Stimulation of cellular signaling and G protein subunit dissociation by G protein βγ subunit binding peptides *

Farida Goubaeva‡§§, Mousumi Ghosh‡§§, Sundeep Malik‡, Jay Yang‡, Patricia M. Hinkle‡, Kathy K. Griendling§, Richard R. Neubig†, and Alan V. Smrcka‡**

§§These authors contributed equally to this work.

*This work was supported by grants from the National Institutes of Health GM60286 and the American Heart Association New York State Affiliate to AVS, NIH DK19974 to PMH, and NIH HL46417 to RRN ‡Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642. §Department of Medicine, Emory University, Atlanta, GA 30322. †Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor, MI 48109. **To whom correspondence should be addressed Phone: 585-275-0892 Fax: 585-273-2652 e-mail: Alan_Smrcka@urmc.rochester.edu Running title: Peptide activation of G βγ signaling

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Summary

We previously developed peptides that bind to G protein βγ subunits and selectively

block interactions between βγ subunits and a subset of effectors in vitro (Scott et al. EMBO J.

20, 767-776). Here, we created cell permeating versions of some of these peptides by N terminal

modification with either myristate or the cell permeation sequence from HIV tat. The

myristoylated βγ binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells

caused rapid activation of ERK1/2 in the absence of an agonist. This activation did not occur if

the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to

eliminate βγ-subunit binding, or if the cells stably expressed the C terminus of βARK1. An HIV

tat modified peptide (tatSIRK), and a myristoylated version of a second peptide (mSCAR) that

binds to the same site on βγ subunits as mSIRK, also caused ERK activation. mSIRK also

stimulated Jun N-terminal kinase (JNK) phosphorylation, p38 MAP kinase phosphorylation,

phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified

G protein subunits in vitro, SIRK promoted α subunit dissociation from βγ subunits without

stimulating nucleotide exchange. These data suggest a novel mechanism by which selective βγ-

binding peptides can release G protein βγ subunits from heterotrimers to stimulate G protein

pathways in cells.

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Introduction

G protein βγ subunits released from αβγ heterotrimers, in response to G protein coupled

receptor activation, regulate a variety of physiological processes including heart rate, neuronal

excitability and neutrophil chemotaxis. This regulation is mediated by interactions between βγ

subunits and a variety target molecules ranging from inwardly rectifying potassium channels to

soluble enzymes such as phospholipase C (see (1) for a review). βγ subunit-dependent

regulation of the majority of its targets is inhibited by theα-GDP subunit. α-GDP is thought to

sterically occlude a binding site on βγ subunits shared by all its effectors. Activation of βγ

dependent signaling occurs when α-GTP dissociates and uncovers this binding site (2). On the

other hand a number of binding sites for effectors outside the βγ-α subunit interface have been

identified by mutagenesis (3) or peptide crosslinking (4). A model emerging from this analysis

predicts βγ subunit effectors may share a common interaction surface at the βγ-α subunit

interface but individual effectors also have unique interaction surfaces on βγ that could be

targeted pharmacologically.

To probe the nature of common and unique binding sites on βγ subunits we screened

random peptide libraries using purified G protein βγ subunits as the target to identify peptide

sequences required for binding to various surfaces on βγ (5). Four divergent families of peptides

were identified that all bound to a single site on the βγ 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 βγ

subunits and effector molecules such as phospholipase C (PLC)1 β and PI 3-kinase γ (PI3Kγ).

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 βγ subunit-target interactions. This indicates that the proposed “hot spot” is not a

common binding surface for all βγ effectors.

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Most inhibitors of G protein βγ subunit-target interactions, such as the C-terminal domain

of β adrenergic receptor kinase (βARK1ct), are thought to be universal βγ subunit inhibitors,

blocking activation of all downstream target molecules. Thus the βARK1ct has been used

extensively in intact cells as a global inhibitor of βγ 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 βγ 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 βγ subunits and promote dissociation of α subunits but

leave a surface available on βγ subunits for activation of MAP kinase pathways.

Experimental Procedures.

Materials. Peptides were synthesized by Alpha Diagnostics International, purified by HPLC to

greater than 90% purity, and the identity of the peptides was confirmed by mass spectrometry

analysis. The sequences of the peptides were as follows: mSIRK: myristate-

SIRKALNILGYPDYD; SIRK: SIRKALNILGYPDYD; mSIRK(L9A): myristate-

SIRKALNIAGYPDYD; mSCAR; myristate-SCARFFGTPCP-amide, tatSIRK: fluorescein-

GGGYGRKKRRQRRRG-SIRKALNILGYPDYD; tatSIRK(L9A): fluorescein-

GGGYGRKKRRQRRRG-SIRKALNIAGYPDYD. mSCAR and mSIRK were dissolved in

DMSO, all other peptides were dissolved in water. Antibodies to phosphorylated ERK1/2, total

ERK1/2, phosphorylated JNK and phosphorylated p38 were obtained from Cell Signaling

Technology Incorporated. The specific EGF receptor kinase inhibitor AG1487; and src inhibitor,

PP2 were from Calbiochem Corporation. Thapsigargin, EGF and lysophosphatidic acid (LPA)

were from Sigma. Pertussis toxin was from List Biological Laboratories.

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Cell culture. All cell culture reagents were obtained from Invitrogen Corporation. All cells

were grown in DMEM supplemented with 10% Fetal Bovine Serum and 1%

Penicillin/Streptomycin at 37oC with 5% CO2. RASM cells were used between passages 5 and

12. Stable cell lines expressing the βARK1ct were prepared and cultured as described

previously (9).

