From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041
Received for publication, November 21, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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The activation of heterotrimeric G proteins is
accomplished primarily by the guanine nucleotide exchange activity of
ligand-bound G protein-coupled receptors. The existence of nonreceptor
guanine nucleotide exchange factors for G proteins has also been
postulated. Yeast two-hybrid screens with G Heterotrimeric guanine nucleotide-binding regulatory proteins
mediate signal transduction between many membrane-bound receptors and
intracellular effectors (1). Traditionally, activation of
heterotrimeric G proteins1 is
accomplished exclusively by the action of GPCRs, seven
transmembrane-spanning proteins that typically reside in the plasma
membrane. These receptors act as guanine nucleotide exchange factors
(GEFs), binding the inactive GDP-bound conformation of G proteins and
stimulating release of GDP from G G protein signaling is attenuated when G Nonreceptor activators of G proteins may operate in lieu of or in
conjunction with GPCRs to enhance signaling, but their physiological role is not well understood (8-11). Activators of G protein signaling AGS1 and AGS3 were identified in a genetic screen in yeast designed to
isolate expressed mammalian cDNAs that encode proteins that bypass
the need for a receptor (12). AGS3 possesses G Other newly appreciated proteins may also act as novel modulators or
effectors of heterotrimeric G proteins. Genetic evidence indicates that
synaptic transmission in Caenorhabditis elegans is
controlled by an antagonistic Gq/Go signaling
network that regulates intracellular concentrations of
diacylglycerol (14-16). G In an effort to identify novel G protein signaling factors, we
initiated yeast two-hybrid screens with G Molecular Cloning and Yeast Two-hybrid Experiments--
The open
reading frames of wild type and the activated (GTPase-deficient, Q227L
of G
The rat RIC-8A prey clone isolated in the two-hybrid screen lacked the
first 9 coding nucleotides of the equivalent mouse and human
RIC-8/synembryn sequences in the data base. These missing nucleotides
were repaired by PCR with codons for the first three amino acids of
mouse RIC-8/synembryn (NP_444424). These residues are identical among
mouse and human Ric-8A and C. elegans Ric-8. The repaired
rat RIC-8A PCR product was cloned into the baculovirus donor construct,
pFastBacGSTTev. pFastBacGSTTev was created by inserting a GST tag and a
Tev protease cleavage site into the vector pFastBac-1 (Invitrogen,
Inc.). The open reading frame of the rat RIC-8B clone obtained in the
two-hybrid screen was amplified by PCR with a 5' oligonucleotide
containing a BamHI linker and an oligonucleotide that
annealed 3' to the gene in the prey vector. This product was digested
with BamHI and SalI and cloned into the
His-tagged baculovirus donor vector pFastBacHTa (Invitrogen).
To clone the human cDNA transcripts that encode the 531 and the
putative 401-amino acid Ric-8A variants (see "Results"), total RNA
was prepared from HeLa cells using an RNeasy Maxiprep kit (Qiagen), and
cDNA was generated using a RETROscript kit (Ambion, Inc.). The
human RIC-8A cDNA encoding the 531-amino acid protein was amplified
using PCR primers derived from the DNA sequence in
GenBankTM (AL390088). Primers designed to amplify the
hypothetical 401-amino acid variant were derived from the incomplete
cDNA sequence (AK022870) found in GenBankTM, but we
were not able to amplify any product from either HeLa or NTERA2 cell
cDNA. This variant was generated using the 531-amino acid
protein-encoding cDNA as PCR template and a 3' PCR primer that
corrected the missing single base pair insertion of this putative
cDNA. Both amplified products contained EcoRI and
SalI linkers added by the primers. The products were
digested with these restriction enzymes and cloned into those
restriction sites in the mammalian expression vector pCMV5 (25), the
two-hybrid prey vector pGADGH (26), pFastBacHTa (Invitrogen, Inc.), and pFastBacGSTTev.
Protein Purification--
Baculoviruses encoding GST-Ric-8A and
His6-Ric-8B were produced and amplified using methods in
the Bac-to-Bac Sf9 cell transfection system (Invitrogen,
Inc.). To prepare the recombinant Ric-8A, Sf9 cells growing
in IPL41 medium with 1% fetal bovine serum, 0.1% pluronic acid, 1%
chemically defined lipid concentrate (Invitrogen), and 10 µg/ml
gentamicin at a density of 2 × 106 cells/ml were
infected for 48 h with amplified GST-Ric-8A baculoviruses. These
cells were pelleted and lysed in lysis buffer (20 mM
NaHepes, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor mixture
(phenylmethylsulfonyl fluoride,
N
Myristoylated G GST-Ric-8A Binding to G Proteins in Membrane Detergent
Extracts--
GST-Ric-8A affinity resin or control resin was prepared
by incubating purified GST-Ric-8A (37 µg) or buffer with
glutathione-Sepharose 4B (bed volume, 25 µl) (Amersham Biosciences)
for 1 h at 4 °C in extraction buffer (20 mM
NaHepes, pH 8.0, 150 mM NaCl, 2 mM MgSO4, 1 mM EDTA, 1 mM
dithiothreitol, and 1% C12E10). The resin was collected by brief
centrifugation and washed twice with 1 ml of extraction buffer.
