Stimulation of Cellular Signaling and G Protein Subunit Dissociation by G Protein {beta}{gamma} Subunit-binding Peptides*

Farida Goubaeva {ddagger} §, Mousumi Ghosh {ddagger} §, Sundeep Malik {ddagger}, Jay Yang {ddagger}, Patricia M. Hinkle {ddagger}, Kathy K. Griendling ¶, Richard R. Neubig || and Alan V. Smrcka {ddagger} **

From the {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously developed peptides that bind to G protein {beta}{gamma} subunits and selectively block interactions between {beta}{gamma} subunits and a subset of effectors in vitro (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). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated {beta}{gamma}-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 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 {beta}{gamma} subunit binding, or if the cells stably expressed the C terminus of {beta}ARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on {beta}{gamma} subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted {alpha} subunit dissociation from {beta}{gamma} subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective {beta}{gamma}-binding peptides can release G protein {beta}{gamma} subunits from heterotrimers to stimulate G protein pathways in cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein {beta}{gamma} subunits released from {alpha}{beta}{gamma} 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 {beta}{gamma} subunits and a variety of target molecules ranging from inwardly rectifying potassium channels to soluble enzymes such as phospholipase C (for review, see Ref. 1). {beta}{gamma} subunit-dependent regulation of the majority of its targets is inhibited by the {alpha}-GDP subunit. {alpha}-GDP is thought to sterically occlude a binding site on {beta}{gamma} subunits shared by all its effectors. Activation of {beta}{gamma}-dependent signaling occurs when {alpha}-GTP dissociates and uncovers this binding site (2). On the other hand, a number of binding sites for effectors outside the {beta}{gamma}-{alpha} subunit interface have been identified by mutagenesis (3) or peptide cross-linking (4). A model emerging from this analysis predicts {beta}{gamma} subunit effectors may share a common interaction surface at the {beta}{gamma}-{alpha} subunit interface, but individual effectors also have unique interaction surfaces on {beta}{gamma} that could be targeted pharmacologically.

To probe the nature of common and unique binding sites on {beta}{gamma} subunits, we screened random peptide libraries using purified G protein {beta}{gamma} subunits as the target to identify peptide sequences required for binding to various surfaces on {beta}{gamma} (5). Four divergent families of peptides were identified that all bound to a single site on the {beta}{gamma} 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 {beta}{gamma} subunits and effector molecules such as phospholipase C (PLC)1 {beta} and phosphatidylinositol 3-kinase {gamma}. 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 {beta}{gamma} subunit-target interactions. This indicates that the proposed hot spot is not a common binding surface for all {beta}{gamma} effectors.

Most inhibitors of G protein {beta}{gamma} subunit-target interactions, such as the C-terminal domain of {beta}-adrenergic receptor kinase ({beta}ARK1ct), are thought to be universal {beta}{gamma} subunit inhibitors, blocking activation of all downstream target molecules. Thus the {beta}ARK1ct has been used extensively in intact cells as a global inhibitor of {beta}{gamma} 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 {beta}{gamma} 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 {beta}{gamma} subunits and promote dissociation of {alpha} subunits but leave a surface available on {beta}{gamma} subunits for activation of MAP kinase pathways.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Peptides were synthesized by Alpha Diagnostics International and purified by high performance liquid chromatography 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; mSIRK(L9A), myristate-SIRKALNIAGYPDYD; mSCAR, myristate-SCARFFGTPCP-amide; TAT-SIRK, fluorescein-GGGYGRKKRRQRRRG-SIRKALNILGYPDYD; TAT-SIRK(L9A), fluorescein-GGGYGRKKRRQRRRG-SIRKALNIAGYPDYD. mSCAR and mSIRK were dissolved in Me2SO; all other peptides were dissolved in water. Antibodies to phosphorylated ERK1/2, total ERK1/2, phosphorylated Jun N-terminal kinase, and phosphorylated p38 were obtained from Cell Signaling Technology Inc. The specific EGF receptor kinase inhibitor AG1487 and src inhibitor PP2 were from Calbiochem. Thapsigargin, EGF, and lysophosphatidic acid (LPA) were from Sigma. Pertussis toxin was from List Biological Laboratories.

Cell Culture—All 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 {beta}ARK1ct were prepared and cultured as described previously (9).

