Ric-8 Enhances G Protein ??-Dependent Signaling in
Response to ??-Binding Peptides in Intact Cells
Sundeep Malik, Mousumi Ghosh, Tabetha M. Bonacci, Gregory G. Tall, and
Alan V. Smrcka
Departments of Pharmacology and Physiology (S.M., M.G., T.M.B., A.V.S.) and Biochemistry and Biophysics (A.V.S.), University
of Rochester School of Medicine and Dentistry, Rochester, New York; and Department of Pharmacology (G.G.T.), University of
Texas Southwestern Medical Center, Dallas, Texas
Received December 7, 2004; accepted March 31, 2005
Peptides derived from a random-peptide phage display screen
with purified G?1?2subunits as the target promote the disso-
ciation of G protein heterotrimers in vitro and activate G protein
signaling in intact cells. In vitro, one of these peptides
(SIRKALNILGYPDYD; SIRK) promotes subunit dissociation by
binding directly to G?? subunits and accelerating the dissoci-
ation of G?GDP without catalyzing nucleotide exchange. The
experiments described here were designed to test whether the
mechanism of SIRK action in vitro is in fact the mechanism of
action in intact cells. We created a mutant of G?1subunits
(?1W332A) that does not bind SIRK in vitro. Transfection of
G?1W332A mutant into Chinese hamster ovary cells blocked
peptide-mediated activation of extracellular signal-regulated
kinase (ERK), but it did not affect receptor-mediated G?? sub-
unit-dependent ERK activation, indicating that G?? subunits
are in fact the direct target in cells responsible for ERK activa-
tion. To determine whether free G? subunits were released
from G protein heterotrimers upon peptide treatment, cells
were transfected with Ric-8A, a guanine nucleotide exchange
factor for free G?GDP, but not heterotrimeric G proteins.
Ric-8A-transfected cells displayed enhanced myristoyl-
SIRKALNILGYPDYD (mSIRK)-dependent inositol phosphate
(IP) release and ERK activation. Ric-8A also enhanced ERK
activation by the Gi-linked G protein coupled receptor ago-
nist lysophosphatidic acid. Inhibitors of G?? subunit function
blocked Ric-8-enhanced activation of ERK and IP release.
These results suggest that one potential function of Ric-8 in
cells is to enhance G protein G?? subunit signaling. Overall,
these experiments provide further support for the hypothesis
that mSIRK promotes G protein subunit dissociation to re-
lease free ?? subunits in intact cells.
G protein-coupled receptors (GPCRs) comprise a large fam-
ily of proteins that bind a diverse array of molecules and
communicate this binding information to alterations of cell
physiology (Gilman, 1987; Hamm, 1998). Activated GPCRs
interact with heterotrimeric G proteins to catalyze the ex-
change of bound GDP for GTP. This process requires the
presence of both G? and G?? subunits, and there is evidence
for direct binding of the receptor to both G? and G?? sub-
units (Taylor et al., 1994, 1996). Binding of GTP to the G?
subunit activates the G protein and is thought to cause
dissociation of G? subunits from G?? subunits, liberating
free G?GTP and G?? subunits to interact with downstream
target proteins and regulate their activities.
It has become apparent that receptor-independent mecha-
nisms exist for G protein activation. AGS proteins, discov-
ered in a yeast screen for activation of the pheromone
pathway, all act to release ?? subunits from ? subunits
(Cismowski et al., 2001). GPR or GoLoco peptides derived
from AGS proteins promote dissociation of G?GDP subunits
from G?? subunits, causing release of G?? (Peterson et al.,
2000; Kimple et al., 2002; Ghosh et al., 2003). In addition, a
novel protein, Ric-8, has been identified that binds specifi-
cally to free G?GDP subunits and promotes GDP release
This work was supported by National Institutes of Health grants GM60286
(to A.V.S.) and GM34497 (to G.G.T. and A.G.G.) and National Institutes of
Health Predoctoral Training grant in Cardiovascular Biology HLT3207949 (to
Article, publication date, and citation information can be found at
ABBREVIATIONS: GPCR, G protein-coupled receptor; AGS, activator of G protein signaling; SIGK, SIGKAFKILGYPDYD; SIRK, SIRKALNIL-
GYPDYD peptide; mSIRK, myristoyl-SIRKALNILGYPDYD peptide; GFP, green fluorescent protein; LPA, lysophosphatidic acid; ERK, extracellular
signaling-regulated kinase; HA, hemagglutinin; HRP, horseradish peroxidase; bG?1, biotinylated G?1; ELISA, enzyme-linked immunosorbent
assay; DMEM, Dulbecco’s modified Eagle’s medium; IP, inositol phosphate; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electro-
phoresis; PLC, phospholipase C; wt, wild-type; PTX, pertussis toxin; GEF, guanine nucleotide exchange factor; ?ARK, ? adrenergic receptor
kinase; ct, C terminus; EE, EYMPTE.
Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 68:129–136, 2005
Vol. 68, No. 1
Printed in U.S.A.
at ASPET Journals on January 4, 2016
(Miller et al., 2000; Tall et al., 2003). Thus, a system poten-
tially exists outside of G protein-coupled receptors for G
protein activation that involves sequential action of proteins
to release ?GDP followed by Ric-8-catalyzed nucleotide ex-
change. Several recent publications suggest a relationship
between AGS proteins and Ric-8 in unconventional G protein
signaling during spindle pole positioning in the initial cell
division events in Caenorhabditis elegans zygotes (Afshar et
al., 2004; Couwenbergs et al., 2004; Hess et al., 2004).
We have identified a mechanism by which G?? binding
peptides can activate G protein signaling by a receptor-inde-
pendent mechanism. Cell-permeant versions of peptides
against G protein ?? subunits promote activation of G pro-
tein ?? subunit-dependent pathways, including mitogen-
activated protein kinase and phospholipase C activation, in
intact cells (Goubaeva et al., 2003). In vitro these peptides
bind directly to G protein ?1?2subunits and accelerate dis-
sociation of G?iGDP subunits from G?i1?1?2heterotrimers
(Ghosh et al., 2003). The structure of G protein ?1?2subunits
bound to one of the peptides (SIGKAFKILGYPDYD; SIGK)
has been solved (T. L. Davis, T. M. Bonacci, S. R. Sprang, and
A. V. Smrcka, submitted). In the structural model, the pep-
tide is bound to a site on G?1?2subunits normally occupied
by the switch II helix of G? subunits (Wall et al., 1995;
Lambright et al., 1996). These data suggest a molecular
mechanism by which these peptides promote G protein sub-
unit dissociation by interfering with G? subunit interactions
with G?? subunits.
Although these in vitro data support a model for peptide-
mediated dissociation of G?GDP from G?? as the mechanism
for (myristoyl-SIRKALNILGYPDYD) (mSIRK) activation of
signaling pathways in intact cells, they do not directly dem-
onstrate this. In this study, we set out to demonstrate that G
protein ?? subunits are the direct target of these cell-perme-
able peptides in cells and that interaction of these peptides
with heterotrimeric G proteins results in release of free
G?GDP in intact cells. As part of our analysis, we studied the
ability of Ric-8 proteins to affect peptide-mediated responses
based on the ability of Ric-8 to selectively activate free
G?GDP subunits. We were surprised to find that Ric-8 can
enhance G protein ?? subunit-mediated responses, probably
by a mechanism that involves sequestration of free G? sub-
Materials and Methods
Materials and Plasmids. GFP-G?1, GFP-G?1W332A, G?2, and
Ric-8A-3HA were in pCI-Neo. EE-?i1and -?twere supplied from
Guthrie cDNA Resource Center (Rolla, MO) in pcDNA 3.1?;
?ARKct, kindly supplied by Dr. Robert Lefkowitz (Duke University,
Durham, NC), was in pRK5 and Ric-8A; and Ric-8B was in pCMV5.
mSIRK and SIGK were synthesized and purified by Alpha Diagnos-
tics International (San Antonio, TX). myo-[3H]Inositol (25 Ci/mmol)
was from PerkinElmer Life and Analytical Sciences (Boston, MA).
Pertussis toxin, lysophosphatidic acid (LPA), and ATP were from
Sigma-Aldrich (St. Louis, MO). Rabbit anti-ERK and anti-phospho-
ERK antisera were from Cell Signaling Technologies Inc. (Beverly,
MA). Anti Ric-8A antiserum was generated in rabbits against holo-
purified Ric-8A protein by Caprologics, Inc. (Hardwick, MA). Mouse
anti-HA and anti-EE antisera were from Covance (Princeton, NJ).
Mouse anti-GFP, goat anti-rabbit IgG-horseradish peroxidase conju-
gate (HRP) and goat anti-mouse IgG-HRP were from Roche Diag-
nostics (Indianapolis, IN).
Construction and Purification of Biotinylated G?? Sub-
units. Construction of baculovirus encoding biotinylated G?1(bG?1)
subunit in the baculovirus transfer vector PDW464 was described
previously (Goubaeva et al., 2003). For other experiments, G protein
?1subunits were tagged at the amino terminus with GFP. We used
GFP-tagged ?1subunits to monitor ? subunit transfection efficiency
by epifluorescence microscopy and to monitor the level of expression
of the transfected protein relative to endogenous ? subunits by
immunoblotting. We (unpublished data) and others have shown that
amino terminal modification of G? with GFP does not alter G??
subunit functions (Azpiazu and Gautam, 2004). Mutants (?W332A
and K337A) were created by overlap extension polymerase chain
reaction (PCR) using standard methods, and the entire protein cod-
ing region was sequenced to confirm the presence of the mutation
and lack of additional mutations.
Phage ELISA. The phage used in this study was from the ran-
dom-peptide phage display screen described previously (Scott et al.,
2001). Phage were propagated and ELISA assays with bG?1?2sub-
units were performed as described previously (Smrcka and Scott,
Measurement of ?-?? Interactions via Flow Cytometry. The
fluorescein-labeled ?i1used in these experiments was prepared as
described previously (Sarvazyan et al., 1998), and competition as-
says were performed as described in detail in Ghosh et al. (2003). In
brief, for competition based assays, 100 to 200 pM fluorescein-labeled
?i1and indicated concentrations of peptides were added to 50 pM
bG?1?2immobilized on 105beads per milliliter of buffer and incu-
bated at room temperature for 30 min to reach equilibrium. The
bead-associated fluorescence was then recorded in the flow cytome-
ter. The data were corrected for nonspecific binding and fit with a
sigmoid dose-response curve using Prism 4 (GraphPad Software Inc.,
San Diego, CA).
