From the
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, ¶Department of Medicine, Emory University, Atlanta, Georgia 30322, and ||Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109
Received for publication, January 3, 2003 , and in revised form, March 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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To probe the nature of common and unique binding sites on subunits, we screened random peptide libraries using purified G protein
subunits as the target to identify peptide sequences required for binding to various surfaces on
(5). Four divergent families of peptides were identified that all bound to a single site on the
subunit surface. We hypothesized this interaction surface represented a protein-protein interaction "hot spot" with the capacity to accommodate a diversity of amino acid sequences. Random peptide library screens often target such protein interaction surfaces (6, 7). The peptides inhibited the interaction between
subunits and effector molecules such as phospholipase C (PLC)1
and phosphatidylinositol 3-kinase
. Interestingly, these peptides did not affect regulation of other effectors such as adenylyl cyclase type I or voltage-gated calcium channels, demonstrating selective interference with particular G protein
subunit-target interactions. This indicates that the proposed hot spot is not a common binding surface for all
effectors.
Most inhibitors of G protein subunit-target interactions, such as the C-terminal domain of
-adrenergic receptor kinase (
ARK1ct), are thought to be universal
subunit inhibitors, blocking activation of all downstream target molecules. Thus the
ARK1ct has been used extensively in intact cells as a global inhibitor of
subunit signaling (8). The goal of this study was to introduce peptides derived from the phage display screen into intact cells to investigate the consequences of selectively interfering with particular G protein
subunit-target interactions in intact cells. To our surprise we found these peptides caused activation of G protein signaling in the absence of receptor activation. We provide evidence for a mechanism where these selective peptides bind to
subunits and promote dissociation of
subunits but leave a surface available on
subunits for activation of MAP kinase pathways.
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EXPERIMENTAL PROCEDURES |
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Cell CultureAll cell culture reagents were obtained from Invitrogen. All cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. Rat arterial smooth muscle (RASM) cells were used between passages 5 and 12. Stable cell lines expressing the ARK1ct were prepared and cultured as described previously (9).
MAP Kinase AssaysRASM or other cells were plated into 35-mm dishes and grown to between 50 and 80% confluency. Serum was removed 16 h before treatment. Peptides in dimethyl sulfoxide or dimethyl sulfoxide vehicle were diluted 100400-fold into the media and incubated at 37 °C for the indicated duration of time. After treatment, the cells were transferred to ice, and the media were quickly aspirated and replaced with 100 µl of 2x SDS sample buffer. The resulting suspension was sonicated briefly in a bath sonicator, and 30 µl was loaded onto a 12% polyacrylamide gel. After SDS-PAGE the proteins were transferred to nitrocellulose for 16 h at 25 V. The transferred proteins were immunoblotted using standard immunoblotting protocols with a 1:1,000 dilution of primary antibody (unless otherwise indicated) and a 1:10,000 dilution of anti-rabbit IgG horseradish peroxidase conjugate. The proteins were visualized by incubation with enhanced chemiluminescence (ECL) reagents (Amersham Biosciences) and exposure to film. Visualization of the shift in molecular weight of ERKs in the total ERK Western blots was somewhat variable. The images for the total ERK Western blots in Fig. 1, A and B, were expanded to emphasize this difference. In other blots the purpose of the total ERK blots was to demonstrate equal sample loading in all lanes of the gels, and conditions were not optimized to resolve the masses of the phosphorylated ERK from ERK.
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Calcium MeasurementsFor Ca2+ imaging, RASM cells were grown on 25-mm glass coverslips and loaded with fura2-AM in Hanks' balanced saline solution plus 15 mM Hepes buffer, pH 7.4. At the start of the experiment, the buffer was switched to Ca2+- and Mg2+-free Hanks' balanced saline solution, and Ca2+ imaging was performed as described previously (10). Between 6 and 20 cells were followed in each experiment, and all experiments were repeated at least three times with similar results. Traces show the responses of individual cells.