MAP Kinase Assays—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 dimethyl sulfoxide vehicle were diluted 100–400-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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FIG. 1.
Cell permeating {beta}{gamma}-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 Me2SO for 10 min before 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, asterisks (*) indicate the presence of a doublet of ERK2 and ERK1. C, substitution of Leu-9 with Ala or removal of the myristoyl group eliminates the ability of mSIRK to activate ERK1/2. RASM cells were treated for 5 min with Me2SO (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, human immunodeficiency virus tat cell permeation sequence also allows the peptide to activate ERK1/2. Cells were treated with 5 µM mSIRK, 10 µM TAT-SIRK, 10 µM LPA, and 10 nM EGF or Me2SO for 5 min. For all the experiments, samples were 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 three times with similar results.

 

Calcium Measurements—For 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 Phosphates—Cells were plated on 35-mm dishes and labeled by adding 3–5 µCi of [3H]inositol for 24–48 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 {beta}{gamma} Subunits—The cDNA for rat {beta}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 {beta}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... {beta}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-{beta}1{gamma}2 was purified from Sf9 cells using hexahistidine-tagged {alpha}i1 following previously published procedures (12). Biotinylation of the purified {beta} subunit was confirmed by SDS-PAGE followed by Western blotting with streptavidin-linked horseradish peroxidase and detection with chemiluminescence. To confirm that the {beta} subunit was fully biotinylated, it was precipitated with streptavidin-agarose, and greater that 90% of the {beta} subunit was removed from the supernatant by this procedure.

Measurement of GTP Hydrolysis and GTP{gamma}S Binding—Steady state GTP hydrolysis was assayed as [32P]Pi release from [{gamma}-32P]GTP by standard procedures (13). GTP{gamma}S binding was measured according to (14), except Sf9 cell membranes expressing the M2 muscarinic acetyl choline receptor were reconstituted with purified recombinant myristoylated {alpha}i and {beta}1{gamma}2.

Measurement of {alpha}-{beta}{gamma} Interactions by Flow Cytometry—Binding of fluorescein isothiocyanate-labeled myristoylated {alpha}i1 (F-{alpha}i) to biotinylated {beta}1{gamma}2 subunits was measured using a flow cytometry assay (15, 16). Biotinylated {beta}{gamma} 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 {beta}{gamma}). For {alpha} subunit dissociation experiments, the beads with bound {beta}{gamma} subunits were premixed with 1 nM F-{alpha}i for 10 min before the addition of competitors. For equilibrium binding measurements, 1 nM F-{alpha}i and competitors were added simultaneously. The amount of F-{alpha}i bound to beads with biotinylated {beta}{gamma} 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-{alpha}i and 50 nM myristoylated {alpha}i, was 10–20% of the total signal and was subtracted from the data.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the consequence of selectively inhibiting {beta}{gamma} 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 N terminus (mSIRK). We tested the ability of this peptide to affect activation of ERK1 and ERK2 in primary cultures of RASM cells. In these cells it has been shown that ERK1 and ERK2 can be activated by stimulation of G protein-coupled LPA receptors. This activation is blocked by pertussis toxin and by an adenovirus expressing the {beta}ARK1ct, implicating Gi and {beta}{gamma} 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 (Fig. 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 {beta}{gamma} 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 Fig. 1, A and B, the conditions were optimized to resolve the phosphorylated from the non-phosphorylated ERK1/2 in a total ERK1/2 Western blot (see the asterisks in the bottom panels, Fig. 1, A and B). 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 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 Ala for Leu-9 increased the IC50 of this peptide for blocking {beta}{gamma}-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 {beta}{gamma} 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 {beta}{gamma} subunits in vitro (5), strongly supports the idea that {beta}{gamma} is the target of the peptides and this targeting is responsible for ERK1/2 activation.

G{beta}{gamma} Is the Target of the Peptides in the Intact Cell—To further support the idea that {beta}{gamma} 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 {beta}{gamma} 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 {beta}{gamma} subunits, the cysteine residues must form an intramolecular disulfide bond. If SCAR is reduced with DTT, it is no longer able to bind to {beta}{gamma} 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 {beta}{gamma} subunits is important to observe activation of ERK1/2, not just the peptide sequence.