Cell Culture and Transfection. All cell culture reagents were
obtained from Invitrogen (Carlsbad, CA). Chinese hamster ovary
cells obtained from American Type Culture Collection (Manassas,
VA) were grown in DMEM supplemented with 10% fetal bovine
serum and 1% penicillin/streptomycin at 37°C with 5% CO2. Cells
were grown in six-well dishes (35-mm wells) for ERK activation
experiments. For these experiments, 200 ng of Ric-8A or Ric-8B was
transfected with or without 800 ng of ?ARK-ct in pRK5, 800 ng of ?t,
or pRK5 empty vector control, using LipofectAMINE Plus (Invitro-
gen) unless otherwise indicated. For inositol phosphate (IP) release
measurements, cells were grown in 12-well plates and 200 ng of
Ric-8A was transfected with 200 ng of the appropriate inhibitor with
a total of 400 ng of DNA transfected in each well. Transfections were
performed 48 h before the final treatment and when multiple plas-
mids were transfected, appropriate amounts of control cDNAs were
added such that the total DNA transfected was constant in each
Measurement of ERK Activation and General Immunoblot-
ting. For measurement of phospho-ERK, serum was removed from
50 to 80% confluent CHO cells 16 h before treatment. Peptides in
dimethyl sulfoxide, dimethyl sulfoxide vehicle, or other agonists
were diluted 100- to 400-fold into the medium and incubated at 37°C
for the indicated times. For all immunoblotting: after treatment,
cells were transferred to ice, and the medium was quickly aspirated
and replaced with 100 ?l of 2? SDS sample buffer. The resulting
suspension was boiled for 5 min, and 5 to 10 ?l was loaded onto a 12%
SDS-polyacrylamide gel. After SDS-PAGE, the proteins were trans-
ferred to nitrocellulose for 16 h at 25 V. The transferred proteins
were immunoblotted using standard protocols with 1:1000 dilution of
primary antibody (unless otherwise indicated) and 1:1000 dilution of
the appropriate IgG-horseradish peroxidase conjugate. The proteins
were visualized by incubation with the chemiluminescence reagent
“Pico” (Pierce Chemical, Rockford, IL) and exposure to film. Film was
quantitated by densitometry. Film was quantitated at different lev-
els of exposure to ensure linearity, and results presented are within
the linear range.
Malik et al.
at ASPET Journals on January 4, 2016
Inositol Phosphate Assays. Cells in 12-well plates were labeled
by adding 3 to 5 ?Ci of [3H]inositol for 24 to 48 h in inositol-free
DMEM. After labeling, the medium was removed and replaced with
1 ml of HEPES-buffered DMEM containing 10 mM LiCl and equili-
brated for 20 min at 37°C. Ligands or peptides were added in a
volume of 50 ?l for 45 min after which the medium was aspirated and
replaced with 1 ml of ice-cold 50 mM formic acid and applied to
Dowex AG1-X8 columns (Bio-Rad, Hercules, CA). The columns were
washed with 50 and 100 mM ammonium formate, followed by elution
of the IP-containing fraction with 1.2 M ammonium formate/0.1 M
formic acid. The eluted fraction was mixed with scintillation fluid
and analyzed by liquid scintillation counting.
Coimmunoprecipitation. CHO cells were plated on 35-mm
dishes and transfected with 250 or 500 ng of each cDNA as indicated.
Forty-eight hours after transfection, cells were lysed in 1% Nonidet
P-40 lysis buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 30 mM sodium
pyrophosphate, 50 mM NaF, 100 ?M phenylmethylsulfonyl fluoride,
and 1% Nonidet P-40). After sonication and centrifugation, the su-
pernatant was incubated overnight with the antibody and protein G
plus agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz, CA)
at 4°C with rocking. Beads were centrifuged for 1 min at 13,000 rpm,
washed twice with 1.0 ml of lysis buffer, once with 1.0 ml of phos-
phate-buffered saline, boiled in 50 ?l of 2? SDS sample buffer, and
loaded onto a 12% SDS polyacrylamide gel. After SDS-PAGE, pro-
teins were transferred to nitrocellulose for 16 h at 25 V followed by
immunoblotting as described above.
Mutation of ?W332 to Alanine Inhibits Interaction of
Peptides with G??. These experiments were designed to
determine whether G protein ?? subunits were indeed the
direct target of mSIRK in intact cells responsible for ERK
activation. We hypothesized that a transfected mutant G?
subunit that could not bind SIRK would not be responsive
to mSIRK treatment and thus not promote ERK activation.