Measurements of Inositol PhosphatesCells were plated on 35-mm dishes and labeled by adding 35 µCi of [3H]inositol for 2448 h in inositol-free Dulbecco's modified Eagle's medium. After labeling, the medium was removed and replaced with 1 ml of Hepes-buffered Dulbecco's modified Eagle's medium containing 10 mM LiCl and equilibrated for 20 min at 37 °C. Ligands or peptides were added in a volume of 50 µl for 30 min, after which the medium was aspirated and replaced with 1 ml of ice-cold perchloric acid. The acidified extracts were neutralized by extraction with 1 ml of 1:1 octylamine:Freon, and the aqueous phase was applied to Dowex AG1-X8 columns (Bio-Rad). The columns were washed with water and 50 mM ammonium formate followed by elution of the inositol phosphate-containing fraction with 1.2 M ammonium formate, 0.1 M formic acid. The eluted fraction was mixed with scintillation fluid and analyzed in a liquid scintillation counter.
Preparation of Biotinylated SubunitsThe cDNA for rat
1 subunit was subcloned into a baculovirus transfer vector for expression of N-terminal fusions of a biotin acceptor peptide (11). The biotin acceptor peptide is a stretch of 20 amino acids that is the substrate for the enzyme biotin holoenzyme synthetase (BirA). When a protein fused to the biotin acceptor peptide is coexpressed with BirA the protein becomes biotinylated in vivo at a specific lysine residue in the acceptor peptide sequence. The rat
1 subunit was subcloned by PCR with Pfu polymerase (Stratagene) using primers 5'-ATAAGGCGCGCCAAGTGAACTTGACCAGCTGC-3' and 5'-CCGGAATTCCGGATCCACCTGCTACTG-3' and cloned into the baculovirus transfer vector PDW464 in-frame with the biotin acceptor sequence between the AscI and EcoRI restriction sites to yield MAGGLNDIFEAQKIEWHEDTGGA...
1 sequence, with the lysine residue the site of biotinylation. Baculovirus was generated via recombination in bacteria as described in the Bac-to-Bac system manual (Invitrogen). Biotin-
1
2 was purified from Sf9 cells using hexahistidine-tagged
i1 following previously published procedures (12). Biotinylation of the purified
subunit was confirmed by SDS-PAGE followed by Western blotting with streptavidin-linked horseradish peroxidase and detection with chemiluminescence. To confirm that the
subunit was fully biotinylated, it was precipitated with streptavidin-agarose, and greater that 90% of the
subunit was removed from the supernatant by this procedure.
Measurement of GTP Hydrolysis and GTPS BindingSteady state GTP hydrolysis was assayed as [32P]Pi release from [
-32P]GTP by standard procedures (13). GTP
S binding was measured according to (14), except Sf9 cell membranes expressing the M2 muscarinic acetyl choline receptor were reconstituted with purified recombinant myristoylated
i and
1
2.
Measurement of -
Interactions by Flow CytometryBinding of fluorescein isothiocyanate-labeled myristoylated
i1 (F-
i) to biotinylated
1
2 subunits was measured using a flow cytometry assay (15, 16). Biotinylated
subunits (250 pM final concentration) were mixed with streptavidin beads in HEDNMLG (20 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM DTT, 150 mM NaCl, 0.2 mM free Mg2+, 0.1% polyoxyethylene 10 lauryl ether, 10 µM GDP). After a 20-min incubation at room temperature, the beads were washed twice by centrifugation in a microcentrifuge with HEDNMLG and resuspended in the same buffer at a concentration of 105 beads/ml (250 pM
). For
subunit dissociation experiments, the beads with bound
subunits were premixed with 1 nM F-
i for 10 min before the addition of competitors. For equilibrium binding measurements, 1 nM F-
i and competitors were added simultaneously. The amount of F-
i bound to beads with biotinylated
was assayed at the times indicated in the figure legends using a BD Biosciences FACscan flow cytometer. Nonspecific binding, determined by the simultaneous addition of 1 nM F-
i and 50 nM myristoylated
i, was 1020% of the total signal and was subtracted from the data.