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FIG. 2.
Activation of ERK1/2 by mSIRK involves a G{beta}{gamma}-dependent signaling pathway. A, other peptides that bind to {beta}{gamma} 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 before the addition to the cells. B, the {beta}ARK1ct inhibits mSIRK-mediated activation of ERK1/2. Stable cell lines expressing the {beta}ARK1ct or vector control cells were treated with the indicated concentrations of mSIRK, 10 µM LPA, or Me2SO (DMSO) control for 5 min and processed as described. This experiment was repeated three times with similar results.

 

To provide further evidence G{beta}{gamma} 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 {beta}ARK that sequesters {beta}{gamma} subunits ({beta}ARK1ct (Gly-495–Leu-689)) (8, 9). We hypothesized that mSIRK binds to {beta}{gamma} but leaves a surface available on {beta}{gamma} to signal to some downstream targets. Based on this hypothesis, we predicted that ERK1/2 activation by mSIRK would be inhibited by the {beta}ARK1ct, a general {beta}{gamma} inhibitor that blocks signaling of {beta}{gamma} to most if not all effectors. The data in Fig. 2B demonstrate that in cells expressing {beta}ARK1ct, activation of ERK1/2 was almost completely inhibited compared with the control RASM cells. This indicates that {beta}{gamma} 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 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 {beta}{gamma}-dependent MAP kinase activation is stronger than others.



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FIG. 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 Me2SO or 10 µM mSIRK for 5 min. All samples were processed and analyzed as described under "Experimental Procedures" and Fig. 1. B, the pathway activated by peptide treatment involves src but not transactivation of the EGF receptor. Cells were treated with 1 µM AG1478 for1hor10 µM PP2 for 30 min before the addition of 5 µM mSIRK or 10 nM EGF for 5 min. Each of the treatments was repeated in at least three separate experiments. C, calcium release from intracellular pools was not responsible for ERK1/2 activation, and the activation is not blocked by pertussis toxin (PTX). Cells were treated with 1 µM thapsigargin (30 min) to empty intracellular calcium pools before the addition of 10 µM 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 Fig. 1.

 

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 (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 {beta}{gamma}-mediated activation of ERK1/2 by G protein-coupled receptors, consistent with a pathway involving {beta}{gamma} 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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FIG. 4.
Multiple signaling pathways are activated by mSIRK treatment. A, p38 MAP kinase and Jun N-terminal kinase are activated by mSIRK. RASM cells were treated with 5 µM mSIRK, mSIRK(L9A), or 1% fetal bovine serum (FBS) for 5 min, and samples were assessed for p38 MAP kinase activation, Jun N-terminal kinase activation, and ERK activation by Western blotting with antibodies specific for phosphorylated (P) p38, phosphorylated Jun N-terminal kinase, and phosphorylated ERK, respectively. Total ERK1/2 is shown as a loading control. DMSO, Me2SO. B, mSIRK causes mobilization of intracellular Ca2+. Fura2-loaded RASM cells were incubated in Ca2+- and Mg2+-free Hanks' balanced saline solution for Ca2+ imaging. At the times noted by the arrows,10 µM mSIRK, 10 µM mSIRK(L9A), or vehicle was added. To estimate the intracellular Ca2+pool, 1 µM ionomycin (Iono) 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 (IP). Total inositol phosphates produced in RASM cells were measured as described under "Experimental Procedures." mSIRK (10 µM), Me2SO (DMSO), or 1% fetal bovine serum (FBS) was added for 30 min, and PP2 treatment was 10 µM. The data presented are representative data showing the S.E. 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 S.E. mSIRK-treated samples were significantly different from Me2SO-treated control (p < 0.0001 using a paired two tailed t test).

 

Activation of Other G Protein-coupled Receptor Signal Transduction Pathways by mSIRK—The effects of mSIRK on other signal transduction pathways downstream of either {beta}{gamma} subunits or {alpha} 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 2–5 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 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 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 {beta}{gamma} subunits and somehow promotes release of {beta}{gamma} subunits, leaving a surface available to signal to downstream pathways. A potential problem with this hypothesis is that SIRK blocks PLC stimulation by {beta}{gamma} in vitro (5) and, thus, should block the {beta}{gamma} 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 {beta}{gamma}, and this residual ability of the {beta}{gamma} 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 {alpha}q/11 may be released, which can bind some GTP once released from {beta}{gamma}.

