©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Small GTP-binding Protein, Rho, Associates with the Platelet-derived Growth Factor Type- Receptor upon Ligand Binding (*)

(Received for publication, April 5, 1995)

Mercedes Zubiaur (1) (2), Jaime Sancho (2) (3), Cox Terhorst (3), Douglas V. Faller (1)(§)

From the  (1)Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, (2)Consejo Superior de Investigaciones Cientficas, Instituto de Biomedicina y Parasitologia, Granada 18001, Spain, and the (3)Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ligand binding to the platelet-derived growth factor (PDGF) receptor initiates a complex and diverging cascade of signaling pathways. GTP-binding proteins with intrinsic GTPase activity (G-proteins) frequently link cell surface receptors to intracellular signaling pathways, but no close associations of the PDGF receptor and any small G-proteins, nor any such associations activated by ligand binding to the receptor have been previously reported. We demonstrate that a small GTP-binding protein binds specifically to the murine and human PDGF type- receptor. In response to PDGF-BB stimulation, there is an increase in the amount of labeled small G-protein associated with the PDGF type- receptor. The GTP-binding protein did not undergo ligand-induced association with a mutant receptor protein that was unable to bind ATP. Proteolytic cleavage analysis, together with two-dimensional separation techniques, identified the small G-protein specifically associating with the PDGF type- receptor after ligand binding as a member of the Rho family. This was confirmed by demonstration that the small G-protein co-immunoprecipitated by the anti-PDGF receptor antibody was a substrate for the ADP-ribosyltransferase C3 exoenzyme. Thus, the PDGF type- receptor may form a complex with one or more small G-proteins upon binding PDGF-BB, and the Rho small G-protein is likely to be an important component of the proteins making up the multimeric signaling complex of the PDGF type- receptor.


INTRODUCTION

Growth factor signal transduction through the platelet-derived growth factor receptor consists of a complex cascade of events. The platelet-derived growth factor (PDGF)() is a 30-kDa homo- or heterodimer that binds to the PDGF receptors, a 180-kDa glycoprotein homo- or heterodimer with at least two classes of subunits: type- and type-(1, 2, 3, 4, 5, 6) . The PDGF type- receptor possesses an intrinsic protein tyrosine kinase activity, which is stimulated upon binding by PDGF-BB(1, 7, 8) . A number of intracellular activities occur rapidly after exposure of a cell to PDGF-BB, including dimerization and autophosphorylation of the PDGF type- receptor and association and activation of specific proteins. Within the PDGF type- receptor, several tyrosine residues have been identified, which function as high affinity binding sites for signaling molecules containing the Src homology 2 domain sequences, such as phospholipase C, p21 GTPase-activating protein (Ras-GAP), the p85 subunit of phosphatidylinositol 3-kinase, and Syp(9, 10, 11, 12, 13) . In addition, increases in phosphatidylinositol turnover and calcium mobilization, activation of protein kinase C and microtubule-associated protein kinase, and induction of a number of growth-related genes, including c-myc, c-fos, JE, c-jun, and egr-1 occur rapidly after ligand binding(7, 14, 15, 16, 17) . A causal or sequential relationship of these PDGF-induced and tyrosine kinase-dependent phenomena to each other and to eventual DNA synthesis is suspected but not well established. We have recently reported that some signals, resulting in the induction of certain growth-related genes, can be transmitted by the PDGF receptor in the absence of tyrosine kinase activity(18) . Therefore, PDGF receptor-activated signaling mechanisms and pathways other than those characterized previously must exist.

GTP-binding proteins with intrinsic GTPase activity (G-proteins) frequently link cell surface receptors to intracellular signaling pathways. They function by cycling between (inactive) GDP-bound and (active) GTP-bound states. The small G-protein p21 has been implicated in mediating signals from a number of tyrosine kinase-type growth factor receptors, including the PDGF type- receptor(19, 20, 21, 22, 23, 24) . p21 may be indirectly linked to these receptors via a pathway involving a number of intermediary adaptor or coupling proteins (22, 24, 25, 26) . No close associations of the PDGF receptor and any small G-proteins, however, and no associations activated by ligand binding to the receptor, have been previously reported.

In the present study we have investigated the involvement of GTP-binding proteins in PDGF-mediated signaling in normal fibroblasts. A technique reported recently for in situ protein labeling of nucleotide-binding proteins in permeabilized cells was utilized (27) . The method involves the introduction of [-P]GTP into permeabilized cells, followed by in situ periodate oxidation. The result was the generation by cross-linking of GTP-labeled G-proteins(28, 29) . The sensitivity and specificity of this method allowed cross-linking of more than 50% of p21 to [-P]GTP in transfected fibroblasts. Heterotrimeric G-proteins (e.g. Gi) can also be labeled by this method(27) . This technique has recently been used to detect a GTP/GDP binding site in the -chain of the T-cell receptor complex(30, 31) .

