From the
Pleckstrin homology (PH) domains are 90-110 amino acid
regions of protein sequence homology that are found in a variety of
proteins involved in signal transduction and growth control. We have
previously reported that the PH domains of several proteins, including
Pleckstrin homology (PH)
The function of PH domains is
unknown. A possible clue to their function comes from the localization
of a PH domain to the carboxyl terminus of
To determine
whether PH domains can function as G
Expression of the
To determine whether PH domain peptides would behave as
G
If PH domains function as
G
The data presented here provide evidence that PH
domain-containing peptides derived from five distinct proteins behave
as G
Recent observations have expanded
the role of G
Appreciation of the broad importance of
G
The solution structures of the PH domains of
pleckstrin
(6) , spectrin (7), and dynamin
(8, 9) bear considerable similarity to proteins that bind small
hydrophobic molecules such as FK506 binding protein and retinal binding
protein, which belong to a structural superfamily termed the calycins.
Indeed, some PH domains can apparently bind phosphatidylinositol
4,5-bisphosphate or phosphatidylinositol 4-phosphate in the cleft of
the
The previously
characterized glutathione S-transferase-PH domain fusion
proteins exhibited a relative order of potency for inhibition of bovine
brain G
Our previous work with PH domain-glutathione
S-transferase fusion proteins in vitro(13) as
well as the data presented here on the behavior of PH domain peptides
expressed in intact cells provide strong evidence that some PH domains
can function as G
We thank S. T. Exum for excellent technical assistance
and D. Addison and M. Holben for secretarial assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ARK1, PLC
, IRS-1, Ras-GRF, and Ras-GAP, expressed as
glutathione S-transferase fusion proteins, can reversibly bind
purified bovine brain G
subunits in vitro with
varying affinity. To determine whether PH domain peptides would behave
as antagonists of G
subunit-mediated signal transduction in
intact cells, plasmid minigene constructs encoding these PH domains
were prepared, which permit transient cellular expression of the
peptides. Pertussis toxin-sensitive, G
subunit-mediated
inositol phosphate (IP) production was significantly inhibited in COS-7
cells transiently coexpressing the
2-C10 adrenergic receptor (AR)
and each of the PH domain peptides. Pertussis toxin-insensitive,
G
subunit-mediated IP production via coexpressed M1
muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the
PLC
PH domain peptide, suggesting that the inhibitory effect of
most of the PH domain peptides was G
subunit-specific.
Stimulation of the mitogen-activated protein (MAP) kinase pathway by
G
-coupled receptors in COS-7 cells has been reported to
require activation of p21
and to be independent
of protein kinase C. Since several proteins involved in activation
contain PH domains, the effect of PH domain peptide expression on
2-C10 AR-mediated p21
-GTP exchange and MAP
kinase activation as well as direct G
subunit-mediated
activation of MAP kinase was determined. In each assay, coexpression of
the PH domain peptides resulted in significant inhibition. Increasing
G
subunit expression surmounted PH domain peptide-mediated
inhibition of MAP kinase activation. These data suggest that the PH
domain peptides behave as specific antagonists of G
-mediated
signaling in intact cells and that interactions between PH domains and
G
subunits or structurally related proteins may play a role
in the activation of mitogenic signaling pathways by G protein-coupled
receptors.
(
)
domains, so
named because of their original description as internal repeats in
pleckstrin, the major protein kinase C substrate in
platelets
(1, 2) , are 90-110 amino acid regions of
protein sequence homology that are found in a variety of proteins
involved in signal transduction and growth control. Included in the
list of approximately 70 known PH domain-containing proteins are
several that participate in the function of Ras including Ras-GRF,
Sos1, and Ras-GAP, the growth factor-binding protein Grb7, the insulin
receptor substrate IRS-1, serine/threonine kinases including Rac-
,
Rac-
, and the
-adrenergic receptor kinases
ARK1 and
ARK2, tyrosine kinases including the Bruton tyrosine kinase (Btk),
Tec, and TSK, phospholipase C species including PLC
and PLC
,
the cytoskeletal protein spectrin, the microtubule-binding GTPase
dynamin, and oxysterol binding protein (3-5). The
three-dimensional structures of the pleckstrin
(6) ,
spectrin
(7) , and dynamin
(8, 9) PH domains
demonstrate that they consist of seven antiparallel
-sheets and a
carboxyl-terminal amphiphilic
-helix. The three structures are
essentially superimposable, indicating that despite relatively low
primary sequence homology, PH domains apparently represent a protein
domain of defined tertiary structure.