SIRK Causes Dissociation of {alpha} Subunits from {beta}{gamma} Subunits without Stimulating Nucleotide Exchange—That all these signaling pathways are being activated in response to a {beta}{gamma} 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 {alpha} subunits. However, these peptides were selected to bind to G protein {beta}{gamma} subunits and, therefore, should not bind to {alpha} subunits. Receptors and receptor-mimicking peptides such as mastoparan act at least in part through binding to {alpha} subunits. It is possible, however, that mSIRK may non-specifically interact with {alpha} subunits or may promote nucleotide exchange by an undefined mechanism through binding to {beta}{gamma} subunits. To determine whether the {beta}{gamma}-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{gamma}S to purified {alpha}i1{beta}1{gamma}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{gamma}S binding was stimulated in a {beta}{gamma} 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 {alpha} and {beta}{gamma} subunits.



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FIG. 5.
SIRK does not stimulate GDP release from G protein heterotrimers. A, binding of [35S]GTP{gamma}S to 200 nM {alpha}i in the presence or absence of 400 nM {beta}1{gamma}2 was measured with 1 µM GTP{gamma}S and [35S]GTP{gamma}S (100,000 cpm/assay) and 4 µM GDP for 10 min in the presence or absence of 10 µg of urea-stripped membranes expressing the M2 muscarinic acetylcholine receptor and 100 µM carbachol. Error bars represent S.E. from triplicate data points, and data are representative of experiments performed three times. B, M2 muscarinic receptor stimulated binding of [35S]GTP{gamma}S 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 pmol of GTP{gamma}S bound minus the basal in the absence of added {beta}{gamma} subunits of 1.3 pmol. Basal was unchanged by peptide as in A and is not shown. Error bars represent S.E. from triplicate data points, and data are representative of experiments performed three times. C, steady state GTP hydrolysis was measured with 5 nM recombinant myristoylated G{alpha}o,10nM {beta}1{gamma}2, sonicated phospholipid vesicles (phosphatidylethanolamine/phosphatidylserine/phosphatidylcholine, 5 µM each), 300 nM GTP, and [{gamma}-32P]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 + mastoparan, p < 0.05, but not significantly different from Go + mSIRK analyzed with a one way analysis of variance 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 Fig. 1.

 

Nucleotide exchange can also be assayed by measuring the steady state rate of GTP hydrolysis on the {alpha} 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 [{gamma}-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 {alpha} from {beta}{gamma} in the absence of nucleotide exchange, we assayed the binding of {alpha} subunits to biotinylated {beta}{gamma} subunits bound to streptavidin-agarose beads. G protein {alpha} subunits (10 nM) were incubated with 10 nM biotinylated {beta}{gamma} 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 {alpha} subunits were estimated by quantitative Western blotting (Fig. 6A). At 10 and 30 µM mSIRK the amount of {alpha} subunit bound to {beta}{gamma} subunits was significantly lower than in the absence of added mSIRK.



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FIG. 6.
SIRK inhibits {alpha}-{beta}{gamma} interactions. A, biotinylated {beta}1{gamma}2 (10 nM) was bound to streptavidin-agarose beads followed by 10 nM myristoylated {alpha}i and the indicated concentrations of mSIRK or (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% polyoxyethylene 10 lauryl ether. After incubation for 30 min, the beads were precipitated, and {alpha}i associated with the {beta}{gamma} beads was assessed by quantitative Western blotting. Error bars represent the S.E. from triplicate experiments. B, 1.5 nM fluorescein isothiocyanate-{alpha}i was mixed with 200 pM biotinylated {beta}1{gamma}2 in the presence of the indicated concentrations of SIRK (•) or SIRK(L9A) ({blacktriangledown}), and the amount of F-{alpha}i bound was assessed by flow cytometry as described under "Experimental Procedures." Data are representative of experiments performed at least three times each.

 

A drawback of the pull-down assay is the high concentrations of {alpha} and {beta}{gamma} 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 {alpha}-{beta}{gamma} interactions of about 1 nM. A more quantitative assay to measure {alpha}-{beta}{gamma} interactions at very low subunit concentrations has recently been developed that uses flow cytometry to assess the amount of fluorescent {alpha} subunit bound to biotinylated {beta}{gamma} subunits immobilized on beads (15, 16). In this assay, where SIRK and fluorescein isothiocyanate-labeled {alpha}i (F-{alpha}i) are simultaneously mixed with {beta}{gamma} subunits, there was a dose-dependent decrease in the amount of {alpha} subunit bound to {beta}{gamma} subunits, whereas SIRK(L9A) had no effect (Fig. 6B). At 10 µM SIRK, F-{alpha}i bound to {beta}{gamma} 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 {alpha}-{beta}{gamma} interaction, making it easier to observe a decrease in binding.