We made single alanine substitutions in the G?1subunit
to identify amino acids important for interaction with
SIRK. We chose to mutate ?W332 to A because this muta-
tion had been shown previously to inhibit activation of
PLC? and not affect inhibition of adenylyl cyclase (Li et
al., 1998), and both of these properties were consistent
with the ability of SIRK to inhibit ??-dependent activation
of PLC? but not ??-dependent inhibition of adenylyl cy-
clase (Scott et al., 2001). Single alanine-substituted mu-
tant biotinylated-G?1subunits were expressed with ?2and
6his-?i1subunits in Sf9 insect cells and partially purified
by nickel-agarose chromatography. That the G?? subunits
bound to the nickel column and eluted with AlF4
that these mutants folded and assembled properly with ?
and ? subunits.
We used a phage ELISA assay to examine peptide binding
to the partially purified bG?1?2mutant. In this assay, we
used a peptide closely related to SIRK, SIGK, that gives a
greater ELISA signal and has a higher affinity for G?? sub-
units than SIRK. Here, SIGK displayed on the surface of an
M13-derived phage (f88) was tested for binding to immobi-
lized wt or mutant bG?1?2subunits. As previously demon-
strated, these phages do not give an appreciable binding
signal in the absence of bG?1?2, and phages that do not
display peptide (f88 control) also do not bind bG?1?2. SIGK-
displaying phages bound strongly to wild-type bG?1?2and
bG?1K337A?2, whereas binding to bG?1W332A?2was negli-
gible (Fig. 1A).
To more quantitatively evaluate the decrease in apparent
affinity of SIGK for G?1W332A, the ability of SIGK to compete
for G?-G?? interactions was tested in a flow cytometry assay
(Fig. 1B). The G?1W332A mutation decreased the apparent
affinity of peptide for bG?1?2by approximately 40-fold. Previ-
ous reports indicate that heterotrimers containing G?W332A
are still capable of interacting with receptors, G protein ? sub-
units, and some effectors (Ford et al., 1998; Li et al., 1998;
Myung and Garrison, 2000). The three-dimensional crystal
structure of G?1?2subunits bound to SIGK has been solved,
demonstrating a direct interaction of this peptide with W332 on
G?1(T. L. Davis, T. M. Bonacci, S. R. Sprang, and A. V. Smrcka,
submitted). Thus, the results showing that G?1W332A binds to
? subunits in the flow cytometry assay, yet has a decreased
Fig. 1. ?1W332A inhibits SIRK binding. A, binding of control phage or a
phage bearing the sequence SIGKAFKILGYPDYD to bG?1?2in a phage
ELISA assay. One hundred nanograms of the indicated wt or mutant
bG?1?2subunits was immobilized and tested for binding of 1 ? 1010
phage particles. Bound phages were detected with anti-phage-HRP anti-
body followed by a colorimetric HRP reaction and optical density mea-
surement at 405 nm as described under Materials and Methods. B,
synthetic SIGK peptide was tested at the indicated concentrations for its
ability to compete for binding of fluorescein isothiocyanate-?i1to either
bG?1?2or bG?1W332A?2in a flow cytometry assay.
G?? Signaling Enhancement by Ric-8
at ASPET Journals on January 4, 2016
affinity for SIGK, are consistent with previously published data
and the crystal structure.
Transfection of ?W332A into Intact Cells Inhibits
mSIRK-Dependent G Protein Activation. G?i1and G?2
were cotransfected into CHO cells with either GFP-G?1or
GFP-G?1W332A. We expected that transfection of the wt
heterotrimer would enhance mSIRK-mediated ERK phos-
phorylation, but it did not (Fig. 2A, lanes 1–4). We were
surprised to find that transfection of the trimer containing
GFP-?1W332A significantly inhibited the response of these
cells to mSIRK (Fig. 2A, compare lanes 1 and 2 with lanes 5
and 6). It is possible that the ? subunit transfected with
G?1W332A was weakened in its interaction with ?1?2con-
taining this mutation and that the excess free G? subunits
could sequester endogenous G?? subunits released upon
peptide addition. To test this, we transfected G?1W332A
with ?2subunits without ? subunits. Transfected G?1W332A
with G?2also inhibited mSIRK-dependent ERK phosphory-
lation, whereas transfection of wild-type G?1and G?2did not
If G?1W332A is acting as a dominant negative inhibitor of
peptide-mediated ERK activation, then transfection of cells
with excess wild-type GFP-?1should overcome the inhibition
by G?1W332A. Cells were transfected with either GFP-
G?1W332A?2, GFP-G?1?2, or GFP-G?1W332A?2 cotrans-
fected with a 2-fold excess of GFP-G?1?2. mSIRK-dependent
ERK activation (lanes 1 and 2) was strongly inhibited by
transfection of mutant ?1W332A?2(lanes 3 and 4), and this
was largely rescued by the cotransfection of the wild-type
?1?2subunit (lanes 5 and 6).