ARK1 and
ARK2,
where it corresponds approximately to that region of the protein which
functions as a specific recognition domain for the G
subunits
of heterotrimeric G proteins. With
ARK, this region is responsible
for agonist-induced translocation of the kinase from the cytosol to the
plasma membrane, where it initiates the process of homologous
desensitization
(10, 11, 12) . We have previously
shown that the PH domains of several other proteins, expressed as
glutathione S-transferase fusion proteins, can reversibly bind
bovine brain G
subunits in vitro with varying
affinity
(13) . This observation, coupled with recent reports
that Ras-dependent activation of MAP kinase by those G protein-coupled
receptors that utilize pertussis toxin-sensitive G proteins is mediated
by G
subunits
(14, 15, 16) , suggests
that the PH domains of other proteins may function as recognition
domains for G
subunits in the intact cell.
subunit-binding domains
in the intact cell, we have prepared a series of plasmid minigene
constructs that permit transient cellular expression of PH
domain-containing peptides. We tested whether expression of peptides
comprising the PH domains of
ARK1, IRS-1, PLC
, Ras-GAP and
Ras-GRF could specifically antagonize G
-coupled
receptor-mediated PLC activation in transiently transfected COS-7
cells, i.e. whether the peptides could act as free G
subunit scavengers within the cell. We also assessed whether PH domain
peptide expression could antagonize G
-coupled
receptor-mediated Ras-GTP exchange and MAP kinase activation as well as
the activation of MAP kinase that resulted directly from overexpression
of G
subunits. Our data suggest that the PH domains of
several proteins can function as G
subunit-binding domains in
the intact cell and that PH domain-directed association of these
proteins with G
subunits or structurally related proteins may
play an important role in the regulation of cellular mitogenesis.
Materials
The cDNA for the human 2-C10 AR
was cloned in our laboratory
(17) . The cDNA for the human M1
AChR
(18) was provided by Dr. Ernest Peralta. The cDNAs encoding
G
1
(19) and G
2
(20) were provided by Dr. Mel
Simon. The cDNA encoding hemagglutinin-tagged p44
(erk1) (p44
)
(21) was
provided by Dr. Jacques Pouyssegur. The sources of cDNA fragments
encoding the PH domains of bovine
ARK1, rat IRS-1, rat PLC
,
bovine Ras-GAP, and rat Ras-GRF were as described
previously
(13) . LipofectAMINE was obtained from Life
Technologies, Inc. Rabbit anti-
ARK carboxyl terminus was prepared
in our laboratory
(11) . Rabbit polyclonal anti-IRS-1 was from
Transduction Laboratories (Lexington, KY). Mixed monoclonal anti-GAP
and anti-PLC
were from Upstate Biotechnology Incorporated (Lake
Placid, NY). Rabbit polyclonal anti-erk2 (C14) agarose
conjugate was from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antibody 12CA5 was from Boehringer Mannheim. Monoclonal
anti-p21
antibody Y13-259, Protein
A-agarose, and Protein G plus-agarose were from Oncogene Science
(Cambridge, MA). Protein G Sepharose 4 Fast Flow was from Pharmacia
Biotech Inc. Sources of other reagents were as described
previously
(14, 22) .
DNA Constructs
The peptide-encoding cDNA fragments
spanning the previously described
(13) G
subunit-binding regions of the PH domains of bovine
ARK1
(Gly
-Leu
), rat IRS-1
(Phe
-Glu
), rat PLC
(Asn
-Lys
), bovine Ras-GAP
(Asp
-Asn
) and rat Ras-GRF
(Phe
-Ser
), shown schematically in
Fig. 1
, were amplified using the polymerase chain reaction to
create minigene peptide coding inserts. Expression constructs were
prepared by subcloning the amplified peptide coding cassette into the
minigene peptide coding region as described previously
(22) . The
full-length minigene DNA fragments were subcloned into the pRK5 vector
for transient eukaryotic cell expression. Receptor cDNAs and cDNAs
encoding G
1, G
2, and p44
were
subcloned into pRK5 or pcDNA eukaryotic expression vectors.