Rat brain membrane extracts were prepared by homogenizing whole rat
brains in 10 mM Tris-HCl, pH 8.0, 11% sucrose, and
protease inhibitor mixture using a Dounce homogenizer. The homogenate
was centrifuged at 500 × g to remove debris, and the
supernatant was sequentially centrifuged at 12,000 × g
and 100,000 × g. The 100,000 × g
pellet (P100 membranes) was solubilized for 1 h in extraction buffer and centrifuged again at 100,000 × g. A volume
of detergent extract corresponding to 8 mg of extracted membrane
protein was then incubated with the GST-Ric-8A and control glutathione
resins. After incubation at 4 °C for 1 h, the resins were
washed twice with 1 ml of extraction buffer, suspended in 45 µl of
extraction buffer and incubated for 16 h at 4 °C with 5 µl of
recombinant Tev protease (Invitrogen). The resins were collected by
centrifugation, and the soluble proteins released by Tev protease were
resolved by SDS-PAGE; the gels also contained purified G protein
subunit standards. The gels were transferred to nitrocellulose and
blotted with the following G protein subunit-specific antisera: W082, G Formation of a G Gel Filtration of the Ric-8A·G Effect of Ric-8A on GTP
Steady-state GTPase reactions with G Mammalian Ric-8A and Ric-8B Interact with Subsets of G
Twenty-three positive clones were obtained using G
To test the interactions of Ric-8A and Ric-8B with various G GST-Ric-8A Interacts with Endogenous Membrane-derived G Ric-8A Interacts Preferentially with Nucleotide-free G
To further explore the identity of any guanine nucleotide associated
with the complex of G Ric-8A Stimulates GTP Ric-8A Stimulates the Steady-state GTPase Activity of
G
GPCRs interact with intact G protein heterotrimers to promote
nucleotide exchange. Ric-8A is capable of promoting such exchange with
individual
To test the effect of Ric-8A on the steady-state GTPase activity of
preformed G protein heterotrimers,
G We report the discovery of Ric-8A and Ric-8B as two homologous
G Mammalian Ric-8A and Ric-8B were identified as G Despite the fact that Ric-8A and GPCRs differ structurally and
mechanistically, understanding the physical mechanism by which Ric-8A
promotes guanine nucleotide exchange could provide important insights
into the mechanism of action of GPCRs. When GDP-bound G An important aspect of Ric-8 function yet to be resolved in terms of
traditional heterotrimeric G protein biology is the inability of Ric-8A
to activate trimeric forms of G proteins in vitro (Fig. 7).
Both GPCRs and the G protein activator AGS1 differ from Ric-8A in this
regard. GPCRs and AGS1 (9) promote exchange on G An intriguing question will be to determine the specific physiological
G An alternative and attractive hypothesis is that the targets of
Ric-8-like exchange factors are not plasma membrane-bound G protein o and
G
s as baits were performed to identify binding partners
of these proteins. Two mammalian homologs of the Caenorhabditis
elegans protein Ric-8 were identified in these screens: Ric-8A
(Ric-8/synembryn) and Ric-8B. Purification and biochemical
characterization of recombinant Ric-8A revealed that it is a potent
guanine nucleotide exchange factor for a subset of G
proteins
including G
q, G
i1, and G
o,
but not G
s. The mechanism of Ric-8A-mediated guanine
nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound
G
proteins, stimulates release of GDP, and forms a stable
nucleotide-free transition state complex with the G
protein; this
complex dissociates upon binding of GTP to G
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. To ensure directionality of
exchange, GEFs stabilize a nucleotide-free transition state of G
that is disrupted by binding of GTP (2, 3). This facilitates
dissociation of G
·GTP from the G
dimer and release of these
proteins from the receptor. Dissociated G protein subunits then
participate in interactions with a variety of effectors.
hydrolyzes the
phosphate of its bound GTP and G
·GDP reassociates with
.
GTPase-activating proteins (GAPs) facilitate the inactivation of many G
proteins. Most of these GAPs contain a regulator of G protein signaling (RGS) domain that binds preferentially to the G
·GTP transition state and accelerates GTPase activity (4, 5). More than 20 unique RGS
domain-containing proteins have been discovered, and the nature of
their G protein specificity and their mode of action in cells are
subjects of intense interest (6, 7).
guanine nucleotide
dissociation inhibitor activity but may activate G proteins by
liberating G
(10, 13). AGS1 encodes a Ras-like small GTPase that,
when bound to GTP, possesses in vitro guanine nucleotide
exchange activity for members of the Gi subfamily of G
proteins (9).
q is thought to enhance
synaptic transmission by activation of phospholipase C
, an enzyme
that cleaves phosphatidylinositol 4,5-bisphosphate into diacylglycerol
and inositol triphosphate (for review, see Ref. 17). Activation of
Go negatively influences synaptic transmission,
hypothetically by lowering concentrations of diacylglycerol (mechanism
unknown). Mutant screening and suppression analyses have revealed
proteins that likely operate in conjunction with G
q or
G
o in this pathway (18, 19). One class of these mutations was termed RIC (resistant to
inhibitors of cholinesterase). They were
selected for their ability to survive the neurotoxic effects of
cholinesterase inhibitors by causing a decrease in the amount of
acetylcholine released at the synapse (18). The gene that complemented
the ric-8 mutant allele, RIC-8/synembryn, appears to promote synaptic
transmission much like G
q. However, the sequence of the
encoded protein was not revealing (20). A more recent study also
demonstrated that reduction of function mutations of C. elegans RIC-8 or GAO-1 affects a pathway that regulates the
movement of centrosomes in dividing embryos (21).
o and
G
s as baits and found that two mammalian homologs of
Ric-8/synembryn, which we have termed Ric-8A and Ric-8B, are in fact
G
-binding proteins. Purification and biochemical characterization of
recombinant Ric-8A revealed that it is a potent guanine nucleotide
exchange factor for G
q, G
i1, and
G
o but not G
s. The mechanism of
Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A
interacts with GDP-bound G
subunits in the absence of G
,
causing release of GDP and formation of a stable, nucleotide-free
G
·Ric-8A complex. GTP, but not GDP, and then binds to G
and
disrupts the complex, releasing Ric-8A and the activated G
protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
s) mutants of G
s long,
G
o, G
q, G
i1, and
G
13 were amplified using the polymerase chain reaction with oligonucleotides containing BamHI and/or
SalI linkers. The respective G
PCR products were digested
and cloned into these same sites in the LexA two-hybrid bait
vector, pVJL11 (22). To initiate two-hybrid screens, the
LexA-G
o and LexA-G
s bait constructs were
transformed into the L40 yeast strain using the TE-lithium
acetate/PEG-4000 transformation method (23). Yeast harboring these bait
plasmids were then transformed with a rat brain embryonic cDNA
library contained in a pVP16 activation domain prey vector (a gift of
Thomas Südhof, University of Texas Southwestern Medical Center).