MAP kinase assays. Rat arterial smooth muscle cells (RASM) 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 dimethylsulfoxide vehicle were diluted 100-400

fold into the media and incubated at 37oC for the indicated duration of time. After treatment, the

cells were transferred to ice, the media 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 volts. The transferred proteins were immunoblotted using standard

immunoblotting protocols with 1:1000 dilution of primary antibody (unless otherwise indicated)

and 1:10,000 dilution of anti-rabbit IgG-horseradish peroxidase conjugate. The proteins were

visualized by incubation with Enhanced Chemiluminescence (ECL) reagents (Amersham

Biosciences Inc.) and exposure to film. Visualization of the shift in molecular weight of ERK’s

in the total ERK western blots was somewhat variable. The images for the total ERK western

blots in figures 1A and 1B 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.

Calcium measurements. For Ca2+ imaging, RASM cells were grown on 25 mm glass coverslips

and loaded with fura2-AM in Hank’s balanced saline solution (HBSS) plus 15 mM Hepes buffer,

pH 7.4. At the start of the experiment, the buffer was switched to Ca2+- and Mg2+-free HBSS,

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 3 times with similar

results. Traces show responses of individual cells.

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Measurements of inositol phosphates (IPs). Cells were plated on 35 mm dishes and labeled by

adding 3-5 µCi of 3H-inositol for 24-48 h in inositol free DMEM. After labeling, the media was

removed and replaced with 1ml of Hepes buffered DMEM containing 10 mM LiCl and

equilibrated for 20 min at 37oC. Ligands or peptides were added in a volume of 50 µl for 30 min

after which the media was aspirated and replaced with 1 ml of ice cold perchloric acid. The

acidified extracts were neutralized by extraction with 1ml of 1:1 octylamine:freon and the

aqueous phase applied to Dowex AG1-X8 columns (BioRad). The columns were washed with

water and 50 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 in a liquid scintillation counter.

Preparation of biotinylated βγ subunits. The cDNA for rat β1 subunit was subcloned into a

baculovirus transfer vector for expression of amino 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 β1 subunit was subcloned by PCR with pfu

polymerase (Stratagene) using primers:

5’-ATAAGGCGCGCCAAGTGAACTTGACCAGCTGC3’

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…β1 sequence, with the lysine residue being the site of

biotinylation. Baculovirus was generated via recombination in bacteria as described in the Bac-

to-Bac system manual (Invitrogen). Biotin-β1γ2 was purified from Sf9 cells using hexahistidine

tagged αi1 following previously published procedures (12). Biotinylation of the purified β

subunit was confirmed by SDS-PAGE followed by western blotting with streptavidin linked

horseradish peroxidase and detection with chemiluminescence. To confirm that the β subunit

was fully biotinylated it was precipitated with streptavidin agarose and greater that 90% of the β

subunit was removed from the supernatant by this procedure.

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Measurement of GTP hydrolysis and GTPγS binding. Steady state GTP hydrolysis was

assayed as [32P]-Pi release from γ[32P]-GTP by standard procedures (13). GTPγS binding was

measured according to (14) except Sf9 cell membranes expressing the M2 muscarinic acetyl

choline receptor were reconstituted with purified recombinant myristoylated αi and β1γ2.

Measurement of α−βγ interactions by flow cytometry. Binding of fluorescein isothiocyanate

(FITC) labeled myristoylated αi1 (F-αi) to biotinylated β1γ2 subunits was measured using a flow

cytometry assay (15;16). Biotinylated βγ subunits (250 pM final concentration) were mixed with

streptavidin beads in HEDNMLG (20 mM Hepes, pH 8.0, 1 mM EDTA, 1mM DTT, 150 mM

NaCl, 0.2 mM free Mg2+, 0.1% C12E10, 10 µM GDP). After 20 min incubation at room

temperature, the beads were washed twice by centrifugation in a microfuge with HEDNMLG

and resuspended in the same buffer at a concentration of 105 beads /mL (250 pM βγ). For α

subunit dissociation experiments, the beads with bound βγ subunits were premixed with 1 nM F-

αi for 10 min prior to addition of competitors. For equilibrium binding measurements 1 nM F-αi

and competitors were added simultaneously. The amount of F-αi bound to beads with

biotinylated βγ was assayed at the times indicated in the figure legends using a Beckman-

Dickson FACscan flow cytometer. Non-specific binding, determined by the simultaneous

addition 1 nM F-αi and 50 nM myristoylated αi, was 10-20% of the total signal and was

subtracted from the data.

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Results To investigate the consequence of selectively inhibiting βγ subunit functions in intact cells,

we created cell permeating peptides by synthesizing one of our phage display selected peptides,

SIRKALNILGYPDYD (SIRK) with myristate (17) at the amino terminus (mSIRK). We tested

the ability of this peptide to affect activation of ERK1 and ERK2 in primary cultures of rat

arterial smooth muscle (RASM) cells. In these cells it has been shown that ERK1 and ERK2 can

be activated by stimulation of G protein-coupled lysophosphatidic acid (LPA) receptors. This

activation is blocked by pertussis toxin and by an adenovirus expressing the βARK1ct,

implicating Gi and βγ subunits in the activation process (18). For these reasons, we predicted

that our cell permeating peptides might block LPA-induced ERK activation. Surprisingly,

application of mSIRK to RASM cells, in the absence of added LPA, caused a rapid and dose

dependent activation of ERK1/2 as detected by western blotting for the phosphorylated forms of

ERK1/2 (Figure 1 A and B). The concentration of mSIRK required for half maximal activation

of ERK1/2 was 2.5-5 µM, equivalent to the estimated Kd of this peptide for G protein βγ

subunits (5). Full activation was achieved after 1 min of application and diminished over the

course of 30 min. Activation of ERK1/2 is also indicated by the upward shift in molecular

weight of ERK1/2 in a western blot for total ERK1/2. For Figures 1A and 1B the conditions

were optimized to resolve the phosphorylated from the non-phosphorylated ERK1/2 in a total

ERK1/2 western blot (See asterisks in bottom panels, figure 1A and 1B). At full activation, all

of the ERK1/2 was shifted to a higher molecular weight phosphorylated form, indicating all of

the ERK in all of the cells was being activated by this peptide.