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RESULTS |
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To analyze the specificity of this effect we created a second myristoylated version of this peptide where leucine 9 was changed to alanine (mSIRK(L9A)). We had previously performed alanine-scanning mutagenesis of SIRK and found substitution of Ala for Leu-9 increased the IC50 of this peptide for blocking -dependent PLC activation by more than 100-fold (from 3 to 300 µM) (5). The results in Fig. 1C demonstrate mSIRK(L9A) was completely unable to enhance ERK1/2 phosphorylation. Additionally, a myristoylated-scrambled version (not shown) and a non-myristoylated version of the peptide (SIRK) (Fig. 1C) were unable to cause ERK1/2 phosphorylation. The necessity for myristoylation of the peptide for cellular activity supports the idea that entry of the peptide into the cell is needed to cause ERK activation. To further support the need for intracellular action, we constructed a peptide where the 11-amino acid cell permeation sequence from human immunodeficiency virus TAT protein (19) was added to the N-terminal end of SIRK (TAT-SIRK). Fluorescein was attached to the N terminus to monitor cellular uptake. TAT-SIRK was efficiently taken up into virtually all of cells on the dish as monitored by fluorescence microscopy (not shown). TAT-SIRK was able to activate ERK1/2 to an extent comparable with mSIRK (Fig. 1D), whereas TAT-SIRK(L9A) had no effect (data not shown). These cell-permeating peptides activated ERK to a greater extent than LPA and to an extent comparable with EGF in this assay paradigm (Fig. 1D). Together, these data indicate that structural requirements in the peptides for
binding need to be maintained to observe ERK1/2 activation and that the peptides need to get inside the cell and are not acting by binding to cell surface receptors. This, in conjunction with the observation that the concentration requirement for ERK1/2 activation correlates with the apparent Kd of SIRK for
subunits in vitro (5), strongly supports the idea that
is the target of the peptides and this targeting is responsible for ERK1/2 activation.
G Is the Target of the Peptides in the Intact CellTo further support the idea that
subunits are the target of the peptides in intact cells, we tested a peptide with a completely different amino acid sequence from SIRK. The peptide, SCARFFGTPCP (SCAR), was also selected in the phage display screen, and we have shown by competition analysis that SCAR peptide binds to
subunits in vitro at the same site as SIRK (5). We predicted a myristoylated version of SCAR (mSCAR) should have the same effect as mSIRK on ERK1/2 activation in RASM cells. We have also shown that for SCAR to bind to
subunits, the cysteine residues must form an intramolecular disulfide bond. If SCAR is reduced with DTT, it is no longer able to bind to
in a phage enzyme-linked immunosorbent assay assay. The results in Fig. 2A show mSCAR activates ERK1/2, pretreatment with DTT eliminates its ability to activate ERK1/2, whereas DTT has no effect on the ability of the linear mSIRK to stimulate ERK1/2. These data emphasize that the structure of the peptide binding to
subunits is important to observe activation of ERK1/2, not just the peptide sequence.
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To provide further evidence G subunits are the target of mSIRK and related peptides, we tested the ability of mSIRK to cause ERK1/2 activation in a RASM cell line stably transfected with the C-terminal region of
ARK that sequesters
subunits (
ARK1ct (Gly-495Leu-689)) (8, 9). We hypothesized that mSIRK binds to
but leaves a surface available on
to signal to some downstream targets. Based on this hypothesis, we predicted that ERK1/2 activation by mSIRK would be inhibited by the
ARK1ct, a general
inhibitor that blocks signaling of
to most if not all effectors. The data in Fig. 2B demonstrate that in cells expressing
ARK1ct, activation of ERK1/2 was almost completely inhibited compared with the control RASM cells. This indicates that
subunit signaling is the target of these peptides.