Approximately 106 individual transformants were screened
with each bait. For studies of pairwise two-hybrid interactions, yeast
co-transformed with specific bait and prey fusion combinations growing
in liquid culture were spotted onto complete synthetic medium plates
lacking tryptophan and leucine, grown, and subjected to a
-galactosidase filter assay as described (24).
-p-tosyl-L-lysine-chloromethyl ketone,
N-tosyl-L-phenylalanine-chloromethyl ketone,
leupeptin, and lima bean trypsin inhibitor; Sigma)) by nitrogen
cavitation. GST-Ric-8A was purified from the 100,000 × g supernatant fraction of these lysates by adsorbtion to
glutathione Sepharose 4B (Amersham Biosciences). The fusion protein was
eluted from the washed glutathione Sepharose with lysis buffer
containing 20 mM reduced glutathione and then exchanged
into lysis buffer. To remove the GST tag, the glutathione resin with
attached GST-Ric-8A protein was washed with lysis buffer and incubated
with Tev protease (Invitrogen) for 16 h at 4 °C. The cleaved,
eluted Ric-8A protein (amino acid sequence G-E-F-Ric-8A) was further
purified on a Hi-trap Q column (Amersham Biosciences) and eluted with a
linear gradient of NaCl (150 mM to 1 M). The
active protein eluted at ~375 mM NaCl and was exchanged
into lysis buffer. Active Tev-digested Ric-8A was resolved by 11%
SDS-PAGE and visualized with Coomassie Blue; it was also
trypsinized and subjected to mass spectrometry. Despite the fact that
Tev-digested Ric-8A co-migrated with protein standards of ~75 kDa on
SDS-PAGE, the predicted mass of the purified protein (>95%) of 60.1 kDa was confirmed by mass spectrometry (C. Thomas, University of Texas
Southwestern Medical Center). Attempts to purify nonaggregated
His6-Ric-8B have been unsuccessful to date.
o and nonmyristoylated G
i1
were purified from Escherichia coli as previously described
(27). Myristoylated G
i1 containing a His6
tag inserted after amino acid 121 was purified from E. coli
and was a gift from Roger K. Sunahara (University of Michigan).
G
s short was purified from E. coli and was a
gift from Mark E. Hatley (University of Texas Southwestern Medical Center). G
q and
1
2 dimers
were purified from Sf9 cells as previously described
(28).
q (29); S214, G
o (30); B087,
G
i1 and G
i2 (31); 584, G
s
(32); B860, G
13 (33); and B600, G
s1-4
(31). To visualize the bands recognized by G protein antisera, the
blots were processed with secondary antibody conjugated to horseradish peroxidase and treated with SuperSignal West Femto chemiluminescent reagent (Pierce) diluted 3-fold in phosphate-buffered saline.
·Ric-8A Complex--
Purified myristoylated
G
i1 (600 nM) was incubated in 500 µl of
HEMNDL buffer (20 mM NaHepes, pH 8.0, 1 mM
EDTA, 2 mM MgSO4, 150 mM NaCl, 1 mM dithiothreitol, and 0.05% C12E10) containing either 30 µM GDP; 30 µM GTP
S; or 10 mM
NaF, 30 µM AlCl3, and 30 µM GDP
for 1 h at 30 °C. Purified GST-Ric-8A was then added to a final
concentration of 200 nM, and these reactions were incubated for an additional 20 min at 20 °C. Glutathione-Sepharose (bed volume, 25 µl) in HEMNDL buffer was added, and the reactions were incubated for 1 h at 4 °C with gentle agitation. The resin was collected and washed consecutively with three 1-ml aliquots of cold
HEMNDL buffer containing the respective nucleotide and/or AlF
i1
Complex--
Nonmyristoylated G
i1 was chosen for gel
filtration analysis in lieu of myristoylated G
i1 protein
because the myristoylated protein and its associated detergent gave
aberrant elution profiles on gel filtration columns. Nonmyristoylated
G
i1 (26 µM) was incubated for 1 h at
30 °C with either 260 µM [35S]GTP
S or
[
-32P]GDP in gel filtration buffer (20 mM
NaHepes, pH 8.0, 150 mM NaCl, 2 mM
dithiothreitol, 2 mM MgSO4, 1 mM
EDTA, and protease inhibitor mixture). The reaction mixtures were then
diluted 2-fold by the addition of Tev-digested Ric-8A to a final
concentration of 10 µM in gel filtration buffer and were
subsequently incubated for 5 min at 22 °C. The mixtures were applied
to tandem Superdex 75/200 columns (HR 10/30) and resolved at a flow
rate of 0.2 ml/min. The amount of protein and radiolabeled nucleotide
in each fraction was determined by Bradford assay and scintillation
counting, respectively. A fixed volume of each odd-numbered fraction
(based on a sample of 750 ng of protein in the peak protein-containing
fraction) was resolved by SDS-PAGE, and the proteins were visualized
with Coomassie Blue.