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 A for L9 increased the IC50 of

this peptide for blocking βγ-dependent PLC activation by over 100 fold (from 3 µM to 300 µM)

(5). The results in figure 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

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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 HIV tat (19) was added to the amino terminal end of SIRK (tatSIRK).

Fluorescein was attached to the amino terminus to monitor cellular uptake. TatSIRK was

efficiently taken up into virtually all of cells on the dish as monitored by fluorescence

microscopy (not shown). TatSIRK was able to activate ERK1/2 to an extent comparable to

mSIRK (Fig. 1D) while tatSIRK(L9A) had no effect (data not shown). These cell permeating

peptides activated ERK to a greater extent than LPA and to an extent comparable to EGF in this

assay paradigm (Fig. 1D). Together, these data indicate structural requirements in the peptides

for βγ 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 βγ subunits in vitro (5), strongly supports the idea that βγ is the target of

the peptides and this targeting is responsible for ERK1/2 activation.

G βγ is the target of the peptides in the intact cell. To further support the idea that βγ 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 βγ

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 βγ subunits the cysteine residues must form an

intramolecular disulfide bond. If SCAR is reduced with DTT, it is no longer able to bind to βγ in

a phage ELISA assay. The results in figure 2A show mSCAR activates ERK1/2, pretreatment

with DTT eliminates its ability to activate ERK1/2, while 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 βγ subunits is important to observe activation of ERK1/2, not just the peptide

sequence.

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To provide further evidence G βγ 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 βARK that sequesters βγ subunits (βARK1ct (Gly495-Leu689))

(8;9). We hypothesized that mSIRK was binding to βγ but leaving a surface available on βγ to

signal to some downstream targets. Based on this hypothesis, we predicted that ERK1/2

activation by mSIRK would be inhibited by the βARK1ct, a general βγ inhibitor that blocks

signaling of βγ to most if not all effectors. The data in figure 2B demonstrate that in cells

expressing βARK1ct, activation of ERK1/2 was almost completely inhibited compared to the

control RASM cells. This indicates that βγ subunit signaling is the target of these peptides.

Cell type specificity of ERK activation by mSIRK. All 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 figure 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 while 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

tatSIRK on HEK293 and Cos7 cells. Similar to mSIRK, tatSIRK had only small effects on

activation of ERK1/2 (not shown). We were able to monitor the uptake of tatSIRK 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 βγ 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 stores. We used a variety of pharmacological inhibitors to examine the pathway

involved in the response to mSIRK. As expected a MEK 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 βγ-mediated activation of ERK1/2 by GPCRs, consistent with a

pathway involving βγ 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 m-

SIRK (Fig. 3B). Thapsigargin treatment, which almost completely emptied the intracellular

calcium stores (data not shown and figure 4B), was unable to block the effect of mSIRK

indicating 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.

Activation of other GPCR signal transduction pathways by mSIRK. The effects of mSIRK

on other signal transduction pathways downstream of either βγ subunits or α subunits were

examined. First we looked at other members of the MAP kinase pathway. p38 MAP kinase

phosphorylation and Jun Kinase (JNK) phosphorylation are increased by addition of mSIRK

(Figure 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 2 to 5 min (figure 4B, top panel). Ca2+

spiking was not stimulated by addition of buffer, or the control peptide, mSIRK(L9A) (0/34

cells) (figure 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 (figure 4B, bottom panel). mSIRK also

stimulated Ca2+ oscillations in HEK293 cells (data not shown). These Ca2+ oscillations resulted

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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

3-8 to 0.5-1 min (data not shown). Pertussis toxin treatment did not inhibit the effects of mSIRK

on intracellular Ca2+ (data not shown). To determine whether IP3 production was responsible

for this increase we examined total inositol phosphate (IP) production in cells labeled with 3H-

inositol. mSIRK caused a reproducible increase in intracellular IP release that was unaffected by

treatment with PP2 (Fig 4C). Since PP2 blocks src activation and the subsequent pathway

leading to ERK1/2 activation, the IP3 release is not an indirect consequence of activation of this

pathway.

mSIRK activation of many G protein dependent pathways suggests mSIRK is binding to

βγ subunits and somehow promoting release of βγ subunits, leaving a surface available to signal

to downstream pathways. A potential problem with this hypothesis is that SIRK blocks PLC

stimulation by βγ in vitro (5) and thus should block the βγ surface required to signal to PLC.

The PLC response caused by mSIRK is quite weak compared to 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 IP

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 βγ

and this residual ability of the βγ to activate PLC, even in the presence of peptide, is responsible

for the weak IP3 and Ca2+ responses observed. Another possibility is that αq/11 may be released,

that can bind some GTP once released from βγ.