Figure 1:
Construction of PH
domain peptide minigenes. A, the cDNA sequences encoding the
indicated regions of ARK1, PLC
, IRS-1, Ras-GRF, and Ras-GAP
were employed in the construction of PH domain peptide minigenes.
Boxedareas represent the relative location of PH
domain sequences within the peptide minigene product and the
approximate position of the G
subunit-binding domains of
ARK1, Ras-GRF, and PLC
as previously determined (13). PH
domain sequences are aligned on the conserved tryptophan residue
(W) in subdomain 6 as shown. B, schematic
representation of the peptide minigene construct employed for PH domain
peptide expression. aa, amino
acids.
Cell Culture and Transfection
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin
at 37 °C in a humidified, 5% CO atmosphere. For
transfection using DEAE-dextran, cells were seeded in 6-well tissue
culture plates and transfected using a total of 1-2 µg of
DNA/well as described previously
(22, 23) . For
transfection using the LipofectAMINE reagent, cells in 6-well tissue
culture plates were incubated at 37 °C with a transfection mixture
composed of 1 ml of serum-free DMEM containing 2 µg of DNA/well and
12 µl of LipofectAMINE. After 5 h, 1 ml of DMEM containing 20%
fetal bovine serum was added, and the cells were incubated overnight.
LipofectAMINE transfection consistently resulted in COS-7 cell
transfection efficiencies of greater than 80% (data not shown). Assays
were performed 48 h after transfection. Empty pRK5 vector was added to
transfections as needed to keep the total mass of DNA added per well
constant within an experiment.
ARK1 PH domain
was determined by protein immunoblotting of whole cell detergent
lysates using rabbit anti-
ARK1 carboxyl terminus antiserum as
described
(24) . Cellular expression of the IRS-1, PLC
, and
Ras-GAP PH domain peptides was documented following metabolic labeling
with [
S]methionine. Transfected COS-7 cells in
100-mm dishes were incubated overnight in 3 ml of methionine-free
medium containing [
S]methionine (100
µCi/ml), lysed in 1 ml of RIPA buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate), and
clarified by centrifugation. Supernatants were incubated with
anti-IRS-1 (4 µg), anti-PLC
(8 µg), or anti-Ras-GAP (8
µg); immune complexes were collected on Protein A-agarose
(anti-IRS-1) or Protein G plus-agarose (anti-PLC
and anti-Ras-GAP)
and resolved by 16% Tricine SDS-polyacrylamide gel electrophoresis.
Labeled peptides were visualized by autoradiography. Receptor
expression was quantitated by saturation binding analysis as
described
(22) .
Inositol Phosphate Production
For measurement of
total inositol phosphates (IPs), transfected COS-7 cells were labeled
for 18-24 h with [H]myo-inositol
(2 mCi/ml) in DMEM supplemented with 3% fetal bovine serum. After
labeling, cells were washed for 20 min at 37 °C with calcium-free
Dulbecco's phosphate-buffered saline, preincubated for 20 min in
Dulbecco's phosphate-buffered saline supplemented with 20
mM LiCl and 1 mM CaCl
and stimulated for
45 min with or without agonist (10 µM UK-14304 for the
2-C10 AR and 100 µM carbachol for the M1 AChR).
Following stimulation, IPs were extracted in 0.4 M perchloric
acid (1 ml/well); 0.8 ml of each sample was neutralized with 0.4 ml of
0.72 M KOH, 0.6 M KHCO3; and 1.0 ml of the
neutralized supernatant was analyzed for total IPs as
described
(22) .