S Binding and Steady-state GTPase
Activity of G
Proteins--
The kinetics of GTP
S binding was
assessed using a filter binding method (34). The reactions with
myristoylated G
i1, myristoylated G
o, and
G
s contained 20 mM NaHepes, pH 8.0, 100 mM NaCl, 10 mM free Mg2+, 1 mM dithiothreitol, and 0.05% C12E10. The reaction mixture for G
q was identical except that 0.05% Genapol C-100
detergent was used instead of C12E10. For each reaction, Tev-digested
Ric-8A (200 nM) was first added to reaction buffer and 10 µM [35S]GTP
S (10,000 cpm/pmol). To
initiate the reactions, each G
protein was added to a final
concentration of 200 nM. Duplicate aliquots were removed at
indicated times, and binding of radioactive nucleotide was stopped by
the addition of ice-cold buffer containing 20 mM Tris-HCl,
pH 7.7, 100 mM NaCl, 2 mM MgSO4,
0.05% C12E10, and 1 mM GTP. The quenched reactions were
passed through BA-85 nitrocellulose filters and washed (20 mM Tris-HCl, pH 7.7, 100 mM NaCl, 2 mM MgSO4); the filters were dried and subjected
to liquid scintillation counting. All of the reactions were conducted at 20 °C with the exception of those with G
i1, which
were performed at 30 °C.
q were conducted at
20 °C in buffer containing 0.05% Genapol C-100, 1 µM
[
-32P]GTP (40,000 cpm/pmol), and the indicated amounts
of Tev-digested Ric-8A and/or G
1
2. The
reactions were initiated by adding purified G
q or
G
q
1
2 to a final
concentration of 45 nM. Aliquots of the reactions were
removed at 2, 4, 6, 8, and 10 min, and the amount of released
Pi was quantified using a charcoal-based method (35).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Proteins
in Yeast Two-hybrid Assays--
L40 yeast expressing LexA bait fusion
proteins of the activated (GTPase-deficient) mutants of
G
o and G
s long were transformed with a
rat brain pVP16-cDNA fusion library. Approximately 106
individual transformants were screened with each bait using the HIS3 reporter of L40 and a
-galactosidase filter assay
(24). Sequence analysis of 240 plasmids rescued from positive
G
o transformants revealed cDNA inserts that encode
portions of several previously identified G
-interacting proteins,
including: GRIN1 (36), GRIN2 (36), Ras GAP III (37), Rap1GAP (38), AGS3
(12), LGN (39), RGS8 (40), and RGS17 (RGSZ2) (38). One of the clones
encoded an open reading frame, less the first three amino acids, that shared 97% amino acid identity with mouse Ric-8/synembryn (NP_444424) and 87% amino acid identity with the predicted sequence of a 531-amino acid human hypothetical protein (NP_068751). We have named these homologs Ric-8A. The rat Ric-8A protein found in the two-hybrid screen
is compared with three different putative human Ric-8A proteins in Fig.
1A. The human RIC-8A cDNAs
that encode proteins of 531 and 537 amino acids are likely the products
of alternative RNA splicing. The sequence of a cDNA encoding the
531-amino acid variant was verified from a clone isolated from HeLa
cells (not shown). Inspection of the complete human RIC-8A genomic
sequence contained in a BAC clone (AC069287) revealed that the 531- and
537-amino acid encoding transcripts could be produced by the use of two
alternative consensus splice acceptor sites of exon 7 (not shown). The
hypothetical RIC-8A transcript predicted to encode the 401-amino acid
Ric-8A protein is derived from insertion of a single base pair at codon
395 of the transcript encoding the 531-residue protein. Interestingly,
this frameshift would result in synthesis of a protein with five
different carboxyl-terminal amino acids and a new stop codon that form
a consensus CAAX box of the type that would be modified with
a geranylgeranyl moiety (Fig. 1A) (41). However, all
attempts to verify the existence of this apparent point mutation using
the cDNA source reported in GenBankTM (AK022870) have
been unsuccessful.
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Fig. 1.
A, three putative human RIC-8A cDNAs
are homologous to the rat RIC-8A clone isolated in the two-hybrid
screen. The 531-amino acid human Ric-8A protein (human Ric-8A (531))
(NP_068751) is the equivalent of the 529-amino acid rat protein. A
predicted 537-amino acid human Ric-8A protein (human Ric-8A (537))
(CAB66869) is identical to the 531-residue protein except for a 6-amino
acid insertion, including a polyproline sequence, at codon 355. The
third putative variant encodes a protein that consists of the
amino-terminal 396 residues of the 531-amino acid protein with the
addition of five amino acid residues that form a consensus
CAAX box (CAAX-Ric-8A). B, the human
RIC-8B cDNA (NM_018157) is predicted to encode a protein of 536 amino acid residues. The amino-terminal 483 amino acids of human Ric-8B
and the protein encoded by the rat RIC-8B cDNA obtained in the
two-hybrid screen are homologous. The remaining divergent 37 carboxyl-terminal amino acids encoded by the rat cDNA are likely
the product of alternative splicing.
s as
bait, and of these, none encoded previously known G
-interacting
proteins. One positive clone shared significant sequence identity with
the human cDNA FLJ10620 (NM_018157). Human FLJ10620 encodes a
protein of 536 amino acid residues that shares ~40% amino acid
identity with mouse Ric-8/synembryn (Ric-8A). The rat clone obtained in the two-hybrid screen encodes a 520-residue protein. The amino-terminal 483 residues of this protein are 86% identical to the amino-terminal 483 amino acids of human FLJ10620. The remaining carboxyl-terminal amino acids of both proteins are completely divergent. We have named
FLJ10620 and its rat homolog Ric-8B (Fig. 1B). Analysis of
the RIC-8B genomic sequence from a compilation of BAC clones revealed
that the Human cDNA FLJ10620 contains an extra 3' splice site that
was not utilized in the Rat RIC-8B cDNA we obtained. This likely
manner of alternative splicing explains the divergence of the carboxyl
termini of rat and human Ric-8B.
proteins in the two-hybrid system, the full-length rat Ric-8A and
Ric-8B preys were individually co-transformed with wild type and
GTPase-deficient (QL) G
baits: G
i1,
G
o, G
q, G
s long, and
G
13. Suspensions of these transformed yeast were spotted onto synthetic medium plates lacking tryptophan and leucine and tested
for positive interactions between bait and prey using a
-galactosidase filter activity assay (24). The Ric-8A prey yielded a
strong positive signal with G
i1, G
o, and
G
q and a weak signal with G
13. No
interaction was observed with G
s long (Fig.