SIRK causes dissociation of α subunits from βγ subunits without stimulating nucleotide

exchange. That all these signaling pathways are being activated in response to a βγ 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 α subunits.

However, these peptides were selected to bind to G protein βγ subunits and therefore should not

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bind to α subunits. Receptors and receptor-mimicking peptides such as mastoparan act at least

in part through binding to α subunits. It is possible, however, that mSIRK may non-specifically

interact with α subunits or may promote nucleotide exchange by an undefined mechanism

through binding to βγ subunits. To determine if the βγ 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]-GTPγS to purified αi1β1γ2 (21) and found it did not (figure 5A). If

the same subunits were reconstituted with urea-stripped Sf9 cell membranes expressing the M2

muscarinic receptor and carbachol, [35S]-GTPγS binding was stimulated in a βγ subunit-

dependent manner. To determine if 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 (figure 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 is acting through

disruption of interactions between α and βγ subunits.

Nucleotide exchange can also be assayed by measuring the steady state rate of GTP

hydrolysis on the α 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 γ[32P]-GTP by Go while mSIRK did not (figure 5C), indicating that mSIRK cannot

stimulate GDP dissociation. Also shown in figure 5D is that mastoparan has virtually no effect

on ERK activation in RASM cells while mSIRK stimulates significant ERK activation.

To determine if SIRK could stimulate dissociation of α from βγ in the absence of

nucleotide exchange, we assayed the binding of α subunits to biotinylated βγ subunits bound to

streptavidin agarose beads. G protein α subunits (10 nM) were incubated with 10 nM

biotinylated βγ subunits bound to streptavidin agarose beads and incubated with mSIRK or AlF4-

for 20 minutes. The beads were centrifuged, the supernatant removed and the bound α subunits

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estimated by quantitative western blotting (Figure 6A). At 10 and 30 µM mSIRK the amount of

α subunit bound to βγ subunits was significantly lower than in the absence of added mSIRK.

A drawback of the pulldown assay is the high concentrations of α and βγ 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 α-βγ interactions of about 1

nM. A more quantitative assay to measure α-βγ interactions at very low subunit concentrations

has recently been developed that uses flow cytometry to assess the amount of fluorescent α

subunit bound to biotinylated βγ subunits immobilized on beads (15;16). In this assay, where

SIRK and FITC labeled αi (F-αi) are simultaneously mixed with βγ subunits, there was a dose

dependent decrease in the amount of α subunit bound to βγ subunits while SIRK(L9A) had no

effect (Figure 6B). At 10 µM SIRK, F-αi bound to βγ is reduced by 75% compared to only 25%

in the pulldown assay. This probably reflects that the G protein subunit concentration in this

assay is near the Kd for the α-βγ interaction making it easier to observe a decrease in binding.

A possible mechanism for the increase in α-βγ dissociation might involve direct

competition between the peptide and the α subunit for the α subunit-binding site on βγ subunits.

In this model, the peptide would increase net subunit separation by preventing rebinding of α-

GDP subunits after dissociation. This process would be inherently slow because it would depend

on spontaneous dissociation of tightly bound α-GDP from βγ. A second possibility is the

peptide promotes dissociation of α from βγ subunits by a mechanism that does not involve direct

competition. These two possibilities can be distinguished by measuring the α subunit

dissociation rate. If the peptide simply competes for the α subunit-binding site on βγ subunits

then the dissociation rate should be equivalent to the intrinsic α subunit dissociation rate. If the

rate of α 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-αi dissociation from βγ by adding a 50 fold excess of unlabeled myristoylated

αi subunit at time 0 and measuring the amount of F-αi bound to βγ at various times using flow

cytometry (Fig. 7A). The intrinsic off rate was slow, with a koff between 0.05 and 0.08 min-1 (t ½

~ 9-14 min) in four separate experiments. These data are similar to previously published results

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(0.047 min-1) with this assay, and consistent with the low apparent Kd for α-βγ interactions (15).

The rate of α dissociation from βγ was also measured at various times after addition of SIRK.

The initial rate of dissociation was rapid, with the majority of the dissociation occurring within 2

min (t ½ ~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 α subunit

dissociation rate. The extent of initial rapid dissociation by peptide was always at least 75% of

the total dissociation that occurred after α subunit alone was added for at least 1h.

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 to the

intrinsic dissociation rate measured with unlabeled αi as the sole competitor (figure 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 α from βγ subunits without

promoting nucleotide exchange on the α subunit.

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Discussion Activation of ERK1/2 by βγ inhibitory peptides

There is extensive evidence in the literature suggesting free βγ subunits released from Gi

heterotrimers, upon α subunit activation and dissociation, can initiate activation of multiple

MAP kinase pathways (22;23). We have done extensive characterization of our peptide system

to show the effects we observe are specifically due to interactions of our peptides with βγ

subunits in cells. The concentration of peptide required to cause ERK1/2 activation in cells is

nearly the same as for inhibition of interactions between βγ subunits and downstream targets in

vitro. A single L-A point mutation previously shown to eliminate the ability of the peptide to

inhibit βγ interactions with downstream targets (5), completely eliminates peptide dependent

ERK1/2 activation. A requirement for intracellular action of the peptide is indicated by the fact

that the peptide has no effect unless it is modified by either a myristate or the tat cell permeation

sequence from HIV. That two completely different chemical modifications of the peptide,

whose unifying feature is their ability to permeate cell membranes, strongly indicates their site of

action is not receptors or other sites on the cell surface. A peptide (mSCAR) with no sequence

homology to the SIRK peptide, but binds to the same surface on βγ subunits, also caused

activation of ERK1/2. The fact that activation of ERK1/2 is blocked by the src inhibitor PP2

indicates our βγ “inhibitory peptides” are acting though a pathway similar to that which has been

previously described as being involved in MAP kinase activation by βγ (20). Finally, if the

peptide is tested in RASM cells that express the βARK1ct, the effect on ERK1/2 activation is

greatly attenuated. All of these data indicate the G protein signaling system, and the βγ subunits

specifically, are the targets for binding these peptides in cells, resulting in the activation of signal

transduction that has been presented.