Measurement of MAP Kinase Activity
Activation of
endogenous p42 (erk2) and
epitope-tagged p44
(erk1) was
measured using a modification of the procedure of Meloche et
al.(21) . Briefly, transfected COS-7 cells in 6-well
plates, preincubated overnight in low serum medium (DMEM, 0.5% fetal
bovine serum), were stimulated for 5 min with the indicated agonist,
washed with ice-cold calcium- and magnesium-free
phosphate-buffered-saline, lysed in 200 µl of ice-cold lysis buffer
(50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM
EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10
mM NaF, 10 mM sodium pyrophosphate, 0.1 mM
phenylmethylsulfonyl fluoride) and clarified by centrifugation.
Endogenous p42
was immunoprecipitated using
anti-erk2 agarose conjugate (8 µg) plus 25 µl of a 50%
slurry of protein A-agarose agitated for 1 h at 4 °C. Coexpressed
p44
was immunoprecipitated using 12CA5
antibody (6.5 µg) and 30 µl of a 50% slurry of protein
A-agarose. Immune complexes were washed twice with ice-cold lysis
buffer and twice more with kinase buffer (20 mM HEPES, pH 7.4,
10 mM MgCl
, 1 mM dithiothreitol). The
beads were resuspended in 40 µl of kinase buffer containing 250
µg/ml myelin basic protein (MBP), 20 µM ATP, 2.5
µCi of [
-
P]ATP and incubated for 20 min
at 30 °C. The reaction was terminated by the addition of 40 µl
of 2
Laemmli sample buffer and
P-labeled MBP
resolved by SDS-polyacrylamide gel electrophoresis. Phosphorylation was
quantitated using a Molecular Dynamics PhosphorImager.
Activation of
p21
Transfected COS-7 cells in 6-well
plates were serum-starved overnight as described, labeled for 2 h in
phosphate-free DMEM containing [P]orthophosphate
(200 µCi/ml), and stimulated for 2 min with or without UK-14304.
Following agonist stimulation, monolayers were lysed in detergent
buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 25
mM MgCl
, 1% Nonidet P-40), and
p21
was immunoprecipitated using monoclonal
antibody Y13-259 with Protein G-Sepharose 4 Fast Flow (75 µl
of a 20% slurry in lysis buffer). p21
-bound GDP
and GTP were resolved by thin-layer chromatography on
polyethylenimine-cellulose plates in 0.75 M KH2PO4 (pH 3.4) as
described
(14, 25) . Labeled GDP and GTP were quantitated
using a Molecular Dynamics PhosphorImager, and data were expressed as
the percentage of GTP over total labeled guanine nucleotides.
Effects of PH Domain Peptide Expression on G
In COS-7 cells, transiently
expressed
Subunit-mediated IP Production
2-C10 AR mediates a Bordetella pertussis toxin-sensitive increase in IP production, which apparently
reflects G
subunit-mediated activation of a
isoform of
PLC
(24) . In contrast, transiently expressed M1 AChR mediates a
pertussis toxin-insensitive increase in IP production that is dependent
upon G
subunits
(26) . We have previously shown
that the G
-binding
ARK1 PH domain (10-12)
specifically antagonizes
2-C10 AR-mediated IP production with no
effect on that mediated by the M1 AChR
(24) in COS-7 cells.
G
subunit-mediated conditional stimulation of type II adenyl
cyclase in vitro(27) as well as in intact
(24) and permeabilized
(27) transfected cells is also
blocked. Thus, it appears that in these cells, the ability to
selectively antagonize these G
-coupled receptor-mediated
events correlates with the ability to sequester free G
subunits.
-binding peptides in the intact cell, we studied the effects
of PH domain minigene expression on both
2-C10 AR- and M1
AChR-mediated IP production in COS-7 cells transiently cotransfected
with each minigene and receptor. Fig. 1depicts schematically the
PH domain-containing regions of
ARK1, Ras-GRF, Ras-GAP, PLC
,
and IRS-1 employed in the construction of minigenes. Expression of PH
domain minigene products was confirmed by immunoprecipitation following
[
S]methionine labeling of transfected cells
(data not shown). Fig. 2depicts the effect of coexpression of
each PH domain-containing peptide on
2-C10 AR-mediated IP
production in transiently cotransfected COS-7 cells. In control (vector
cotransfected) cells, agonist-stimulated IP production increased
directly as a function of the level of receptor expression.