To determine whether this dominant negative effect was
specific to mSIRK-mediated ERK activation, we tested
whether transfection of G?1W332A?2-affected LPA recep-
tor-dependent ERK activation. In contrast to its effects
G?1W332A?2had no effect on LPA-mediated ERK activation
(Fig. 3A). To confirm that LPA-dependent ERK activation in
CHO cells was mediated by G?? subunits, we tested the
effects of pertussis toxin (PTX) pretreatment. PTX is thought
to inhibit GPCR-dependent ERK activation by preventing
the release of free G?? from Gi heterotrimers (Luttrell et al.,
1997). PTX strongly inhibited LPA-mediated responses, in-
dicating that ERK activation by LPA in these cells is through
a G?? subunit-dependent pathway (Fig. 3B). mSIRK-depen-
dent ERK activation was not inhibited by PTX because
mSIRK works through a non–receptor-dependent mecha-
nism. These data indicate that the W332A mutation does not
affect the ability of the ? subunit to activate ERK through
GPCRs and that it is specific for peptide-mediated ERK ac-
tivation. Thus, a binding site containing W332 on the G?
subunit is probably the direct target of peptide-mediated
ERK activation in intact cells. The exact mechanism for the
dominant negative effect of G?W332A on peptide-mediated
activation of endogenous ERK pathways is unknown, but we
suspect that the overexpressed mutant replaces endogenous
G?? subunits in endogenous G protein heterotrimers and
activation, transfection of
Fig. 2. ?1W332A?2inhibits peptide-dependent activation of ERK in CHO
cells. A, 200 ng of each of the indicated cDNAs was transfected into CHO
cells in a six-well plate. Forty-eight hours later, 10 ?M mSIRK was added
for 5 min. Cells were processed and immunoblotted for phospho-ERK
(pERK), total ERK, EE-?i1(anti-EE antibody), and GFP-?1(anti-GFP
antibody) as discussed under Materials and Methods. B, either GFP-
?1W332A and ?2(200 ng each) or GFP-?1and ?2(200 ng each) were
transfected into CHO cells and treated 48h later with 10 ?M mSIRK for
5 min. Cells were processed and immunoblotted for pERK, total ERK, and
GFP-?1as described in A. In A and B, 1 ?g of total DNA was transfected
and balanced with pRK5 empty vector. C, CHO cells were transfected
with 250 ng of each of the indicated constructs, except for the lanes
labeled W332A GFP-?1?2? GFP-?1?2, where 500 ng of each GFP-?1and
G?2were transfected along with W332A GFP-?1?2. In each case, the total
DNA transfected in each well was made up to 1.5 ?g with pRK5 vector.
Treatments were done in the same manner as in A and B. Samples were
immunoblotted for pERK, ERK, and GFP-?1as described in A.
Fig. 3. W332A?1?2does not inhibit ligand-mediated ERK activation in
CHO cells. A, CHO cells transfected with 200 ng of each GFP?1and G?2
or GFP-?1W332A, and G?2were treated with the indicated concentra-
tions of LPA for 5 min. Samples were prepared and separated by SDS-
PAGE, and proteins were transferred and immunoblotted for pERK as in
Fig. 2. B, CHO cells were treated for 16 h with 100 ng/ml PTX and treated
with either 10 ?M mSIRK or 5 nM LPA for 5 min. Samples were prepared
and analyzed as described in A.
Malik et al.
at ASPET Journals on January 4, 2016
these heterotrimers are resistant to mSIRK activation but
not to receptor-mediated activation.
Ric-8A Enhances mSIRK-Mediated IP Release. Ric-
8A, a recently described G protein guanine nucleotide ex-
change factor (GEF) for G?q,11,i,o, and12/13, exchanges GDP
for GTP on free G?GDP but not G?GDP?? (Tall et al., 2003).
We reasoned that if free G?GDP subunits were released from
G protein heterotrimers by mSIRK in cells transfected with
Ric-8A, the G?GTP subunit-mediated responses to mSIRK
would be enhanced. Because G?qGDP is a substrate for
Ric-8A in vitro, we predicted that cells expressing Ric-8A
would have enhanced mSIRK-dependent IP production be-
cause of an increased level of G?qGTP. mSIRK alone causes
a small but reproducible increase in IP release in cells trans-
fected with vector control DNA, similar to what we have
reported previously (Goubaeva et al., 2003). mSIRK-depen-
dent IP production was enhanced in a dose-dependent man-
ner with transfection of increasing amounts of Ric-8A cDNA
(Fig. 4, A and B). On the other hand, Ric-8A had no signifi-
cant effect on basal IP release (data not shown) or IP release
mediated by the GPCR agonists ATP or LPA (Fig. 4, C and D)
consistent with previous reports (Tall et al., 2003). Pretreat-
ment with PTX inhibited ATP-dependent IP release by 50%
and LPA-dependent IP release by 80% (data not shown),
indicating that ATP activates PLC? through a combination
of Gqand Gi/?? pathways, whereas LPA is entirely through
Gi/?? in these CHO cells.