Hot spot binding peptides promote α-βγ dissociation.

We had previously characterized SIRK and other phage display-selected βγ binding

peptides in vitro and demonstrated they could selectively interfere with the activation of some

effectors by βγ subunits. We also postulated they interact with a protein-protein interaction “hot

spot” on the surface of βγ subunits because all the selected peptides, with diverse sequence

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characteristics, bound to the same site on βγ subunits. Here we demonstrate these effector-

selective βγ blocking peptides initiate activation of certain G-βγ dependent signaling pathways in

intact cells in a receptor-independent fashion. We show the peptides can promote dissociation of

α subunits from βγ subunits without inducing nucleotide exchange. In order to explain the

ability of these peptides to activate cellular signaling, they must bind to βγ subunits to decrease

the binding of α to βγ, but leave the surface on βγ subunits required to activate MAP kinase

pathways unoccupied. A defining characteristic of these peptides is they block interactions with

some downstream targets but not others (5). This unique characteristic of these βγ-blocking

peptides makes this proposed mechanism possible. The precise molecular target of βγ 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 βγ release, then why is there cell type specificity to ERK

activation as shown in figure 3A? We believe this reflects the different degrees to which free βγ

can activate ERK in different cell types. RASM cells have previously been demonstrated to have

a robust βγ dependent activation of ERK (18). We suspect that βγ is probably released by

peptide treatment in all the cells tested, but they may have different levels of the signaling

molecules necessary for robust βγ stimulated ERK1/2 activation.

It is becoming increasingly clear, particularly through the identification of AGS proteins

(24), there are multiple mechanisms for G protein activation that do not depend on receptor-

dependent nucleotide exchange. The data we present here indicate binding to a previously

postulated “hot spot” on βγ subunits can promote subunit dissociation in vitro and activate G

protein βγ subunit dependent pathways in cells. This suggests a potential mechanism for

regulating G protein βγ subunit dependent signaling by proteins (either effectors or other types of

proteins such as RGS proteins) interacting directly with the peptide binding “hot spot”. A

protein with properties similar to our peptides is a protein designated AGS2 (activator of G

protein signaling 2) that was found in a yeast screen for receptor-independent mechanisms for G

protein activation. AGS2 was shown to bind to βγ subunits, but not α subunits, and could

activate the pheromone response pathway in yeast. AGS2 is identical to Tctex 1, a component of

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the cytoplasmic motor protein dynein. A possible interpretation of the ability to Tctex 1 to cause

separation of α from βγ is that Tctex1 is a βγ subunit effector that can cause separation of α from

βγ but leaves surfaces available on βγ 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 βγ subunit that promotes subunit

dissociation. Generally it is not thought that the βγ subunits undergo significant conformational

changes because the conformations of βγ crystallized with and without α subunits are very

similar (25-28). On the other hand the structure of βγ crystallized in the presence of phosducin

shows significant conformational alteration (29;30). Interestingly, a peptide derived from

phosducin that apparently binds outside of the α subunit-binding site alters α-βγ subunit

interactions suggesting that binding to βγ can transmit information to the α subunit interface

(31). The peptides we derived from phage display have similarity to a region of phosducin that

binds outside of the α subunit interface. We previously postulated that the site on βγ subunits

where this region of phosducin binds is where our peptide binds. If true, it suggests a truly novel

mechanism for G protein activation involving conformational alteration of βγ.

An alternative hypothesis is since α-βγ interactions involve two parts of α (i.e. switch

regions and N-terminus) (26;27), the slow dissociation of α and βγ may depend on the

requirement for simultaneous breaking of these two contacts. If each contact were dynamic,

mSIRK peptide binding to βγ (for example at the site where βγ contacts the N-terminus) would

leave only one contact, which would generate a complex with a lower stability and lifetime. In

contrast, addition of excess α subunit could only prevent reassociation following full release of α

from βγ due to steric interference between the incoming and outgoing α subunits. Identification

and characterization of the peptide-binding site is critical to determination of this mechanism.

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References Reference List

1. Clapham, D. E. and Neer, E. J. (1997) Ann.Rev.Pharmacol.Toxicol. 37, 167-203

2. Hamm, H. E. (1998) J.Biol.Chem. 273, 669-672

3. Panchenko, M. P., Saxena, K., Li, Y., Charnecki, S., Sternweis, P. M., Smith, T. F., Gilman, A. G., Kozasa, T., and Neer, E. J. (1998) J.Biol.Chem. 273, 28298-28304

4. Yoshikawa, D. M., Bresciano, K., Hatwar, M., and Smrcka, A. V. (2001) J.Biol.Chem. 276, 11246-11251

5. Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776

6. Wells, J. A. and de Vos, A. M. (1996) Ann.Rev.Biochem. 65, 609-634

7. Fairbrother, W. J., Christinger, H. W., Cochran, A. G., Fuh, G., Keenan, C. J., Quan, C., Shriver, S. K., Tom, J. Y., Wells, J. A., and Cunningham, B. C. (1998) Biochem. 37, 17754-17764

8. Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M., and Lefkowitz, R. J. (1994) J.Biol.Chem. 269, 6193-7:

9. Ushio-Fukai, M., Alexander, R. W., Akers, M., Lyons, P. R., Lassegue, B., and Griendling, K. K. (1999) Mol Pharmacol 55, 142-149

10. Romoser, V. A., Graves, T. K., Wu, D., Jiang, H., and Hinkle, P. M. (2001) Mol Endocrinol. 15, 125-135

11. Duffy, S., Tsao, K. L., and Waugh, D. S. (1998) Analytical Biochemistry 262, 122-128

12. Kozasa, T. and Gilman, A. G. (1995) J.Biol.Chem. 270, 1734-1741

13. Higashijima, T., Burnier, J., and Ross, E. M. (1990) J.Biol.Chem. 265, 14176-14186

14. Hartman IV, J. L. and Northup, J. K. (1999) J.Biol.Chem. 271, 22591-22597

15. Sarvazyan, N. A., Remmers, A. E., and Neubig, R. R. (1998) J.Biol.Chem. 273, 7934-7940

16. Sarvazyan, N. A., Lindau, M., and Neubig, R. R. (2002) Biochem. 41, 12858-12867

17. Eichholtz, T., de Bont, D. B., de Widt, J., Liskamp, R. M., and Ploegh, H. L. (1993) J.Biol.Chem. 268, 1982-1986

18. Iaccarino, G., Smithwick, L. A., Lefkowitz, R. J., and Koch, W. J. (1999) Proc.Natl.Acad.Sci.USA 96, 3945-3950

19

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.jbc.org/D

ownloaded from

19. Schwarze, S. R., Ho, A., Vocera-Akbani, A., and Dowdy, S. F. (1999) Science 285, 1569-1572

20. Luttrell, L. M., Della, R. G., van Biesen, T., Luttrell, D. K., and Lefkowitz, R. J. (1997) J Biol.Chem. 272, 4637-4644

21. Northup, J. K., Smigel, M. D., and Gilman, A. G. (1982) J.Biol.Chem. 257, 11416-11423

22. Luttrell, L. M., van Biesen, T., Hawes, B. E., Koch, W. J., Krueger, K. M., Touhara, K., and Lefkowitz, R. J. (1997) Adv.Second Messenger Phosphoprotein Res. 31, 263-277

23. Gutkind, J. S. (2001) Sci STKE. 2000, RE1

24. Cismowski, M. J., Takesono, A., Bernard, M. L., Duzic, E., and Lanier, S. M. (2001) Life Sci. 68, 2301-2308

25. Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374

26. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319

27. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058

28. Sprang, S. R. (1997) Ann.Rev.Biochem. 66, 639-678

29. Loew, A., Ho, Y. K., Blundell, T., and Bax, B. (1998) Structure 6, 1007-1019

30. Gaudet, R., Bohm, A., and Sigler, P. B. (1996) Cell 87, 577-588

31. Bluml, K., Schnepp, W., Schroder, S., Beyermann, M., Macias, M., Oschkinat, H., and Lohse, M. J. (1997) EMBO J. 16, 4908-4915

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1Abbreviations used: PLC, Phospholipase C; PI3K, Phosphoinositide 3-kinase; RGS, regulator of G proteins signaling; RASM, rat arterial smooth muscle; JNK, Jun N-terminal kinase; LPA, lysophosphatidic acid; EGF, epidermal growth factor βARKct, C-terminus of β-adrenergic receptor kinase; DTT, dithiothreitol; C12E10, polyoxyethylene 10-lauryl ether. Acknowledgements. I would like to thank Mamata Hatwar and John Puskas for excellent technical assistance, Dr. Brad Berk’s laboratory for preparation of rat arterial smooth muscle cells and Tabetha Bonacci for kindly providing biotinylated βγ subunits.

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Figure Legends

Figure 1. Cell permeating βγ binding peptides activate ERK1/2 in vascular smooth muscle cells.

A) Concentration dependence. RASM cells were treated with the indicated concentrations of

mSIRK, added in 5 µl of DMSO for 10 min prior to extraction into sample buffer. Asterisks (*)

indicate the presence of doublets of ERK2 (42) and ERK1 (44) that appear with peptide

treatment. B) Time course. RASM cells were incubated with 10 µM mSIRK for the indicated

times. Again (*) indicate the presence of a doublet of ERK2 and ERK1. C) Substitution of L9

with A, or removal of the myristoyl group eliminates the ability of mSIRK to activate ERK1/2.

RASM cells were treated for 5 min with DMSO, 10 µM mSIRK, 10 µM mSIRK with alanine

substituted for leucine at the 9 position, mSIRK(L9A), or 10 µM SIRK with no myristate. D)

HIV tat cell permeation sequence also allows the peptide to activate ERK1/2. Cells were treated

with 5 µM mSIRK, 10 µM tatSIRK, 10 µM LPA and 10 nM EGF or DMSO for 5 min. For all

experiments, samples were all applied to 12 % gels and analyzed by western blotting for either

phosphorylated ERK1/2; p44 and p42 or total ERK1/2 protein; labeled 44 and 42. Each of these

experiments was repeated at least 3 times with similar results.