2). The Ric-8A prey also showed a clear
preference for interaction with the wild type G
i1 bait
compared with its GTPase-defective counterpart but did not show this
preference for the other G
baits tested. The Ric-8B prey showed a
strong signal with G
q and a weak signal with the
G
s long bait (Fig. 2). Much like the
Ric-8A/G
i1 interaction, the Ric-8B prey interacted
preferentially with the wild type G
s long bait.
Intriguingly, the common two-hybrid interaction partner for these two
mammalian Ric-8 homologs is G
q, the G
that was first
shown to interact genetically with RIC-8 in C. elegans
(20).
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Fig. 2.
Ric-8A and Ric-8B interact with different
subsets of G proteins in the yeast two-hybrid
system. L40 yeast were co-transformed with rat Ric-8A or Ric-8B
prey plasmids and the indicated wild type (WT) or activated
(GTPase-deficient) mutant (QL) G
bait plasmids. The
transformants were grown on a minimal medium plate lacking tryptophan
and leucine and assayed for
-galactosidase activity to assess
positive interactions between bait and prey. Vec,
vector.
Proteins--
To investigate whether Ric-8A interacts directly with
membrane-derived G
proteins, a GST-full-length rat Ric-8A fusion
protein was expressed and purified from baculovirus-infected
Sf9 cells and used to bind G proteins present in a detergent
extract of rat brain membranes. Three controls were used in this
experiment: GST-Ric-8A incubated with glutathione Sepharose alone (Fig.
3, lane 1), GST-Ric-8A
incubated with rat brain membrane extract and glutathione Sepharose
(Fig. 3, lane 2), and brain membrane extract incubated with
glutathione Sepharose alone (Fig. 3, lane 3). Proteins
adsorbed to Sepharose were released by incubation with Tev protease,
resolved by SDS-PAGE, and either transferred to nitrocellulose for
Western blotting with G protein subunit-specific antisera or stained
with Coomassie Blue. Specific binding of a protein to Ric-8A was
indicated when unique protein bands were found only in the experiments
where GST-Ric-8A and extract were incubated together (Fig. 3,
lane 2). These bands were isolated from the gels,
trypsinized, and analyzed by mass spectrometry. Unique protein bands of
~40 kDa were identified as G
o and a mixture of
G
i1, G
i2, or G
i3 (Hongjun
Shu, University of Texas Southwestern Medical Center). The immunoblots
shown in Fig. 3 confirmed the mass spectrometry and also revealed the
presence of immunoreactive G
q and G
13.
Neither G
s nor G protein
subunits were detected (Fig. 3, lane 2).
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Fig. 3.
Ric-8A binds a subset of brain membrane
G proteins. Glutathione-Sepharose was
incubated with either GST-Ric-8A (lanes 1 and 2)
or buffer (lane 3) and then washed. A detergent extract from
rat brain membranes was incubated with the GST-Ric-8A beads (lane
2) or the buffer-treated beads (lane 3), and the beads
were washed. Proteins bound to the glutathione Sepharose in each
condition were released with Tev protease. The eluants from each
condition were resolved by SDS-PAGE, transferred to nitrocellulose, and
probed with G protein subunit-specific antisera as described under
"Experimental Procedures." Std, standard.
--
To
understand the biochemical consequences of the interaction between
Ric-8A and G
proteins, we first determined whether association was
dependent on the identity of the guanine nucleotide bound to G
.
Purified myristoylated G
i1 was first incubated with GDP,
GDP and AlF
S. These proteins were then
further incubated with GST-Ric-8A and bound to glutathione Sepharose.
The beads were extensively washed, and the bound proteins were eluted
and resolved by SDS-PAGE (Fig. 4).
GST-Ric-8A interacted preferentially with GDP-bound G
i1
(lanes 5 and 6), less well with
G
i1·GDP·AlF
i1·GTP
S
(lanes 1 and 2).
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Fig. 4.
Ric-8A binds to the GDP-bound form of
myristoylated G i1. Duplicated
aliquots of purified myristoylated G
i1 (600 nM) were incubated for 1 h with GTP
S, GDP, or GDP
and AlF
i1 and Ric-8A, we gel-filtered Ric-8A·G
i1 complexes prepared by incubation of Ric-8A
with G
i1 bound to radiolabeled GDP or GTP
S. The
fractions were analyzed by SDS-PAGE and subjected to Bradford analysis
and scintillation counting to quantify the amounts of complex,
monomeric Ric-8A, monomeric G
i1, and guanine nucleotide
present in each fraction (Fig. 5).
SDS-PAGE showed that Ric-8A forms a stable, apparently stoichiometric
complex with G
i1 that had been prelabeled with [
-32P]GDP (Fig. 5A) but not with
[35S]GTP
S (Fig. 5B). All of the detectable
[
-32P]GDP and [35S]GTP
S were
associated with uncomplexed, monomeric G
i1. The Ric-8A·G
i1 complex was free of nucleotide (Fig.
5A). When the fractions containing the
Ric-8·G
i1 complex shown in Fig. 5A (GDP experiment) were pooled, concentrated, and rerun over the gel filtration columns, the complex remained intact (data not shown). However, when the complex was incubated with GTP
S and then
gel-filtered again, the majority of the complex was dissociated (data
not shown). Together, these gel filtration experiments suggest strongly
that Ric-8A acts as a G
protein guanine nucleotide exchange factor, interacting with the GDP-bound form of G
to form a stable nucleotide free complex that is disrupted upon binding of GTP.
View larger version (30K):
[in a new window]
Fig. 5.