Coexpression of a minigene encoding the third intracellular (3i) domain
of the
1B AR, which antagonizes G
-coupled
receptor-mediated IP production
(22) , had no effect on the
2-C10 AR-mediated response (Fig. 2A). In contrast,
coexpression of each of the five PH domain-containing peptides
inhibited the
2-C10 AR-mediated response (Fig. 2,
B-F), suggesting that each could antagonize
G
subunit-mediated PLC activation.
Figure 2:
Effect
of PH domain peptides on 2-C10 AR-mediated IP production. COS-7
cells were transiently cotransfected with plasmid DNA encoding the
2-C10 AR (0.02-2 µg/well) plus empty pRK5 vector (
)
or the indicated peptide minigene (2 µg/well) (
) by the
DEAE-dextran method. Receptor expression and basal and maximal
agonist-stimulated IP production were determined as described. Each
data point represents the mean of triplicate binding and IP
determinations performed on an independent transfection. Vector
cotransfected cell data are shown in each panel for comparison
with
1B 3i domain (A),
ARK1 PH domain (B),
PLC
PH domain (C), IRS-1 PH domain (D), Ras-GRF
PH domain (E), and Ras-GAP PH domain (F)
cotransfected cells.
To determine whether
the inhibition was specific for G subunit-mediated PLC
activation, the effects of coexpression of the
1B 3i domain
peptide and the PH domain-containing peptides on
2-C10 AR- and M1
AChR-mediated IP production were compared, as shown in Fig. 3. At
equal levels of receptor expression, coexpression of each of the PH
domain peptides resulted in a 50-80% attenuation of
2-C10
AR-mediated IP production (Fig. 3A). The
1B 3i
domain peptide had no significant effect. When assayed for the ability
to antagonize M1 AChR-mediated IP production, only the
1B 3i
domain peptide and PLC
PH domain exhibited significant activity
(Fig. 3B). Thus, the
1B 3i domain peptide
selectively antagonized the G
subunit-mediated M1 AChR
signal
(22) ; the
ARK1, IRS-1, Ras-GAP, and Ras-GRF PH
domains selectively antagonized the G
subunit-dependent
2-C10 AR signal; and the PLC
PH domain was inhibitory to both
pathways.
Figure 3:
Comparison of the effects of PH domain
peptides on IP production mediated by 2-C10 AR and M1 AChR. COS-7
cells were transiently cotransfected with plasmid DNA encoding the
2-C10 AR (A) or M1 AChR (B) (0.2 µg/well)
plus empty pRK5 vector or the indicated peptide minigene (2
µg/well) by the DEAE-dextran method. Receptor expression and basal
and maximal agonist-stimulated IP production were determined as
described. Mean levels of receptor expression were not significantly
different between vector cotransfected and minigene cotransfected
cells. IP production is presented as percentage of the maximal
agonist-induced stimulation observed in vector cotransfected cells in
each experiment. Each bar represents mean ± S.E. values
for six to eight separate experiments performed with the
2-C10 AR
and three separate experiments performed with the M1 AChR. *, value
less than control, p < 0.05.
Effects of PH Domain Peptide Expression on G
Several receptors that couple to
pertussis toxin-sensitive G proteins, including the
Subunit-mediated Activation of MAP Kinase and
p21
2 AR, the M2
AChR, and the receptors for lysophosphatidic acid and
-thrombin,
have been shown to activate MAP kinase in various cell
types
(16, 28, 29, 30, 31, 32, 33) .
In COS-7 cells
(16, 29) and Rat 1 fibroblasts
(30-33) this pathway is dependent upon the activation of
p21
and is independent of protein kinase C.
