, respectively, contain
one-tenth and one-fifth of the lysate volume used in each interaction
assay.
|
|
The interaction of AGS3 with G
may actually stabilize the GDP-bound
or nucleotide-free conformation of G
and "free up" G
for
downstream signaling. Indeed, this conjecture would account for the
biological activity of AGS3 in the yeast functional assay (4), where
G
is responsible for subsequent activation of the pheromone
response pathway. To address this issue, we asked if AGS3 influenced
the guanine nucleotide binding properties of G
i.
AGS3-GPR blocked the binding of GTP
S to G
i1
(IC50 ~ 0.1 µM) (Fig.
9A). We had previously
identified key amino acid residues in GPR-IV that disrupted binding of
AGS3-CT to G
i (4), and we then examined the effect of
this series of AGS3-CT mutants on GTP
S to G
i1. The
AGS3-CT peptides containing GPR mutations that resulted in a loss of
binding to G
i in protein interaction assays (F609R,
R624F) (4) were also ineffective at inhibiting GTP
S binding to
G
i1 (Fig. 9B). These data, and the results
obtained in protein interaction experiments where AGS3 preferentially
binds G
GDP versus
G
GTP
S, suggest that AGS3 actually
stabilizes the G
GDP or nucleotide-free conformation and
functions as an inhibitor of guanine nucleotide exchange on G
. These
biochemical data are consistent with the functional properties of AGS3
in S. cerevisiae in that the action of AGS3 did not require
the generation of G
GTP and it was not antagonized by
overexpression of the GTPase activating protein RGS4
(4).4

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Fig. 9.
Effect of AGS3-GPR on nucleotide binding to
G . A, GTP S binding (1.0 µM GTP S, 4 × 104 dpm/pmol) to
G i1 (100 nM) was measured in the presence of
increasing concentrations of GST or GST-GPR as described under
"Experimental Procedures." Data are expressed as the percentage of
specific binding (~5 pmol) observed in the absence of GST or GST-GPR.
B, GTP S binding (0.5 µM GTP S, 4 × 104 dpm/pmol) to G i1 (100 nM)
was measured in the absence or presence (1 µM) of GST,
AGS3-GPR, AGS3-CT, and AGS3-CT constructs containing mutations that
disrupt AGS3-CT binding to G in protein interaction assays (4). Data
are expressed as the percentage of specific binding (~3.1 pmol)
observed in the absence of GST. Data in A and B
are presented as the mean ± S.E. of two experiments.
|
|
 |
DISCUSSION |
A large number of diverse signaling mechanisms within the cell
utilize guanine nucleotide-binding proteins as a molecular switch to
process biological signals. Due to the central place of these events in
signal propagation, several mechanisms have evolved to turn this switch
on and off. Such mechanisms include the regulation of guanine
nucleotide exchange (e.g. guanine nucleotide exchange
factors, guanine nucleotide dissociation inhibitors), hydrolysis
(GTPase-activating proteins) and the subcellular targeting of
G-proteins themselves. In general, signal processing by
G-protein-regulated systems, as is the case for single membrane span
receptors, likely operates within the context of a dynamic signal
transduction complex. Such a multiprotein complex may provide
coordinated and integrated functionality for heterotrimeric G-protein
signaling systems, which process a myriad of external stimuli via
G-protein-coupled receptors. Within such a complex there are likely
accessory proteins distinct from receptor, G-protein and effector that
influence various aspects of signal propagation. Such proteins may: 1)
determine the specific pathway that the signal travels, 2) provide a
cell-specific mechanism for signal amplification, 3) influence the
population of activated G-protein/effector within the cell independent
of receptor activation, 4) be "effectors" subject to receptor
regulation providing attractive targets for cross-talk between diverse
signaling systems, 5) provide alternative modes of input to
G-protein-regulated signaling pathways independent of a classical
G-protein-coupled receptor, and/or 6) serve as scaffolding proteins to
organize a signal transduction complex.