Ric-8A interacts with
GDP·G i1 to form a stable,
nucleotide-free complex. Purified nonmyristoylated
G
i1 (13 µM) bound to
[
-32P]GDP (A) or [35S]GTP
S
(B) was incubated with 10 µM purified
Tev-digested Ric-8A for 5 min. The reaction mixtures were then resolved
by gel filtration chromatography over tandem Superdex 75/Superdex 200 columns (HR 10/30). Fractions from the column were analyzed by SDS-PAGE
to visualize proteins, by Bradford assay to assess the protein
concentration of each fraction, and by scintillation counting to
determine the amount of radiolabeled nucleotide in each fraction.
S Binding to a Subset of G
Proteins--
To determine whether Ric-8A acts as a GEF for G
proteins, the kinetics of GTP
S binding to purified G
proteins was
determined in the presence and absence of purified Tev-digested Ric-8A.
Binding of guanine nucleotide to G
proteins is limited by the rate
of dissociation of GDP. Ric-8A dramatically stimulated the rate of GTP
S binding to G
q from immeasurable values to 0.1 mol of GTP
S·mol G
q
1·min
1 (Fig.
6A), to G
i1
from 0.02 to 0.15 mol GTP
S·mol
G
i1
1·min
1 (Fig.
6B), and to G
o from 0.16 to 0.32 mol
GTP
S·mol G
o
1·min
1
(Fig. 6C) but did not stimulate binding to
G
s short appreciably (0.085-0.1 mol GTP
S·mol
G
s
1·min
1) (Fig.
6D). Ric-8A also stimulates the rate of GTP
S binding to
G
13.2
View larger version (11K):
[in a new window]
Fig. 6.
Ric-8A is a guanine nucleotide exchange
factor for a subset of G proteins.
Purified G
q (A), G
i1
(B), G
o (C), and
G
s short (D) (200 nM each) were
incubated with [35S]GTP
S in reactions containing 0 (
) or 200 (
) nM Tev-digested Ric-8A. Duplicate
aliquots of these reaction mixtures were taken at the indicated time
points, quenched, and filtered to adsorb nucleotide-bound protein. The
amount of G protein-bound [35S]GTP
S was determined by
scintillation counting.
q--
The rate-limiting step in G
protein-catalyzed steady-state GTPase reactions in vitro is
the release of GDP from the G
. Inclusion of a GEF such as Ric-8A in
such reactions should enhance the observed rate of GTPase activity in a
manner similar to that observed with GTP
S binding. Steady-state
GTPase reactions containing varying amounts of Tev-digested Ric-8A were
initiated by the addition of G
q (45 nM)
(Fig. 7A). Ric-8A maximally
accelerated the GTPase activity of G
q from immeasurable
values to 0.29 mol Pi·mol of G
q
1·min
1. The
EC50 for Ric-8A was ~160 nM. Ric-8A also
stimulated G
i1 steady-state GTPase activity in a manner
consistent with the observed kinetics of Ric-8A-stimulated
G
i1 GTP
S binding shown in Fig. 6 (data not
shown).
View larger version (13K):
[in a new window]
Fig. 7.
A, Ric-8A stimulates the steady-state
GTPase activity of G q. G
q (45 nm) was
incubated with the indicated concentrations of Tev-digested Ric-8A and
[
-32P]GTP. Duplicate aliquots from each reaction were
taken at 2-min intervals from 0 to 10 min. The amount of
32Pi in each aliquot was determined, and the
reaction rates were calculated. B,
1
2 inhibits Ric-8A-stimulated
G
q GTPase activity. G
q (45 nM) was used to initiate GTPase reactions containing
Tev-digested Ric-8A (500 nM) and the indicated
concentrations of
1
2. The samples were
processed as described for A. C, Ric-8A does not
stimulate the steady-state GTPase activity of heterotrimeric
G
q
1
2. G
q
(45 nM) was incubated with 200 nM (
) or 500 nM (
)
1
2. This
heterotrimeric G
q
1
2 was
then used to initiate GTPase reactions containing the indicated amounts
of Tev-digested Ric-8A. The samples were processed as described for
A.
subunits. The indicated amounts of purified G protein
1
2 subunits and an amount of Ric-8A
corresponding to approximately three times the EC50 value
(500 nM) were mixed and used to initiate steady-state
GTPase reactions containing 45 nM G
q.
G
1
2 clearly inhibited Ric-8A-stimulated
GTPase activity at concentrations comparable with that of
G
q in the assay (Fig. 7B). However, the
inhibition did not appear to be complete.
q
1
2 was first formed by
incubating purified G
q (45 nM) with 200 and
500 nM concentrations of purified
1
2 for 30 min at 20 °C. These
heterotrimer preparations were then used to initiate steady-state
GTPase reactions containing the indicated concentrations of Ric-8A
(Fig. 7C). Ric-8A was not capable of interacting with
preformed G protein heterotrimers to facilitate nucleotide exchange
(Fig. 7C).
apparently competes with Ric-8A for
binding to G
proteins. Ric-8A functions as a GEF for monomeric G
proteins, which clearly differs from the mechanism used by conventional GPCRs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-binding proteins. Ric-8A is a unique guanine nucleotide exchange
factor for a subset of G
proteins, unlike a GPCR because it does not
contain transmembrane spanning sequences and because it activates only
monomeric G protein
subunits in vitro. Ric-8A is a G
protein activator and not just an exchange factor, because the
Ric-8A-mediated exchange process appears to proceed in one direction
(exchange of GDP for GTP). When G
i1 is bound to GDP, binding of Ric-8A causes rapid release of GDP. Ric-8A does not measurably bind the GTP-bound form of G
i1. Upon release
of GDP, Ric-8A stabilizes an otherwise extremely unstable
nucleotide-free conformation of G
i1. GTP, but not GDP,
then binds to G
i1, which causes the complex to
dissociate and release the activated G
protein.