Recently, G
subunits have been implicated in this direct
Ras-dependent activation of MAP kinase. Coexpression of G
1
2
subunits in COS-7 cells leads directly to MAP kinase
activation
(15, 16) . Furthermore, the G
subunit-binding
ARK1 PH domain specifically antagonizes
2-C10
AR-, M2 AChR- and lysophosphatidic acid receptor-stimulated Ras-GTP
exchange and MAP kinase activation, with no effect on
G
subunit-mediated, protein kinase C-dependent,
1B AR-mediated signaling
(14) .
subunit-binding domains in the intact cell, then expression
of PH domain peptide minigenes should result in selective inhibition of
G
-coupled receptor-mediated activation of MAP kinase. To
test this hypothesis, COS-7 cells were cotransfected with the
G
-coupled
2-C10 AR or the G
-coupled M1
AChR, plus each of the PH domain peptide minigenes. Receptor-stimulated
MAP kinase activity was determined following immunoprecipitation of
p42
. As shown in Fig. 4, stimulation of
both the G
-coupled
2-C10 AR and the
G
-coupled M1 AChR produced a severalfold increase in MAP
kinase activity. Coexpression of each of the PH domain peptides
resulted in a 55-75% attenuation of
2-C10 AR-mediated MAP
kinase activation (Fig. 4A). The
1B 3i domain
peptide had no effect (data not shown). When assayed for the ability to
antagonize M1 AChR-mediated MAP kinase activation, which proceeds via a
p21
-independent pathway
(14, 16) ,
only the PLC
PH domain peptide exhibited significant activity
(Fig. 4B). This probably reflects inhibition of
G
-mediated, PLC-dependent activation of protein kinase
C (Fig. 3B). As we have previously shown for the
ARK1 PH domain
(14) , none of the PH domain peptides
inhibited epidermal growth factor-stimulated MAP kinase activation
(data not shown). The effect of PH domain peptide expression on
2-C10 AR-mediated activation of p21
-GTP
exchange, an event known to be involved upstream of MAP kinase in the
pathway of G
subunit-mediated mitogenic signaling, was also
determined. As shown in Fig. 4C, COS-7 cells
coexpressing
2-C10 AR and the
ARK1, IRS-1 and Ras-GRF PH
domain peptides showed impaired agonist-stimulated
p21
-GTP exchange compared with controls.
Differences in the apparent activity of the PH domain constructs were
similar to, but more striking than, that observed for inhibition of MAP
kinase activation, with the
ARK1 and Ras-GRF PH domains exhibiting
the greatest effects.
Figure 4:
Effect of PH domain peptides on G
protein-coupled receptor-mediated MAP kinase activation and p21-GTP
exchange. A, COS-7 cells were transiently cotransfected with
plasmid DNA encoding the 2-C10 AR (0.2 µg/well) plus empty
pRK5 vector or the indicated PH domain peptide minigene (2 µg/well)
by the LipofectAMINE method. Basal and maximal agonist-stimulated MAP
kinase activity were determined as described. MAP kinase activity is
presented as the -fold increase in p42-catalyzed
[
P]phosphate incorporation into MBP. Bars represent mean ± S.E. values for five separate experiments
performed in duplicate. B, COS-7 cells were transiently
cotransfected with plasmid DNA encoding the M1 AChR (0.2 µg/well)
plus empty pRK5 vector or the indicated PH domain peptide minigene (2
µg/well) and assayed as described. MAP kinase activity is presented
as the -fold increase in p42-catalyzed
[
P]phosphate incorporation into MBP. Bars represent mean ± S.E. values for three separate experiments
performed in duplicate. C, COS-7 cells were transiently
cotransfected with plasmid DNA encoding the
2-C10 AR (0.2
µg/well) plus empty pRK5 vector or the indicated PH domain peptide
minigene (2 µg/well). Following agonist stimulation, guanine
nucleotides bound to p21 were quantitated as described. Data are
expressed as the -fold increase in the ratio of GTP to total guanine
nucleotides following agonist stimulation. Bars represent mean
± S.E. values for three separate experiments performed in
duplicate. In each panel, * signifies value less than control,
p < 0.05.