AGS3 is one of three mammalian cDNAs isolated in an expression
cloning system in S. cerevisiae as receptor-independent
activators of heterotrimeric G-protein signaling (3-5). Epistasis
experiments in the yeast system indicated that the three cDNAs
activated the pheromone response pathway at the level of G-protein, and
the proteins were therefore termed activators of G-protein signaling (AGS1-3). Both cellular and/or in vitro studies indicated
that these proteins exhibited selectivity for G-proteins and used
different mechanisms to activate G-protein signaling. AGS1 is a novel
Ras-related protein that directly increases GTP
S binding to G
(3,
5). AGS2 is identical to mouse Tctex1, a protein that exists as a light
chain component of the cytoplasmic motor protein dynein and subserves
as yet undefined functions in cell signaling pathways.
The two domain structure (TPR and GPR motifs) of AGS3 is highly
conserved. TPR motifs serve a range of functions for diverse proteins
(9, 10, 22, 23). The TPR domains of Rapsyn, which contains an
organization of TPR motifs most closely related to the AGS3 TPR
domains, are involved in clustering of nicotinic receptors at the
neuromuscular junction (23). Studies with the D. melanogaster AGS3/LGN homolog PINS suggest a role for the TPR domains in trafficking of AGS3 within the cell (9, 10). In D. melanogaster PINS binds to Inscuteable, and this interaction is
required for placement of key proteins (Inscuteable and Bazooka) involved in polarization and proper orientation of mitotic spindles of
neuroblasts during asymmetric cell division of neuroblasts (9, 10).
G
i/G
o is apparently complexed with the
PINS/Inscuteable complex, where it presumably plays a signaling
function. In mammalian tissues, AGS3 is expressed at highest levels in
brain where it is primarily found in a 100,000 × g
supernatant following homogenization. AGS3 may oscillate between
cytosol and membrane compartments as observed for PINS (AGS3/LGN
ortholog) in D. melanogaster.
AGS3 selectively binds to G
i in the presence of GDP, and
protein interaction assays with AGS3-GPR indicate that the
AGS3-GPR-G
complex is free of G
suggesting the following
possibilities. First, AGS3 binds to G-protein heterotrimer (G

)
and actively promotes subunit dissociation, while maintaining G
in
the GDP-bound state. Second, AGS3 "catches" a transient
nucleotide-free conformation of G
i and this interaction
is stabilized by binding of GDP to G
with AGS3 replacing G
as
a G
binding partner. Third, during "basal" cycling of the G
through its various states of activation/inactivation, there is a
period when G
is free of G
, allowing AGS3 to bind G
and
exclude rebinding of G
. Each possibility could account for the
activity of AGS3 in the yeast functional screen, where the pheromone
response pathway is activated by G
(4), and each is consistent
with the biochemical data indicating that G
and AGS3 compete with
each other for interaction with G
. Thus, the activity of AGS3 as a
receptor-independent activator of G-protein signaling may actually
involve dissociation of G
and G
in the absence of nucleotide
exchange "releasing" G
from G
GDP to activate downstream effectors. In such a scenario, G
bound to AGS3 may be
functionally inert and signal termination would require dissociation of
AGS3 and G
GDP with rebinding of G
and
G
GDP. Each of these scenarios are also of note relative
to the role of AGS3 (PINS)-G
i/G
o complexes in neuroblast processing in D. melanogaster and
the defect in orientation of the mitotic spindle observed in the
absence of PINS (9, 10). In the latter situation, interaction of AGS3
(PINS) with G
i(GDP)/G
o(GDP) could
"release" G
for effector regulation, which may involve the
apparent localization of G
to microtubules and/or mitotic
spindles (24, 25). The detailed analysis of the interaction of AGS3
with G
presented in this report should greatly facilitate efforts to
further define the role of AGS3 in signal processing in mammalian systems.
Another possible explanation for the detection of AGS3-G
complexes
that do not contain G
is that there is a population of
G
i in the cell that exists free of G
. In such a
case, AGS3 may regulate the activation state of G
in much the same
way as does G
. AGS3-G
complexes may be regulated by unexpected
modes of signal input, which promote nucleotide exchange on the
AGS3-G
GDP complex and release "activated" or
"functional" AGS3 and G
i(GTP). An interaction of
AGS3 with G
in the absence of G
may also function to hold G
in the right place so that the signal input is more effectively
processed and as such the two-domain structure of AGS3 may serve as a
type of scaffold within a larger signal transduction complex. By virtue
of its potential to bind up to three and possibly four G
subunits,
AGS3 could "seed" oligomeric structures of G
i (26,
27), which might mesh with arrays of other signaling molecules. AGS3
may actually be complexed with a mixture of G
i1-3.