A possible mechanism for the increase in {alpha}-{beta}{gamma} dissociation might involve direct competition between the peptide and the {alpha} subunit for the {alpha} subunit-binding site on {beta}{gamma} subunits. In this model, the peptide would increase net subunit separation by preventing rebinding of {alpha}-GDP subunits after dissociation. This process would be inherently slow because it would depend on spontaneous dissociation of tightly bound {alpha}-GDP from {beta}{gamma}. A second possibility is that the peptide promotes dissociation of {alpha} from {beta}{gamma} subunits by a mechanism that does not involve direct competition. These two possibilities can be distinguished by measuring the {alpha} subunit dissociation rate. If the peptide simply competes for the {alpha} subunit-binding site on {beta}{gamma} subunits, then the dissociation rate should be equivalent to the intrinsic {alpha} subunit dissociation rate. If the rate of {alpha} 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-{alpha}i dissociation from {beta}{gamma} by adding a 50-fold excess of unlabeled myristoylated {alpha}i subunit at time 0 and measuring the amount of F-{alpha}i bound to {beta}{gamma} 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 (t1/2 ~ 9–14 min) in four separate experiments. These data are similar to previously published results (0.047 min–1) with this assay and consistent with the low apparent Kd for {alpha}-{beta}{gamma} interactions (15). The rate of {alpha} dissociation from {beta}{gamma} 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 {alpha} subunit dissociation rate. The extent of initial rapid dissociation by peptide was always at least 75% of the total dissociation that occurred after {alpha} subunit alone was added for at least 1 h.



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FIG. 7.
SIRK promotes {alpha}i dissociation from {beta}{gamma} subunits in the absence of nucleotide exchange. A,1nM F-{alpha}i was preincubated with streptavidin beads bound to 250 pM {beta}1{gamma}2 for 10 min. Either 50 nM unlabeled myristoylated {alpha}i alone ({blacktriangleup}) or 50 nM myristoylated {alpha}i plus 25 µM SIRK ({blacksquare}) was added to the preformed F-{alpha}i-{beta}{gamma} complex. The rate of dissociation of F-{alpha}i was measured using flow cytometry to assess the amount of F-{alpha}i bound at various times after the 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-{alpha}i in the absence of {beta}{gamma} bound to the beads). Curves were best fit with a two-exponent decay function using Graph-Pad Prism data analysis software. To test for statistical significance, the F-{alpha}i fluorescence bound after the addition of {alpha}i alone was compared with {alpha}i 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-{alpha}i bound 1 min after the addition of 100 nM {alpha}i as the competitor was 98.25% ± 1.0 S.E., and that for {alpha}i plus SIRK was 69.15% ± 0.77 S.E.; they were significantly different, p < 0.0001. In a separate experiment at 2 min after competitor addition the values were 90.0% ± 1.77 S.E. and 65.71% ± 0.82 S.E. (means were significantly different, p < 0.0001) for {alpha}i and {alpha}i plus SIRK respectively. B, real time measurement of {alpha} 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. {triangleup}, SIRK added without the addition of myristoylated {alpha}i. Curves were best fit with a single-exponent decay function using GraphPad Prism data analysis software yielding a koff for {alpha}i of 0.02 min–1, peptide 0.28 min–1 and peptide + {alpha}i of 0.24 min–1. This experiment was repeated five times with qualitatively similar results.

 