To confirm that the PH domain peptide effects
on 2-C10 AR-mediated MAP kinase activation resulted from the
sequestration of G
subunits, the effect of PH domain peptide
expression on direct G
1
2 subunit-mediated MAP kinase
activation was determined. COS-7 cells were cotransfected with plasmid
DNA encoding hemagglutinin-tagged p44
and
G
1 and G
2 subunits, plus each of the PH domain peptide
minigenes. Activity of immunoprecipitated p44
was determined to assess MAP kinase activation in the transfected
cell pool. As shown in Fig. 5A, p44
activity increased as a function of the amount of G
1
2
subunit expression. Coexpression of increasing amounts of the
ARK1
PH domain peptide resulted in progressive inhibition of this
G
1
2 subunit-mediated effect. Conversely, as shown in
Fig. 5B, increasing G
1
2 subunit expression
diminished the
ARK1 PH domain peptide-mediated inhibition,
suggesting that the observed inhibition resulted from the competitive
interaction of G
subunits and the PH domain peptide. As shown
in Fig. 5C, coexpression of the PLC
, IRS-1,
Ras-GAP, and Ras-GRF PH domain peptides also resulted in significant
attenuation of p44
kinase activity, with the
greatest inhibition observed in cells expressing the
ARK1,
PLC
, and Ras-GRF PH domains.
Figure 5:
Effect of PH domain peptides on direct
G subunit-mediated MAP kinase activation. A, COS-7
cells were transiently cotransfected with plasmid DNA encoding p44 (0.1
µg/well) plus increasing amounts of plasmid DNA encoding G
1
and G
2 subunits (0.01-2.0 µg of each/well) by the
LipofectAMINE method, and basal MAP kinase activity was determined as
described. MAP kinase activity is presented as the -fold increase in
p44-catalyzed [
P]phosphate incorporation into
MBP. Data shown represent mean values for duplicate determinations in
one of two separate experiments. B, COS-7 cells were
transiently cotransfected with the
ARK1 PH domain minigene (0.1 or
0.5 µg/well) and plasmid DNA encoding p44 (0.1 µg/well) plus
increasing amounts of plasmid DNA encoding G
1 and G
2 subunits
(0.01-2.0 µg of each/well), and basal MAP kinase activity was
determined as described. Data are presented as the percent inhibition
of MAP kinase activation in
ARK1 PH domain-expressing cells
compared with control cells expressing only p44 plus each amount of
G
1
2 subunit plasmid DNA. Data shown represent mean values for
duplicate determinations in one of two separate experiments.
C, COS-7 cells were transiently cotransfected with plasmid DNA
encoding G
1 and G
2 subunits (1.0 µg of each/well) and
plasmid DNA encoding p44 (0.1 µg/well) plus empty pRK5 vector or
the indicated PH domain peptide minigene (2 µg/well), and basal MAP
kinase activity was determined as described. MAP kinase activity is
presented as the -fold increase in p44-catalyzed
[
P]phosphate incorporation into MBP. Data shown
represent mean ± S.E. values for three separate experiments
performed in duplicate. *, value less than control, p <
0.05.
subunit-binding domains when expressed in intact cells.
Each peptide, when expressed in COS-7 cells, impaired
2-C10
AR-mediated stimulation of IP production, a G
subunit-dependent process. Four of the peptides (the
ARK1, IRS-1,
Ras-GAP, and Ras-GRF PH domains) were specific for G
subunit-mediated PLC activation in that G
subunit-mediated PLC activation via the M1 AChR was not significantly
attenuated. When these peptides were assayed for the ability to
antagonize
2-C10 AR-mediated p21
-GTP
exchange and MAP kinase activation, two steps in a pathway of
G
-coupled receptor-mediated mitogenic signaling suspected
to involve G
subunits, similar effects were observed.
Furthermore, direct G
subunit-mediated activation of MAP
kinase was attenuated by the peptide minigenes, providing the most
direct evidence that these PH domain peptides act as G
sequestrants in the intact cell.
subunits in cellular signal transduction by G
protein-coupled receptors
(34, 35) . In addition to
activating
ARK1 and
ARK2, G
subunits mediate the
conditional stimulation of types II and IV adenyl cyclase, the PLC
2 and
3 isozymes, phospholipase A2, a
phosphatidylinositol-3`-kinase
(36) , and the atrial K
channel as well as the inhibition of type I adenyl cyclase and
the PLC
1 isozyme. Through as yet undefined pathways, G
subunits apparently mediate the p21
-dependent
activation of MAP kinase, independent of any effects on PLC and protein
kinase C (14, 16).