Figure 2. Activation of ERK1/2 by mSIRK involves a G βγ-dependent signaling pathway. A)

Other peptides that bind to βγ also activate ERK1/2. RASM cells were treated with 5 µM

mSIRK for 5 min or 5 µM mSCAR for 15 min. For the DTT treatment, the peptides in media at

5 µM were treated with 10 mM DTT for 30 min prior to addition to the cells. B) The βARK1ct

inhibits mSIRK mediated activation of ERK1/2. Stable cell lines expressing the βARK1ct or

vector control cells were treated with the indicated concentrations of mSIRK, 10 µM LPA or

DMSO control for 5 min and processed as described. This experiment was repeated 3 times with

similar results.

Figure 3. Cell type specificity and pathway for ERK1/2 activation. A) The magnitude of

activation of ERK1/2 is dependent upon the cell type. The indicated cell types were treated with

DMSO or 10 µM mSIRK for 5 min. All samples were processed and analyzed as described in

“Experimental Procedures” and figure 1. B) The pathway activated by peptide treatment involves

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src but not transactivation of the EGF receptor. Cells were treated with 1 µM AG1478 for 1h or

10 µM PP2 for 30 min prior to addition of 5 µM mSIRK or 10 nM EGF for 5 min. Each of the

treatments was repeated in at least 3 separate experiments. C) Calcium release from

intracellular pools was not responsible for ERK1/2 activation and the activation is not blocked

by pertussis toxin. Cells were treated with 1 µM thapsigargin (30 min) to empty intracellular

calcium pools prior to addition of 10µM mSIRK. For pertussis toxin treatments, cells were

treated for 16 h with 100 ng/ml pertussis toxin followed by treatment with either 5 µM mSIRK

(5 min) or 10 µM LPA (5 min). Measurement of ERK1/2 is as in figure 1.

Figure 4. Multiple signaling pathways are activated by mSIRK treatment. A) p38 MAP kinase

and JNK are activated by mSIRK. RASM cells were treated with 5 µM mSIRK, mSIRK(L9A) or

1% FBS for 5 min and samples were assessed for p38 MAP kinase activation, JNK activation

and ERK activation by western blotting with antibodies specific for phosphorylated p38,

phosphorylated JNK and phosphorylated ERK respectively. Total ERK1/2 is shown as a loading

control. B) mSIRK causes mobilization of intracellular Ca2+. Fura2-loaded RASM cells were

incubated in Ca2+- and Mg2+-free HBSS for Ca2+ imaging. At the times noted by the arrows, 10

µM mSIRK, 10 µM mSIRK(L9A) or vehicle was added. In order to estimate the intracellular

Ca2+pool, 1 µM ionomycin was added at the end of the experiment. In the bottom panel, cells

were exposed to 100 nM thapsigargin during loading and imaging. C) mSIRK causes an

increase in inositol phosphates. Total inositol phosphates produced in RASM cells were

measured as described in “Experimental Procedures”. mSIRK (10 µM), DMSO, or 1% fetal

bovine serum was added for 30 min and PP2 treatment was 10 µM. The data presented are

representative data showing the standard errors of duplicate data points. In 19 experiments the

fold activation by mSIRK ranged from 1.4 to 4 fold with an average of 2.1 fold ± 0.17 SEM.

mSIRK treated samples were significantly different from DMSO treated control (P < 0.0001

using a paired two tailed t-test).

Figure 5. SIRK does not stimulate GDP release from G protein heterotrimers. A. Binding of

[35S]-GTPγS to 200 nM αi in the presence or absence of 400 nM β1γ2 was measured with 1 µM

GTPγS and [35S]-GTPγS (100,000 cpm/assay) and 4 µM GDP for 10 min in the presence or

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absence of 10 µg of urea stripped membranes expressing the M2 muscarinic acetylcholine

receptor and 100 µM carbachol. Error bars represent standard error from triplicate data points

and data are representative of experiments performed 3 times. B. M2 muscarinic receptor

stimulated binding of 35S-GTPγS binding at various concentrations of SIRK. Assay conditions

were as in A in the presence of urea stripped M2 muscarinic receptor expressing membranes and

carbachol. Results are pmols GTPγS bound minus the basal in the absence of added βγ subunits

of 1.3 pmol. Basal was unchanged by peptide as in A and not shown. Error bars represent

standard error from triplicate data points and data are representative of experiments performed 3

times. C. Steady state GTP hydrolysis was measured with 5 nM recombinant myristoylated Gαo,

10 nM β1γ2, sonicated phospholipid vesicles (PE:PS:PC 5 µM each) 300 nM GTP and [γ32P]-

GTP (30,000 cpm/assay) with 100 µM mastoparan (Mas) or 10 µM mSIRK as indicated. Data

are the combined normalized data from four separate experiments. Control (Go alone) is

significantly different from Go+Mas, P < 0.05, but not significantly different from Go+mSIRK

analyzed with a one way ANOVA and Tukey’s multiple comparison test. D. RASM cells were

treated with vehicle (H2O), 100 µM mastoparan or 10 µM mSIRK for 5 min and assayed for

ERK activation as in figure 1.

Figure 6. SIRK inhibits α-βγ interactions. A) Biotinylated β1γ2 (10 nM) was bound to

streptavidin agarose beads followed by 10 nM myristoylated αi and the indicated concentrations

of mSIRK or AlF4- (30 µM AlCl3, 10 mM NaF, 5 mM MgCl2) in 20 mM Hepes pH 8.0, 1 mM

EDTA, 1 mM DTT, 100 mM NaCl, 10 µM GDP and 0.1% C12E10. After incubation for 30 min

the beads were precipitated and αi associated with the βγ beads was assessed by quantitative

western blotting. Error bars represent standard error from triplicate experiments. B) 1.5 nM

FITC-αi was mixed with 200 pM biotinylated β1γ2 in the presence of the indicated concentrations

of SIRK ( • ) or SIRK(L9A) ( ▼ ) and the amount of F-αi bound was assessed by flow cytometry

as described in Experimental Procedures. Data are representative of experiments performed at

least 3 times each.