Cell Type Specificity of ERK Activation by mSIRKAll of the experiments described so far used rat arterial smooth muscle cells. To examine the generality of this response, mSIRK was tested in a variety of cell lines. The results in Fig. 3A show that the effect of mSIRK is dependent on the cell type. RASM and Rat2 cells show dramatic activation of ERK1/2 by mSIRK, whereas other smooth muscle cells such as ddtMF2 cells show very little, but some response. These differences could be due to a variety of factors. One possibility is that the myristoylated peptide is only able to enter certain cell types. We, therefore, tested the effects of TAT-SIRK on HEK293 and Cos7 cells. Similar to mSIRK, TAT-SIRK had only small effects on activation of ERK1/2 (not shown). We were able to monitor the uptake of TAT-SIRK into these cells with a fluorescein label and confirmed that this peptide did indeed enter the cells. This indicates that the cell type-specific responses are not due to differential uptake of the peptides, and differences in the endogenous MAP kinase signaling systems may be responsible, i.e. in some cell types -dependent MAP kinase activation is stronger than others.
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Activation of ERK1/2 Requires src but Not EGF Receptor Transactivation or Calcium Release from Internal StoresWe used a variety of pharmacological inhibitors to examine the pathway involved in the response to mSIRK. As expected, a MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor, PD98059, was able to completely block the effect of the peptide (not shown). PP2, a specific src family tyrosine kinase inhibitor, was able to completely block mSIRK-mediated activation of ERK1/2 (Fig. 3B). Src is thought to be involved in -mediated activation of ERK1/2 by G protein-coupled receptors, consistent with a pathway involving
subunits (20). The involvement of EGF receptor transactivation was tested using a specific inhibitor of the EGF receptor, AG1478. This inhibitor completely blocked the ability of the EGF receptor to activate ERK1/2 but had no effect on ERK1/2 activation by mSIRK (Fig. 3B). Thapsigargin treatment, which almost completely emptied the intracellular calcium stores (data not shown and Fig. 4B), was unable to block the effect of mSIRK, indicating that release of calcium from intracellular stores is not responsible for ERK1/2 activation (Fig. 3C). Finally, treatment with pertussis toxin did not block the effect of mSIRK (Fig. 3C), supporting the idea that receptor activation is not involved in the peptide dependent activation of ERK signaling.
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Activation of Other G Protein-coupled Receptor Signal Transduction Pathways by mSIRKThe effects of mSIRK on other signal transduction pathways downstream of either subunits or
subunits were examined. First we looked at other members of the MAP kinase pathway. p38 MAP kinase phosphorylation and Jun kinase phosphorylation are increased by the addition of mSIRK (Fig. 4A) but not by the control peptide, mSIRK(L9A).
The effect of mSIRK on intracellular Ca2+ was followed in RASM cells loaded with the cytoplasmic Ca2+ indicator fura2. At 10 µM, mSIRK caused pronounced oscillations in cytoplasmic Ca2+ in 22/27 cells, often after a lag of 25 min (Fig. 4B, top panel). Ca2+ spiking was not stimulated by the addition of buffer or the control peptide, mSIRK(L9A) (0/34 cells) (Fig. 4B, middle panel). Ca2+ spikes were completely eliminated by thapsigargin, which depletes intracellular Ca2+ stores by inhibiting the SERCA ATPase, responsible for maintaining high Ca2+ concentrations in the endoplasmic reticulum (Fig. 4B, bottom panel). mSIRK also stimulated Ca2+ oscillations in HEK293 cells (data not shown). These Ca2+ oscillations resulted from the release of intracellular Ca2+, because they were eliminated by thapsigargin and observed in Ca2+-free media. In HEK293 cells, as the dose of mSIRK was increased from 0.1 to 10 µM, the fraction of cells responding increased from 30 to 100%, and the lag time decreased from 38 to 0.51 min (data not shown). Pertussis toxin treatment did not inhibit the effects of mSIRK on intracellular Ca2+ (data not shown). To determine whether inositol 1,4,5-trisphosphate production was responsible for this increase, we examined total inositol phosphate production in cells labeled with [3H]inositol. mSIRK caused a reproducible increase in intracellular inositol phosphate release that was unaffected by treatment with PP2 (Fig 4C). Because PP2 blocks src activation and the subsequent pathway leading to ERK1/2 activation, the inositol 1,4,5-trisphosphate release is not an indirect consequence of activation of this pathway.