subunits in cellular signaling has focused attention on
other proteins that might interact with G
subunits. We have
previously shown that glutathione S-transferase fusion
proteins containing the PH domains of nine proteins,
ARK1,
Ras-GRF, Ras-GAP, PLC
, IRS-1, Rac
, Atk, spectrin, and
oxysterol binding protein, reversibly bound bovine brain G
subunits in vitro to varying extents
(13) . G
subunit binding to the fusion proteins was inhibited by
G
subunits, indicating that the binding of G
subunits to the G
subunits and the PH domains was
mutually exclusive. Deletion mapping of the G
subunit-binding
regions of
ARK1 and Ras-GRF
(13) and Btk
(37) has
indicated that only sequences in and just distal to the
carboxyl-terminal half of the PH domain were required for G
subunit binding. In each case, the most highly conserved region of the
PH domain, subdomain 6, which contains the carboxyl-terminal
-helix, was required for in vitro G
subunit
binding.
-barrel
(38) . Other molecules may associate with PH
domains under physiologic conditions as well. The Btk PH domain has
been reported to bind protein kinase C
(39) as well as
G
subunits
(37) , and the PH domain of
-spectrin
has been reported to be the site of membrane interaction
(40) .
Since G
subunit-binding by PH domains requires only the
carboxyl-terminal subdomains, it is tempting to speculate that the
binding of G
subunits or structurally similar proteins to PH
domains may be regulated in complex ways by other molecules, such as
phosphatidylinositol phosphates, or by protein phosphorylation. Thus,
PH domains that exhibit weak or absent G
-binding as
glutathione S-transferase fusion proteins in vitro,
e.g. glutathione S-transferase-spectrin
(13) and glutathione S-transferase-dynamin
(
)
may be modulated in vivo.
subunit-mediated translocation of
ARK1 in
vitro;
ARK1 > PLC
oxysterol binding protein >
GRF > spectrin
(13) , suggesting differences in the apparent
affinity of these peptides for a mixed population of G
subunits. It is difficult to make such direct comparisons of the
relative potency of the PH domain peptides for inhibition of
G
subunit-mediated signaling in transfected cell systems,
since differences in metabolic labeling efficiency and antibody
affinity make accurate comparisons of cellular expression impossible.
It is noteworthy that those PH domain constructs most effective at
inhibiting
2-C10 AR-mediated IP production in the intact cell were
not necessarily as effective at inhibiting
2-C10 AR- or direct
G
subunit-mediated MAP kinase activation. Whereas the
ARK1 PH domain was about equally effective in both assays, the PH
domain of IRS-1 was apparently a better inhibitor of G
subunit-mediated PLC activation, while the Ras-GRF PH domain was a
better inhibitor of G
subunit-mediated MAP kinase activation.
Such differences suggest that while the ability to bind G
subunits may be a property shared by each of the PH domains studied,
these PH domains may recognize different endogenous ``pools''
of G
subunits with different affinity, thereby imparting
additional specificity. PH domains may also recognize other members of
the G
-transducin superfamily of WD40 motif-containing proteins,
which includes more than 11 nonorthologous
proteins
(41, 42, 43) , among them the receptor
for activated C kinase, RACK1, and phospholipase A2 activating
protein
(44) .
subunit-binding domains. The presence of PH
domains in several proteins involved in signal transduction and growth
control along with the growing appreciation that G
subunits
are involved in modulating the activity of several of these pathways,
from the conditional stimulation of PLC species to the direct
activation of p21
, suggests that PH domains may
play a major role in the assembly of membrane-associated protein
complexes that trigger signal transduction cascades in a manner
analogous to the well characterized G
subunit-mediated
translocation of
ARK through its PH domain.
ARK,
-adrenergic receptor kinase; G protein,
heterotrimeric guanine nucleotide-binding regulatory protein;
G
, inhibitory G protein regulator of adenyl cyclase;
G
, stimulatory G protein regulator of phospholipase C;
G
, complex formed by the
and
subunits of G
proteins; IRS-1, insulin receptor substrate 1; MAP, mitogen-activated
protein; MBP, myelin basic protein; PLC, phospholipase C; Ras-GAP,
GTPase-activating protein regulator of p21; Ras-GRF, guanine
nucleotide-releasing factor of p21; DMEM, Dulbecco's modified
Eagle's medium; IP, inositol phosphate; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.