Figure 7. SIRK promotes αi dissociation from βγ subunits in the absence of nucleotide

exchange. A) 1 nM F-αi was preincubated with streptavidin beads bound to 250 pM β1γ2 for 10

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25

min. Either 50 nM unlabeled myristoylated αi alone (▲) or 50 nM myristoylated αi plus 25 µM

SIRK ( ■ ) were added to the preformed F-αi-βγ complex. The rate of dissociation of F-αi was

measured using flow cytometry to assess the amount of F-αi bound at various times after

addition of the competitors. For each time point 3000 beads were assessed. Initial binding was

35 (mean channel number) for both curves (After subtraction of a background of 13 measured

for F-αi in the absence of βγ bound to the beads). Curves were best fit with a two-exponent

decay function using GraphPad Prism data analysis software. To test for statistical significance,

the F-αi fluorescence bound after addition of αi alone was compared with αi plus SIRK (25 µM)

at 1 and 2 min using an unpaired two tailed t-test (n=6 for each treatment). The average % initial

F-αi bound 1 min after addition of 100 nM αi as the competitor was 98.25 % ± 1.0 SEM and for

αi plus SIRK was 69.15% ± 0.77 SEM, and were significantly different, P < 0.0001. In a

separate experiment at 2 min after competitor addition the values were 90.0% ±1.77 SEM and

65.71% ±0.82 SEM (means were significantly different P < 0.0001) for αi and αi plus SIRK

respectively. B) Real time measurement of α dissociation. Same as A except after SIRK (25

µM) addition the fluorescence associated with the beads was analyzed using the continuous “list

mode” data acquisition option of the flow cytometer. More than 10,000 data points were

collected over the total time course and randomly selected groups of 200-300 events were

averaged for the indicated times and plotted. ( ) is SIRK added without addition of

myristoylated αi. Curves were best fit with a single-exponent decay function using GraphPad

Prism data analysis software yielding a koff for αi of 0.02 min-1, peptide 0.28 min-1 and peptide +

αi of 0.24 min-1. This experiment was repeated 5 times with qualitatively similar results.

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BA

p44p44 p42p42

** **

**

** **** ** 4444

42 42

0 1.2 2.5 5 10 30[mSIRK] (µM)

0 1 2 5 10 15 30Time (min)26

C D p44p44p42p42

4444

DMSO mSIRK mSIRK SIRK (L9A)

42 42

DMSO mSIRK tatSIRK EGF LPA

Fig. 1

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p44p42A

mSIRK - - - + + DTT - - + - +mSCAR - + + - -

4442

BDMSO mSIRK mSIRK LPA

2 µM 5 µM

p44p42

4442

βARK1ct - + - + - + - +

Fig. 2

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A ddtMF2 MDA Rat 2 RASM HEK 293 COS 7

mSIRK - + - + - + - + - + - +

p44p42

4442

B

- + + - + + - -- - + - - - - -- - - - - - + +- - - - - + - +

mSIRKPP2EGFAG1478

p44p42

C

- + + - + + - -- - + - - - - -- - - - - - + +- - - - - + - +

p44p42

mSIRKThaps.LPAPTX

Fig. 3

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0 100 200 300 400 500 600 700Time (sec)

[Cyt

opla

smic

Ca2+

] (34

0/38

0nm

ratio

)

0.20.40.60.8

0.20.40.60.8

0.20.40.60.8

Thapsigargin-pretreated

mSIRK

Iono

mSIRK (L9A)

mSIRK

Iono

Iono

A BP-JNK

P-p38

p44p42P-ERK

4442Total

ERK

DMSO FBS mSIRK mSIRK(L9A)29

IP re

leas

ed (c

pm)

0

500

1000

1500

2000

2500C

DMSO mSIRK mSIRK FBS+PP2 Fig. 4

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-7 -6 -5 -40

1

2

3

Log [SIRK]

pmol

GTP

γ S b

ound

B

αiβγ

SIRKM2

+ + + ++ + - +- + - -- - + +

[35S]

-GTP

γS b

ound

(cpm

) x 1

0-4

0

2

4

6

8

10

12

14A

C D

Go Go+Mas

Go+mSIRK

0

2

4

6

Fold

stim

ulat

ion

Con Mas mSIRK

p44p42

Fig. 5

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A

% α

Bou

nd

0

20

40

60

80

100

120

Con. AlF4 10µM 30µM

Peptide concentration (µM)

0 5 10 15 20 25 30

Spec

ific

F-α

bin

ding

(mea

n ch

anne

l num

ber)

0

5

10

15

20

25

30

35B

Fig. 6

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A

0 5 10 15 2040

50

60

70

80

90

100

Time (min)

% In

itial

F- α

i bou

nd

B

0 100 200 30050

60

70

80

90

100

Time (sec)

% in

itial

F-α

i bou

nd

Fig. 7

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Griendling, Richard R. Neubig and Alan V. SmrckaFarida Goubaeva, Mousumi Ghosh, Sundeep Malik, Jay Yang, Patricia M. Hinkle, Kathy K.

subunit binding peptidesγβStimulation of cellular signaling and G protein subunit dissociation by G protein

published online March 20, 2003J. Biol. Chem. 

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