As noted earlier, the GPR motif is also found in other proteins that
interact with or regulate G-protein
subunits, and one would
hypothesize that the GPR motif has evolved to serve as an anchor for
proteins to bind to G
subunits.5 The GPR domains
are found in proteins that have apparently different effects on the
activation state of G-protein. The GTPase-activating proteins RGS12 and
RGS14 both contain a GPR motif, as does the Purkinje cell-specific
protein Pcp2. The latter protein was reported to activate brain
G-protein by accelerating guanine nucleotide exchange on heterotrimeric
G-proteins (28). The functional and biochemical studies with the
AGS3-GPR motifs indicate that this motif actually behaves as an
inhibitor of guanine nucleotide exchange on G
. As a relatively small
discrete structure that binds to G-protein
subunits, the GPR motif
may serve as a template for rational design of peptides/small molecules
that directly influence the activation state of G-protein.
 |
ACKNOWLEDGEMENTS |
S. M. L. thanks Dr. Starr Hazard
(Medical University of South Carolina, Charleston, SC) for discussions
on the AGS3 protein motifs and Dr. Shigeo Ohno (Yokohama City
University School of Medicine, Yokohama, Japan) for helpful discussions
on cell polarity/development. S. M. L. appreciates the
preprint of Ref. 10 provided by Dr. Knobich. S. M. L. appreciates the continued thoughtful input of Drs. Emir Duzic
(Millenium Pharmaceuticals) and Mary Cismowski (Neurocrine Sciences).
We thank John D. Hildebrandt (Medical University of South Carolina) for
providing G-protein antisera and brain G-protein 
subunits. We
thank Elliot Ross (University of Texas Southwestern Medical Center) for
G
s/G
q, Stephen Graber (West Virginia
University School of Medicine, Morgantown, WV) for
G
i1-3 and G
o, Heidi Hamm (Northwestern
University Medical School, Chicago, IL) for purified G
t
and G

t, Thomas W. Gettys (Medical University of
South Carolina) for G
i3 antisera, and Drs. Goldsmith,
Shenkar, and Spiegel for GA antibody. We appreciate the discussions of this work with Drs. Graber, Hildebrandt, Ross, and Artemyev and the
technical support of Lena Spangler. We are very grateful to Drs. Robert
Gourdie, Robert Thompson, Steven Rosenzweig, and Tim McQuinn
(Medical University of South Carolina) for assistance with microscopy.
 |
FOOTNOTES |
*
This work was supported in part by Grants NS24821 and MH5993
(to S. M. L.) from the National Institutes of Health.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.
Recipient of a Medical Scientist Training Program fellowship,
supported by National Institutes of Health Grant T32-GM08716.
§
To whom correspondence should be addressed: Dept. of Pharmacology,
Medical University of South Carolina, 173 Ashley Ave., Charleston,
SC 29403. Tel.: 843-792-2574; Fax: 843-792-2475; E-mail: laniersm@musc.edu.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M005291200
2
The GPR motif was also termed the GoLoco motif
(6).
3
M. L. Bernard and S. M. Lanier,
unpublished observations.
4
These data contrast with the properties of AGS1,
isolated in the same functional screen, in that the action of AGS1 is
blocked by overexpression of RGS4 and requires the formation of G
GTP (3, 4). In further contrast to AGS3, AGS1 functions as a guanine
nucleotide exchange factor promoting the exchange of GTP for GDP on
G
(5).
5
RNA blot analysis indicated that the mRNA
transcript encoding the 650-amino acid AGS3 protein was selectively
enriched in rat and human brain (N. Pizzinat, A. Takesono, and S. M. Lanier, submitted for publication).
 |
ABBREVIATIONS |
The abbreviations used are:
AGS, activator of
G-protein signaling;
TPR, tetratricopeptide repeat motif;
GPR, G-protein regulatory motif;
PINS, Partner of Inscuteable;
CWS, cell washing solution;
GST, glutathione S-transferase;
CHO, Chinese hamster ovary;
GTP
S, guanosine
5'-3-O-(thio)triphosphate.
 |
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