Ric-8A Enhances mSIRK-Dependent IP Production
and ERK Activation through a ??-Dependent Mecha-
nism. To test whether the Ric-8A enhancement of mSIRK-
dependent IP production was through ?qGTP or ?? subunits,
we determined whether Ric-8A-enhanced IP production
could be suppressed by inhibitors of G protein ?? subunit
signaling. CHO cells were transfected with Ric-8A or Ric-8A
and either the C terminus from ?ARK (?ARK-ct) or the G?
subunit of transducin, ?t. These reagents have been exten-
sively used to sequester free G?? subunits without interfer-
ing directly with receptor catalyzed G protein activation
(Koch et al., 1994). Both transducin and the ?ARK-ct inhib-
ited responses by mSIRK and mSIRK/Ric-8A to similar levels
(Fig. 5, A and B). This indicates that mSIRK-mediated IP
release is through free G?? subunits and that Ric-8A en-
hances this ??-dependent response.
We had previously shown that mSIRK peptides activate
ERK in a manner that was blocked by the ?ARK-ct, strongly
suggesting that this response was dependent upon the re-
lease of free G?? subunits in rat arterial smooth muscle cells
(Goubaeva et al., 2003; data not shown). Here, we tested
whether G??-dependent ERK activation in CHO cells could
be enhanced by transfection of Ric-8A to further explore the
Fig. 4. Ric-8A enhanced mSIRK-dependent IP release in CHO cells. A, CHO cells were transfected with the indicated amounts of Ric-8A pCMV5 [or
enhanced green fluorescent protein (pEGFP) control vector, 400 ng total] for 48 h in 12-well dishes. Cells were then treated with mSIRK for 60 min
and total IP was measured. IP released is expressed as cpm/well minus basal with no mSIRK treatment. Assays were performed in triplicate, and data
are expressed as mean ? S.E.M. The assay was repeated four times. B, pooled data from four experiments with either 200 ng of EGFP or Ric-8A
transfected and 10 ?M mSIRK was added. mSIRK alone (?Ric-8A) was taken as the 100% control, and data are expressed as mean ? SEM. ?Ric-8A
is statistically different from ?Ric-8A, P ? 0.05 in a paired t test. C, effect of Ric-8A on hormone-dependent IP release. Cells were transfected with
the 200 ng of Ric-8A or pEGFP control DNA and assayed and treated with 0.2 ?M ATP, 5 nM LPA, or 10 ?M mSIRK for 30 min. D, data from seven
experiments as described in C were pooled and analyzed for statistical significance. To facilitate pooling, the data were normalized by subtracting the
unstimulated cpm from either ATP-, LPA-, or mSIRK-stimulated cpm in the absence of Ric-8 (100%). Data are expressed as mean ? SEM. ATP alone
was not statistically significantly different from ATP ? Ric-8A, and LPA alone was not statistically significantly different from LPA ? Ric-8A.
G?? Signaling Enhancement by Ric-8
at ASPET Journals on January 4, 2016
idea that Ric-8A can enhance G??-mediated responses. As
shown in Fig. 6, A and B, ERK phosphorylation was in-
creased in the presence of mSIRK, and the response was
significantly enhanced in cells transfected with Ric-8A or
Ric-8B. mSIRK/Ric-8A-dependent ERK activation was signif-
icantly attenuated by transducin (Fig. 6, A and B) and
?ARK-ct expression (data not shown), indicating that Ric-8A
enhancement of mSIRK-dependent ERK activation is medi-
ated by G?? subunits and not G? subunits.
We also examined whether Ric-8A or Ric-8B could alter
ERK activation in CHO cells in response to the GPCR ago-
nists LPA or ATP. LPA is coupled to ERK activation primar-
ily through Gi/G??, whereas ATP is coupled partially
through Gi/?? and partially through a PTX-insensitive G
protein, presumably G?q. Ric-8A and Ric-8B both enhanced
ERK activation in response to LPA (Fig. 7, A and B) and ATP
(Fig. 8, A and B). LPA-dependent ERK activation was com-
pletely blocked by PTX (Fig. 7, A and B), whereas ATP-
dependent ERK activation was partially inhibited by PTX
(Fig. 8, A and B). These data are consistent with a partial and
complete dependence on Gi/?? pathways for ATP- and LPA-
dependent ERK activation, respectively. The enhancement of
mSIRK-, LPA-, and ATP-dependent ERK activation by
Ric-8A or Ric-8B is modest (a 50–100% increase). For this
reason, the results from multiple experiments were quanti-
tated, pooled, and presented in Figs. 6B, 7B, and 8B with
Fig. 5. Inhibitors of ?? subunit signaling inhibit mSIRK and mSIRK/Ric-
8A-dependent IP release. A, cells were transfected with pRK5 control
DNA (400 ng) or Ric-8A (200 ng) plus pRK5 control vector (200 ng); or
Ric-8A (200 ng) plus ?t(200 ng) or ?ARK-ct (200 ng) followed by treat-
ment with 10 ?M mSIRK for 60 min. B, pooled data from seven experi-
ments (?t) and three experiments (?ARKct) expressed as percentage of
mSIRK-dependent IP release as in Fig. 4D.
Fig. 6. Ric-8 enhances mSIRK-dependent ERK activation in a ?? sub-
unit-dependent manner. A, cells transfected with 1 ?g of vector control or
Ric-8A or B DNA (200 ng) and pRK5 (800 ng) or ?t(800 ng) were treated
for 5 min with or without 10 ?M mSIRK and assayed for phospho-ERK,
total ERK, and Ric-8A expression by immunoblotting. B, phosphorylated
ERK immunoblots for mSIRK-treated samples were scanned by densi-
tometry, and data were pooled from five experiments and normalized to
mSIRK-dependent activation at 100%. Data were analyzed by a one-way
analysis of variance followed by Bonferroni’s post test; ??, P ? 0.01
compared with mSIRK alone (column 1).