mSIRK activation of many G protein-dependent pathways suggests mSIRK binds to subunits and somehow promotes release of
subunits, leaving a surface available to signal to downstream pathways. A potential problem with this hypothesis is that SIRK blocks PLC stimulation by
in vitro (5) and, thus, should block the
surface required to signal to PLC. The PLC response caused by mSIRK is quite weak compared with G protein-coupled receptor activation of PLC in RASM cells. It takes minutes for significant Ca2+ spiking to be observed, and the cells respond asynchronously in response to mSIRK. The observed increase in total inositol phosphate production in 30 min was very small. This is reminiscent of very weak activation by a G protein-coupled receptor and contrasts with the rapid strong activation of ERK in the same cells by these peptides. One possibility is that the peptide may not completely block PLC activation by free
, and this residual ability of the
to activate PLC, even in the presence of peptide, is responsible for the weak inositol 1,4,5-trisphosphate and Ca2+ responses observed. Another possibility is that
q/11 may be released, which can bind some GTP once released from
.
SIRK Causes Dissociation of Subunits from
Subunits without Stimulating Nucleotide ExchangeThat all these signaling pathways are being activated in response to a
subunit-binding peptide suggests the peptide is a general activator of G protein signaling. One possibility is that the peptide directly activates the heterotrimer by promoting GTP binding to
subunits. However, these peptides were selected to bind to G protein
subunits and, therefore, should not bind to
subunits. Receptors and receptor-mimicking peptides such as mastoparan act at least in part through binding to
subunits. It is possible, however, that mSIRK may non-specifically interact with
subunits or may promote nucleotide exchange by an undefined mechanism through binding to
subunits. To determine whether the
-binding peptide directly activates the G protein, promoting GDP release and GTP binding, we tested whether the peptide caused an increase in binding of [35S]GTP
S to purified
i1
1
2 (21) and found it did not (Fig. 5A). If the same subunits were reconstituted with urea-stripped Sf9 cell membranes expressing the M2 muscarinic receptor and carbachol, [35S]GTP
S binding was stimulated in a
subunit-dependent manner. To determine whether SIRK could influence M2 receptor-stimulated nucleotide exchange in this assay, SIRK was tested at various concentrations in the presence of M2 receptor and carbachol. Here, SIRK inhibited receptor-dependent nucleotide exchange in a dose-dependent manner (Fig. 5B). Relatively high concentrations of SIRK were required to observe this inhibition (IC50 25 µM), probably because of the relatively high concentration of G protein subunits used in this assay. We hypothesize the peptide inhibits receptor-dependent nucleotide exchange either by interfering with receptor-G protein interactions or G protein subunit interactions. Experiments described later in this section suggest the peptide acts through disruption of interactions between
and
subunits.
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Nucleotide exchange can also be assayed by measuring the steady state rate of GTP hydrolysis on the subunit. In this assay, the rate of hydrolysis is limited by the rate of exchange of GDP for GTP. The amphipathic peptide mastoparan stimulated the rate of hydrolysis of [
-32P]GTP by Go, whereas mSIRK did not (Fig. 5C), indicating that mSIRK cannot stimulate GDP dissociation. Also shown in Fig. 5D is that mastoparan has virtually no effect on ERK activation in RASM cells, whereas mSIRK stimulates significant ERK activation.
To determine whether SIRK could stimulate dissociation of from
in the absence of nucleotide exchange, we assayed the binding of
subunits to biotinylated
subunits bound to streptavidin-agarose beads. G protein
subunits (10 nM) were incubated with 10 nM biotinylated
subunits bound to streptavidin-agarose beads and incubated with mSIRK or
for 20 min. The beads were centrifuged, the supernatant was removed, and the bound
subunits were estimated by quantitative Western blotting (Fig. 6A). At 10 and 30 µM mSIRK the amount of
subunit bound to
subunits was significantly lower than in the absence of added mSIRK.