-binding proteins in
yeast two-hybrid screens designed to search for novel interaction
partners of activated, GTP-bound forms of G
o and G
s (Figs. 1 and 2). Despite the fact that Ric-8A and B
were detected with these presumably GTP-bound proteins, subsequent
biochemical experiments showed that Ric-8A interacts preferentially
with the GDP-bound or nucleotide-free forms of G
proteins (Figs. 5
and 6). This discrepancy is reconciled somewhat by the fact that Ric-8 showed a clear preference for the wild type (GDP-bound) G
proteins in the case of the Ric-8A/G
i1 and
Ric-8B/G
s long two-hybrid interactions (Fig. 2). Ric-8A
and B were found to interact with unique subsets of G
proteins (Fig.
2). Of the G
s tested, G
q was the common interaction
partner of Ric-8A and B.
i1 and Ric-8A are mixed, a stable nucleotide-free
complex is formed in detergent-free solution (Fig. 5). The ability to
isolate substantial quantities of this nucleotide-free complex provides a unique opportunity for crystallization and determination of the
structure of a G protein
subunit in a nucleotide-free conformation; this has been an elusive goal.
subunits when they
exist as
heterotrimers. The function of the Ric-8 proteins could thus be as signal amplifiers. After a heterotrimeric G
protein is activated by a GPCR and subsequently hydrolyzes GTP but
before the signal is completely attenuated by rebinding to G
, a
Ric-8 protein could bind a monomeric G protein
subunit and
reactivate it. This mode of action would serve to amplify the duration
of a signal that comes from an individual G protein.
target(s) of Ric-8A and B in vivo. Genetic studies in
C. elegans show that mutation of the RIC-8 gene negatively affects aspects of synaptic transmission that are positively controlled by G
q. C. elegans Ric-8 was predicted to act
as an upstream activator of G
q or to operate in a
pathway parallel with G
q (20). To date, we have not been
able to observe effects of mammalian Ric-8A on G
q
signaling in intact cells. Overexpression of G
q in
cultured cells led to an increase in steady-state and
hormone-stimulated production of inositol trisphosphate, whereas
overexpression of full-length or CAAX Ric-8A or
co-overexpression of G
q and full-length or
CAAX Ric-8A did not lead to a further increase in
steady-state or hormone-stimulated inositol trisphosphate
accumulation.3 These results
certainly do not preclude Ric-8A from activating G
q
in vivo, but they are consistent with other cellular targets or other sites of action of mammalian Ric-8A. Recent work with C. elegans RIC-8 revealed that in addition to its role in promoting synaptic transmission via a G
q-coupled pathway, mutation
of RIC-8 influences centrosome movements during early embryogenesis
(21). This aspect of C. elegans RIC-8 function was shown
genetically to be independent of the action of G
q and
more likely to involve G
o or another G protein family
member. Because mammalian Ric-8A potently activates both
G
o and G
i in vitro,
examination of the effects Ric-8A has on signaling pathways controlled
by these G proteins should also be undertaken. However, our preliminary
steps in this direction have also not been encouraging.
subunits, which we expect to be heterotrimeric. Increasing evidence
points to G proteins that reside on internal cellular membranes that
might not contain G
subunits (42-44). It is unclear how these G
proteins become activated, but proteins like Ric-8A and B are now
obvious candidates. The possible existence of the 401-amino acid Ric-8A
variant is intriguing in this regard, because it contains a consensus
CAAX box at its carboxyl terminus (Fig. 1). Expression of
the recombinant engineered protein in cultured cells revealed that it
can reside on membranes in a CAAX box-dependent manner.3 However, we have not been able to confirm the
existence of a transcript encoding this protein, and a sequencing error
is a possible explanation for its existence in GenBankTM.
The 531-amino acid Ric-8A protein appears to be mostly
cytosolic3 and would be accessible to the cytosolic face of
most organellar membranes in addition to the plasma membrane. Other
questions of major importance include elucidation of the identity of
the signaling pathways that are regulated by Ric-8 in vivo
and the possible existence of signaling inputs that control the
activities of Ric-8 proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Gilman laboratory for
helpful discussions, Linda Hannigan for technical assistance, Dr. Mark
Hatley for the gift of Gs, Dr. Roger Sunahara for the
gift of Myr-G
i1, and Drs. Celestine Thomas and Hongjun
Shu for mass spectroscopy analyses.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM34497 and the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology (to A. G. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY177754 and AY177755.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Blvd., Dallas, TX 75390-9041. Tel.: 214-648-2370; Fax:
214-648-8812; E-mail: Alfred.gilman@utsouthwestern.edu.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M211862200
2 W. D. Singer and P. C. Sternweis, personal communication.
3 G. G. Tall and A. G. Gilman, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
G protein, guanine
nucleotide-binding regulatory protein;
GTPS, guanosine
5'-3-O-(thio)triphosphate;
C12E10, polyoxyethylene 10-lauryl
ether;
Tev, tobacco etch virus;
GST, glutathione
S-transferase;
GPCR, G protein-coupled receptor;
GEF, guanine nucleotide exchange factor;
GAP, GTPase-activating protein;
RGS, regulator of G protein signaling.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615-649[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bornancin, F., Pfister, C., and Chabre, M. (1989) Eur. J. Biochem. 184, 687-698[Abstract] |
3. | Coleman, D. E., Berghuis, A. M., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412[Medline] [Order article via Infotrieve] |
4. | Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) J. Biol. Chem. 86, 445-452 |
5. | Yowe, D., Yu, K., Wilkie, T. M., and Popov, S. (2002) Methods Enzymol. 344, 647-657[Medline] [Order article via Infotrieve] |
6. | De Vries, L., Zheng, B., Fischer, T., Elenko, E., and Farquhar, M. G. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 235-271[CrossRef][Medline] [Order article via Infotrieve] |
7. | Ross, E. M., and Wilkie, T. M. (2000) Annu. Rev. Biochem. 69, 795-827[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Sato, M.,
Ribas, C.,
Hildebrandt, J. D.,
and Lanier, S. M.