Fig. 7. Ric-8 enhances LPA-dependent ERK activation. A, CHO cells
transfected with 200 ng of the indicated constructs were treated with 5
nM LPA for 5 min with or without prior treatment with 100 ng/ml PTX
for 16 h. B, phosphorylated ERK immunoblots for LPA-treated samples
were scanned by densitometry and data pooled from five experiments and
normalized to mSIRK-dependent activation at 100%. Data were analyzed
by a one-way analysis of variance followed by Bonferroni’s post test; ??,
P ? 0.01; ?, P ? 0.05 compared with mSIRK alone (column 1).
Malik et al.
at ASPET Journals on January 4, 2016
analysis for statistical significance. For mSIRK and LPA, the
data clearly show a significant enhancement of ERK activa-
tion by Ric-8A and Ric-8B. For ATP, there is a trend toward
enhancement that it is not statistically significant. This
could be because not all of the ATP-dependent ERK activa-
tion is mediated by G?? subunits. Overall, these data suggest
that Ric-8A enhances the responses to these agonists by
enhancing G protein ??-dependent signaling.
Ric-8A Binds ? Subunits in Transfected CHO Cells.
We were surprised that Ric-8A enhanced ??-dependent
rather than ? subunit-dependent responses. To explain this,
we hypothesized that excess Ric-8A transfected in cells could
bind and sequester the endogenous ? subunits, thereby en-
hancing signaling by ?? subunits. To determine whether
Ric-8A stably binds ? subunits in CHO cells, we transfected
the cells with HA-tagged Ric-8A and either EE-?i1or the
empty vector. Cell lysates were prepared, followed by immu-
noprecipitation with anti-EE antibody. The immunoprecipi-
tate was probed with anti-HA antibody (Fig. 9). Ric-8A-3HA
only coimmunoprecipitated from cell lysates containing
expressed EE-?i1subunits. Similar results were seen when
Ric-8A-3HA was cotransfected with EE-G?q (data not
shown). Together, these results show that in CHO cells
Ric-8A can efficiently bind and sequester G? subunits.
We have previously shown that phages display derived
peptides that bind to G protein ?? subunits that can activate
several signaling pathways in intact cells and promote G
protein subunit dissociation in vitro. The cocrystal structure
of the peptide bound to G protein ?? subunits was recently
solved, with the peptide bound at a position occupied by the
switch II helix of G?i1(T. L. Davis, T. M. Bonacci, S. R.
Sprang, and A. V. Smrcka, submitted). This provides a plau-
sible explanation at the molecular level of how the peptide
causes G protein activation. Here, we present evidence that
the peptide binds directly to G?? subunits in intact cells and
causes ? subunits to dissociate from G?? subunits to promote
First, the W332A mutant of G?1, but not wt G?1, blocked
mSIRK-dependent ERK activation in intact cells. G?1W332A
does not bind to SIRK and should not respond to mSIRK
treatment. We expected the G?W332A mutation would alter
the behavior of the transfected G protein heterotrimer (both
G? and G?? transfected) and were surprised to find that it
behaved as a dominant negative inhibitor of peptide-depen-
dent activation of endogenous G protein signaling. We do not
fully understand the mechanism of action of this dominant
negative inhibition but hypothesize that the overexpressed
G?1mutant incorporates into and replaces at least part of
the endogenous G protein signaling pool. Regardless of the
mechanism, it is clear that transfection of this mutant G?1
subunit specifically inhibits ERK activation by mSIRK
but not by LPA. The fact that signaling to ERK by endoge-
nous GPCRs remains intact indicates that the ability of
G?1W332A to activate ERK is not impaired. This is not
entirely surprising because this is a binding site for SIRK,
and SIRK does not inhibit ERK activation in cells (Goubaeva
et al., 2003). In addition, mutation of ?W332 to A has previ-
ously been shown to selectively inhibit its ability to interact
with effectors and does not interfere with its ability to inter-
act with certain receptors (Ford et al., 1998; Li et al., 1998;
Myung and Garrison, 2000). This demonstrates that direct
binding of mSIRK to G?? subunits is required for mSIRK to
activate ERK in transfected cells.
Although this result strongly supports the idea that the
G?? subunits of G protein heterotrimers are the target of
these peptides in intact cells, it does not necessarily indicate
that binding of the peptide to G?? causes subunit dissocia-
tion in intact cells. To test this, cells were transfected with
Ric-8A, with the idea that it would convert free G?GDP
released by mSIRK to G?GTP, which could then activate
Fig. 8. Ric-8 marginally enhances ATP-dependent ERK activation. A,
CHO cells transfected with 200 ng of the indicated constructs were
treated with 0.2 ?M ATP for 5 min with or without prior treatment with
100 ng/ml PTX for 16 h. B, phosphorylated ERK immunoblots for ATP-
treated samples were scanned by densitometry and data pooled from five
experiments and normalized to mSIRK-dependent activation at 100%.