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A drawback of the pull-down assay is the high concentrations of and
required to detect binding and the relatively crude nature of the quantitation. At 10 nM, each of the subunits was used at concentrations significantly above the estimated Kd for
-
interactions of about 1 nM. A more quantitative assay to measure
-
interactions at very low subunit concentrations has recently been developed that uses flow cytometry to assess the amount of fluorescent
subunit bound to biotinylated
subunits immobilized on beads (15, 16). In this assay, where SIRK and fluorescein isothiocyanate-labeled
i (F-
i) are simultaneously mixed with
subunits, there was a dose-dependent decrease in the amount of
subunit bound to
subunits, whereas SIRK(L9A) had no effect (Fig. 6B). At 10 µM SIRK, F-
i bound to
is reduced by 75% compared with only 25% in the pull-down assay. This probably reflects that the G protein subunit concentration in this assay is near the Kd for the
-
interaction, making it easier to observe a decrease in binding.
A possible mechanism for the increase in -
dissociation might involve direct competition between the peptide and the
subunit for the
subunit-binding site on
subunits. In this model, the peptide would increase net subunit separation by preventing rebinding of
-GDP subunits after dissociation. This process would be inherently slow because it would depend on spontaneous dissociation of tightly bound
-GDP from
. A second possibility is that the peptide promotes dissociation of
from
subunits by a mechanism that does not involve direct competition. These two possibilities can be distinguished by measuring the
subunit dissociation rate. If the peptide simply competes for the
subunit-binding site on
subunits, then the dissociation rate should be equivalent to the intrinsic
subunit dissociation rate. If the rate of
subunit dissociation in the presence of peptide is greater than the intrinsic dissociation rate, it would suggest the peptide can promote G protein subunit dissociation. We measured the intrinsic rate of F-
i dissociation from
by adding a 50-fold excess of unlabeled myristoylated
i subunit at time 0 and measuring the amount of F-
i bound to
at various times using flow cytometry (Fig. 7A). The intrinsic off rate was slow, with a koff between 0.05 and 0.08 min1 (t1/2
914 min) in four separate experiments. These data are similar to previously published results (0.047 min1) with this assay and consistent with the low apparent Kd for
-
interactions (15). The rate of
dissociation from
was also measured at various times after the addition of SIRK. The initial rate of dissociation was rapid, with the majority of the dissociation occurring within 2 min (t1/2
1 min or less). The dissociation with peptide was in two phases, an initial rapid phase followed by a slower dissociation, with a rate constant similar to the intrinsic
subunit dissociation rate. The extent of initial rapid dissociation by peptide was always at least 75% of the total dissociation that occurred after
subunit alone was added for at least 1 h.
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To examine the initial dissociation phase in more detail, data were collected in real time using the continuous data collection mode of the flow cytometer. Over the time course of 300 s, the peptide-treated samples had a significantly higher rate of dissociation compared with the intrinsic dissociation rate measured with unlabeled i as the sole competitor (Fig. 7B). The peptide-dependent dissociation rate ranged from 5- to 14-fold faster than the intrinsic dissociation rate in 7 separate experiments. The variation resulted primarily from difficulty in getting an accurate quantitative estimate of the intrinsic koff over the short time course of the measurement. Overall, these data indicate SIRK can promote rapid dissociation of
from
subunits without promoting nucleotide exchange on the
subunit.
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DISCUSSION |
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Hot Spot Binding Peptides Promote -
DissociationWe had previously characterized SIRK and other phage display-selected
-binding peptides in vitro and demonstrated they could selectively interfere with the activation of some effectors by
subunits. We also postulated they interact with a protein-protein interaction hot spot on the surface of
subunits because all the selected peptides, with diverse sequence characteristics, bound to the same site on
subunits. Here we demonstrate these effector-selective
-blocking peptides initiate activation of certain G-
-dependent-signaling pathways in intact cells in a receptor-independent fashion. We show the peptides can promote dissociation of
subunits from
subunits without inducing nucleotide exchange. To explain the ability of these peptides to activate cellular signaling, they must bind to
subunits to decrease the binding of
to
but leave the surface on
subunits required to activate MAP kinase pathways unoccupied. A defining characteristic of these peptides is that they block interactions with some downstream targets but not others (5). This unique characteristic of these
-blocking peptides makes this proposed mechanism possible. The precise molecular target of
subunits in ERK1/2 activation has not been defined, so we cannot test the effects of these peptides on this particular interaction directly.