(1996)
J. Biol. Chem.
271,
30052-30060 |
9. |
Cismowski, M. J.,
Ma, C.,
Ribas, C.,
Xie, X.,
Spruyt, M.,
Lizano, J. S.,
Lanier, S. M.,
and Duzic, E.
(2000)
J. Biol. Chem.
275,
23421-23424 |
10. |
Bernard, M. L.,
Peterson, Y. K.,
Chung, P.,
Jourdan, J.,
and Lanier, S. M.
(2001)
J. Biol. Chem.
276,
1585-1593 |
11. | Kroslak, T., Koch, T., Kahl, E., and Hollt, V. (2001) J. Biol. Chem. 276, 38772-39778 |
12. |
Takesono, A.,
Cismowski, M. J.,
Ribas, C.,
Bernard, M.,
Chung, P.,
Hazard, S., III,
Duzic, E.,
and Lanier, S. M.
(1999)
J. Biol. Chem.
274,
33202-33205 |
13. |
De Vries, L.,
Fischer, T.,
Tronchere, H.,
Brothers, G. M.,
Strockbine, B.,
Siderovski, D. P.,
and Farquhar, M. G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
14364-14369 |
14. | Lackner, M. R., Nurrish, S. J., and Kaplan, J. M. (1999) Neuron 24, 335-346[Medline] [Order article via Infotrieve] |
15. | Nurrish, S., Segalat, L., and Kaplan, J. M. (1999) Neuron 24, 231-242[Medline] [Order article via Infotrieve] |
16. | Miller, K. G., Emerson, M. D., and Rand, J. B. (1999) Neuron 24, 323-333[Medline] [Order article via Infotrieve] |
17. | Singer, W. D., Brown, H. A., and Sternweis, P. C. (1997) Annu. Rev. Biochem. 66, 475-509[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Miller, K. G.,
Alfonso, A.,
Nguyen, M.,
Crowell, J. A.,
Johnson, C. D.,
and Rand, J. B.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12593-12598 |
19. |
Hajdu-Cronin, Y. M.,
Chen, W. J.,
Patikoglou, G.,
Koelle, M. R.,
and Sternberg, P. W.
(1999)
Genes Dev.
13,
1780-1793 |
20. | Miller, K. G., Emerson, M. D., McManus, J. R., and Rand, J. B. (2000) Neuron 27, 289-299[Medline] [Order article via Infotrieve] |
21. |
Miller, K. G.,
and Rand, J. B.
(2000)
Genetics
156,
1649-1660 |
22. | Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214[Medline] [Order article via Infotrieve] |
23. | Sherman, F., Fink, G. R., and Lawrence, L. W. (1979) Methods in Yeast Genetics: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. |
Hama, H.,
Tall, G. G.,
and Horazdovsky, B. F.
(1999)
J. Biol. Chem.
274,
15284-15291 |
25. |
Andersson, S.,
Davis, D. N.,
Dahlback, H.,
Jornvall, H.,
and Russell, D. W.
(1989)
J. Biol. Chem.
264,
8222-8229 |
26. | Hannon, G. J., Demetrick, D., and Beach, D. (1993) Genes Dev. 7, 2378-2391[Abstract] |
27. | Lee, E., Linder, M. E., and Gilman, A. G. (1994) Methods Enzymol. 237, 146-163[Medline] [Order article via Infotrieve] |
28. | Kozasa, T. (1999) in G Proteins: Techniques of Analysis (Manning, D. R., ed) , pp. 23-38, CRC Press, Baco Raton, FL |
29. |
Pang, I. H.,
and Sternweis, P. C.
(1990)
J. Biol. Chem.
265,
18707-18712 |
30. | Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-269[Abstract] |
31. | Linder, M. E., Middleton, P., Hepler, J. R., Taussig, R., Gilman, A. G., and Mumby, S. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3675-3679[Abstract] |
32. | Johnson, R. A., and Corbin, J. D. (1991) Methods Enzymol. 1991, 215-233 |
33. |
Singer, W. D.,
Miller, R. T.,
and Sternweis, P. C.
(1994)
J. Biol. Chem.
269,
19796-19802 |
34. |
Sternweis, P. C.,
and Robishaw, J. D.
(1984)
J. Biol. Chem.
259,
13806-13813 |
35. | Wang, J., Tu, Y., Mukhopadhyay, S., Chidiac, P., Biddlecome, G. H., and Ross, E. M. (1999) in G Proteins: Techniques of Analysis (Manning, D. R., ed) , pp. 123-151, CRC Press, Boca Raton, FL |
36. |
Chen, L. T.,
Gilman, A. G.,
and Kozasa, T.
(1999)
J. Biol. Chem.
274,
26931-26938 |
37. | Jiang, Y., Ma, W., Wan, Y., Kozasa, T., Hattori, S., and Huang, X. (1998) Nature 395, 808-813[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Jordan, J. D.,
Carey, K. D.,
Stork, P. J. S.,
and Iyengar, R.
(1999)
J. Biol. Chem.
274,
21507-21510 |
39. | Mochizuki, N., Cho, G., Wen, B., and Insel, P. A. (1996) Gene (Amst.) 181, 39-43[CrossRef][Medline] [Order article via Infotrieve] |
40. | Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529[CrossRef][Medline] [Order article via Infotrieve] |
41. | Zhang, F. L., and Casey, P. J. (1996) Annu. Rev. Biochem. 65, 241-269[CrossRef][Medline] [Order article via Infotrieve] |
42. | Wilson, B. S., Komuro, M., and Farquhar, M. G. (1994) Endocrinology 134, 233-244[Abstract] |
43. |
Lou, X.,
McQuistan, T.,
Orlando, R. A.,
and Farquhar, M. G.
(2002)
J. Am. Soc. Nephrol.
13,
918-927 |
44. | Denker, S. P., McCaffery, J. M., Palade, G. E., Insel, P. A., and Farquhar, M. G. (1996) J. Cell Biol. 133, 1027-1040[Abstract] |