Data were analyzed by a one-way analysis of variance followed by Bon-
ferroni’s post test, and Ric-8A and B enhancements were not found to be
Fig. 9. Ric-8A coimmunoprecipitates with G?i1. CHO cells transfected
with 500 ng each of Ric-8A-3HA and EE-?i1(lanes 1 and 2) or Ric-8A-3HA
and pCI-Neo vector (lanes 3 and 4) were immunoprecipitated with an-
ti-EE antibody and probed with anti-HA antibody. Lanes 1 and 3 show
expression of Ric-8A-3HA in the transfected cell lysate (1/15 loaded
relative to the immunoprecipitate). Lanes 2 and 4 show the Ric8-A 3HA
protein that only coimmunoprecipitates when expressed in the presence
of EE-?i1(lane 2 compared with lane 4).
G?? Signaling Enhancement by Ric-8
at ASPET Journals on January 4, 2016
signal transduction pathways downstream of G?GTP. We
had previously shown that mSIRK causes increases in IP
production in RASM cells. It was not clear whether this was
caused by free G?? subunits or by free G?qGDP released that
spontaneously exchanged GDP for GTP (Higashijima et al.,
1987). If free ?qGDP was released by mSIRK and this was a
potential substrate for Ric-8A, then we predicted Ric-8A
would enhance mSIRK-mediated IP release. This is in fact
what was observed; to our surprise, however, the enhanced
IP release seems to be dependent on G?? rather than G?q.
This is based on the observation that the IP production in
response to mSIRK/Ric-8A can be almost completely abro-
gated by treatment with transducin and the ?ARK-ct.
The surprising result that Ric-8A can enhance G??-depen-
dent responses is supported by the observation that Ric-8A
also enhances mSIRK-dependent ERK activation. We had
reported previously, and confirm here in CHO cells, that
mSIRK-dependent ERK activation is entirely dependent on
G?? subunits. Similar results were seen with activation of G
protein-coupled receptor agonists where Ric-8A or Ric-8B
enhanced the ligand-dependent ERK activation. The en-
hancement in these cases is modest yet reproducible. For
LPA in particular, the entire response was blocked by PTX,
indicating that Ric-8A enhanced a G??-dependent pathway.
These data are among the first to show that transfected
Ric-8 has a biological effect. Previous work noted that trans-
fected Ric-8A had no effect on G?q-dependent signaling in
intact cells (Tall et al., 2003). In those studies, there were
multiple possible reasons that transfected Ric-8 was either
inactive or unable to access the G protein. The studies pre-
sented here show that that Ric-8A binds G protein ? subunits
in cells and enhances ?? subunit-dependent signaling, yet
does not seem to enhance ? subunit-mediated responses. If
the Ric-8 can access and bind to endogenous G protein ?
subunits, why is no ?GTP subunit-dependent signaling ob-
served? A possibility is that at the high concentrations of
Ric-8 expressed in these cells, the excess Ric-8 can bind
G?GTP attenuating G?GTP-dependent signaling. Such a
possibility is suggested by the observation that Ric-8A stim-
ulates steady-state GTP hydrolysis at low concentrations of
Ric-8A, but it inhibits at higher concentrations (G. G. Tall
and A. G. Gilman, unpublished observations).
Demonstration that Ric-8 can enhance G??-dependent
pathways was unexpected but not entirely inconsistent with
its known function. Ric-8A binds to G?i, G?o, G?12/13, and
G?qGDP subunits and catalyzes exchange of GDP for GTP.
After hydrolysis of G?GTP to G?GDP, the G?GDP might
preferentially bind to the expressed Ric-8 over free G?? and
another round of exchange could occur. Neither free G?GTP,
Ric-8:G?GTP, or Ric-8:G?GDP would be expected to rebind
to G protein ?? subunits, thus the presence of excess Ric-8
would extend the lifetime of free G protein ?? subunits in the
cell. Overall, the data support the notion that free G?GDP
subunits are generated in the cell upon treatment with
mSIRK because Ric-8 enhances the mSIRK effects.
Several articles have been published suggesting a role for
Ric-8 in asymmetric cell division in C. elegans (Afshar et al.,
2004; Couwenbergs et al., 2004; Hess et al., 2004). Because
deletion of G? subunits in these animals enhances the G
protein-dependent effects on spindle positioning, presumably
by raising the level of free G? subunits in cells, it is unlikely
that G?? is directly involved in this process. Thus, it is also
unlikely that there is a role for Ric-8 in generating free G??
subunits in this system. Although it is not entirely clear that
release of free G?? subunits is a mechanism that occurs with
these endogenous Ric-8/G protein signaling systems, our
data suggest the possibility that Ric-8 may enhance G??
effects through a novel mechanism in more conventional G
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Address correspondence to: Dr. Alan V. Smrcka, Department of Pharma-
cology and Physiology, University of Rochester School of Medicine and
Dentistry, Box 711, Rochester, NY 14642. E-mail: alan_smrcka@urmc.
Malik et al.
at ASPET Journals on January 4, 2016