If these peptides promote release, then why is there cell type specificity to ERK activation as shown in Fig. 3A? We believe this reflects the different degrees to which free
can activate ERK in different cell types. RASM cells have previously been demonstrated to have a robust
-dependent activation of ERK (18). We suspect that
is probably released by peptide treatment in all the cells tested, but they may have different levels of the signaling molecules necessary for robust
-stimulated ERK1/2 activation.
It is becoming increasingly clear, particularly through the identification of AGS (activator of G protein signaling) proteins (24), that there are multiple mechanisms for G protein activation that do not depend on receptor-dependent nucleotide exchange. The data we present here indicate that binding to a previously postulated hot spot on subunits can promote subunit dissociation in vitro and activate G protein
subunit-dependent pathways in cells. This suggests a potential mechanism for regulating G protein
subunit-dependent signaling by proteins (either effectors or other types of proteins such as RGS (regulator of G proteins signaling) proteins) interacting directly with the peptide binding hot spot. A protein with properties similar to our peptides is a protein designated AGS2 that was found in a yeast screen for receptor-independent mechanisms for G protein activation. AGS2 was shown to bind to
subunits, but not
subunits, and could activate the pheromone response pathway in yeast. AGS2 is identical to Tctex 1, a component of the cytoplasmic motor protein dynein. A possible interpretation of the ability to Tctex 1 to cause separation of
from
is that Tctex1 is a
subunit effector that can cause separation of
from
but leaves surfaces available on
to interact with the yeast signaling machinery.
Still remaining to be resolved is how SIRK peptide promotes subunit dissociation. One hypothesis is the peptide causes a conformational change in the subunit that promotes subunit dissociation. Generally it is not thought that the
subunits undergo significant conformational changes because the conformations of
crystallized with and without
subunits are very similar (25, 26, 27, 28). On the other hand the structure of
crystallized in the presence of phosducin shows significant conformational alteration (29, 30). Interestingly, a peptide derived from phosducin that apparently binds outside of the
subunit-binding site alters
-
subunit interactions, suggesting that binding to
can transmit information to the
subunit interface (31). The peptides we derived from phage display have similarity to a region of phosducin that binds outside of the
subunit interface. We previously postulated that the site on
subunits where this region of phosducin binds is where our peptide binds. Overall, our data suggest a novel mechanism for G protein activation that may involve conformational alteration of
.
An alternative hypothesis is, Because -
interactions involve two parts of
(i.e. switch regions and N terminus) (26, 27), the slow dissociation of
and
may depend on the requirement for simultaneous breaking of these two contacts. If each contact were dynamic, mSIRK peptide binding to
(for example at the site where
contacts the N terminus) would leave only one contact, which would generate a complex with a lower stability and lifetime. In contrast, the addition of excess
subunit could only prevent reassociation after full release of
from
due to steric interference between the incoming and outgoing
subunits. Identification and characterization of the peptide-binding site is critical to the determination of this mechanism.
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FOOTNOTES |
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Both authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-0892; Fax: 585-273-2652; E-mail: Alan_Smrcka{at}urmc.rochester.edu.
1 The abbreviations used are: PLC, phospholipase C; RASM, rat arterial smooth muscle; LPA, lysophosphatidic acid; EGF, epidermal growth factor; ARK1ct, C terminus of
-adrenergic receptor kinase; DTT, dithiothreitol; MAP, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; F-
i, fluorescein isothiocyanate-labeled myristoylated
i1; TAT-SIRK, human immunodeficiency virus TAT cell permeation sequence fused to SIRK.
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ACKNOWLEDGMENTS |
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REFERENCES |
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