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 {alpha}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 {alpha} from {beta}{gamma} subunits without promoting nucleotide exchange on the {alpha} subunit.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of ERK1/2 by {beta}{gamma} Inhibitory Peptides—There is extensive evidence in the literature suggesting free {beta}{gamma} subunits released from Gi heterotrimers upon {alpha} 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 that the effects we observe are specifically due to interactions of our peptides with {beta}{gamma} 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 {beta}{gamma} subunits and downstream targets in vitro. A single Leu-Ala point mutation, previously shown to eliminate the ability of the peptide to inhibit {beta}{gamma} 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 protein cell permeation sequence from human immunodeficiency virus. 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 binding to the same surface on {beta}{gamma} subunits also caused activation of ERK1/2. The fact that activation of ERK1/2 is blocked by the src inhibitor PP2 indicates our {beta}{gamma} "inhibitory peptides" act though a pathway similar to that which has been previously described as being involved in MAP kinase activation by {beta}{gamma} (20). Finally, if the peptide is tested in RASM cells that express the {beta}ARK1ct, the effect on ERK1/2 activation is greatly attenuated. All of these data indicate the G protein-signaling system and the {beta}{gamma} 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 {alpha}-{beta}{gamma} Dissociation—We had previously characterized SIRK and other phage display-selected {beta}{gamma}-binding peptides in vitro and demonstrated they could selectively interfere with the activation of some effectors by {beta}{gamma} subunits. We also postulated they interact with a protein-protein interaction hot spot on the surface of {beta}{gamma} subunits because all the selected peptides, with diverse sequence characteristics, bound to the same site on {beta}{gamma} subunits. Here we demonstrate these effector-selective {beta}{gamma}-blocking peptides initiate activation of certain G-{beta}{gamma}-dependent-signaling pathways in intact cells in a receptor-independent fashion. We show the peptides can promote dissociation of {alpha} subunits from {beta}{gamma} subunits without inducing nucleotide exchange. To explain the ability of these peptides to activate cellular signaling, they must bind to {beta}{gamma} subunits to decrease the binding of {alpha} to {beta}{gamma} but leave the surface on {beta}{gamma} 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 {beta}{gamma}-blocking peptides makes this proposed mechanism possible. The precise molecular target of {beta}{gamma} 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 {beta}{gamma} 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 {beta}{gamma} can activate ERK in different cell types. RASM cells have previously been demonstrated to have a robust {beta}{gamma}-dependent activation of ERK (18). We suspect that {beta}{gamma} is probably released by peptide treatment in all the cells tested, but they may have different levels of the signaling molecules necessary for robust {beta}{gamma}-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 {beta}{gamma} subunits can promote subunit dissociation in vitro and activate G protein {beta}{gamma} subunit-dependent pathways in cells. This suggests a potential mechanism for regulating G protein {beta}{gamma} 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 {beta}{gamma} subunits, but not {alpha} 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 {alpha} from {beta}{gamma} is that Tctex1 is a {beta}{gamma} subunit effector that can cause separation of {alpha} from {beta}{gamma} but leaves surfaces available on {beta}{gamma} 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 {beta}{gamma} subunit that promotes subunit dissociation. Generally it is not thought that the {beta}{gamma} subunits undergo significant conformational changes because the conformations of {beta}{gamma} crystallized with and without {alpha} subunits are very similar (25, 26, 27, 28). On the other hand the structure of {beta}{gamma} crystallized in the presence of phosducin shows significant conformational alteration (29, 30). Interestingly, a peptide derived from phosducin that apparently binds outside of the {alpha} subunit-binding site alters {alpha}-{beta}{gamma} subunit interactions, suggesting that binding to {beta}{gamma} can transmit information to the {alpha} subunit interface (31). The peptides we derived from phage display have similarity to a region of phosducin that binds outside of the {alpha} subunit interface. We previously postulated that the site on {beta}{gamma} 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 {beta}{gamma}.

An alternative hypothesis is, Because {alpha}-{beta}{gamma} interactions involve two parts of {alpha} (i.e. switch regions and N terminus) (26, 27), the slow dissociation of {alpha} and {beta}{gamma} may depend on the requirement for simultaneous breaking of these two contacts. If each contact were dynamic, mSIRK peptide binding to {beta}{gamma} (for example at the site where {beta}{gamma} 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 {alpha} subunit could only prevent reassociation after full release of {alpha} from {beta}{gamma} due to steric interference between the incoming and outgoing {alpha} subunits. Identification and characterization of the peptide-binding site is critical to the determination of this mechanism.


    FOOTNOTES
 
* This work was supported by National Institutes of Health (NIH) Grant GM60286 and American Heart Association, New York State Affiliate (to A. V. S.) and by NIH Grants DK19974 (to P. M. H.) and HL46417 (to R. R. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

** 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; {beta}ARK1ct, C terminus of {beta}-adrenergic receptor kinase; DTT, dithiothreitol; MAP, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; F-{alpha}i, fluorescein isothiocyanate-labeled myristoylated {alpha}i1; TAT-SIRK, human immunodeficiency virus TAT cell permeation sequence fused to SIRK. Back


    ACKNOWLEDGMENTS
 
We 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 {beta}{gamma} subunits.



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