In this report, we demonstrate that a small GTP-binding protein associates with the PDGF type- receptor upon PDGF-BB stimulation. We have identified the small G-protein specifically associating with the PDGF type- receptor after ligand binding as a member of the Rho family of G-proteins. On the basis of these results, this Rho G-protein is likely to be an important component of the proteins making up the multimeric signaling complex of the PDGF type- receptor.


MATERIALS AND METHODS

Cells

Balb/c-3T3 fibroblasts were obtained from the American Type Culture Collection. Kirsten v-ras-transformed Balb/c-3T3 fibroblasts (KBalb) were described previously(32) . Cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% heat-inactivated donor calf serum (Sigma). When quiescent cells were required, monolayers were grown to confluence and then starved for 48 h in Dulbecco's modified Eagle's medium with 0.5% donor calf serum. Dog kidney epithelial cells (TRMP) expressing wild type or a mutant human PDGF type- receptor (L635R) were the generous gift of A. Kazlauskas and J. Cooper (33) . The L635R mutation made the receptor unable to bind ATP and therefore kinase-negative. These cells were maintained with a mixture 1:1 of Dulbecco's modified Eagle's medium and F-12 medium supplemented with 10% heat-inactivated fetal calf serum, including 400 mg/ml G418 (Geneticin, Life Technologies, Inc.). All media were supplemented with 100 units/ml penicillin and 100 units/ml streptomycin.

Antibodies and Reagents

Antiserum specific for the human PDGF type- receptor, directed against amino acid sequence 1013-1025, and cross-reacting with mouse receptors was purchased from Upstate Biotechnology, Inc. (UBI). Antibody 538, directed against the PDGF type- receptor, was a gift from Tom Daniel, Vanderbilt University. Anti-p21 monoclonal antibody Y13-259 (rat mAb-IgG) directed against residues 63-76 was obtained from Oncogene Science. The following affinity-purified rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc.. Anti-Rap1A/B/Krev-1 was raised against a synthetic peptide corresponding to residues 121-137, which are conserved between Rap1A/B proteins. Anti-RhoA was raised against a synthetic peptide corresponding to amino acids 119-132 of the RhoA protein. Anti-RhoB was raised against a synthetic peptide corresponding to amino acids 119-132 of the RhoB protein. Anti-phosphotyrosine mAb 4G10 was purchased from UBI. Affinity-purified rabbit anti-mouse Vectastain ABC kit was from Vector Laboratories. Recombinant protein A- and protein G-Sepharose were purchased from Sigma and Pharmacia Biotech Inc. Recombinant human PDGF-BB homodimer (PDGFh-BB) was purchased from R& Systems. [-P]GTP was purchased from DuPont NEN or Amersham Corp. (3000 Ci/mmol; final specific activity 100-400 Ci/mmol). [-P]NAD (1000 Ci/mmol) was purchased from Amersham. Purified exoenzyme C3 fusion protein containing full-length 24.5-kDa Clostridium botulinum (D strain) C3, expressed in Escherichia coli, was purchased from UBI.

PDGF Receptor in Vivo Stimulation

Balb, Kbalb, and TRMP cells were grown in 10-cm diameter dishes until confluence. They were serum-starved in Dulbecco's modified Eagle's medium plus 0.5% donor calf serum for 24-48 h. Starved cells were washed twice with phosphate-buffered saline (PBS), at 37 °C, then twice with the isotonic wash (120 mM KCl, 30 mM NaCl, 2 mM MnCl, 10 mM HEPES, pH 7.4), at 37 °C. For ``in vivo'' stimulation with PDGF-BB, washed cells were overlaid with 3 ml of isotonic wash buffer at 37 °C in 5% CO incubator, without or with PDGFh-BB (R& Systems), to a final concentration of 30 ng/ml for 2 or 5 min at 37 °C, as indicated.

Cell Permeabilization

The cells were washed twice with ice-cold PBS and twice with ice-cold labeling buffer (ICB buffer), containing 20 mM Hepes, pH 7.8, 140 mM KCl, 10 mM NaCl, and 2.5 mM MgCl, and then permeabilized with 50 µg/ml L--lysophosphatidylcholine (LPC) (Sigma) for 5 min on ice. The cells were then dislodged with a cell scraper and transferred to a microcentrifuge tube. Under these conditions, 95% of the treated cells were permeable with trypan blue. To remove excess LPC, permeabilized cells were centrifuged at 6,000 rpm for 15 s in a microcentrifuge and resuspended in the labeling buffer.

In Situ Labeling with [-P]GTP Nucleotides

Permeabilized cells in a final volume of 500 µl of labeling buffer were incubated with 1 µM [-P]GTP for 30 min at 37 °C, to allow the added guanine nucleotide to bind or to exchange for endogenous nucleotide, after which nucleotides were oxidatively cleaved with 1 mM NaIO (Sigma) for 1 min at 37 °C. Condensation products were rapidly stabilized by reduction with 20 mM NaCNBH (Sigma) for 1 min at 37 °C. To remove free reactive GTP nucleotides, cells were treated with 20 mM NaBH (Sigma) for 1 min at 37 °C, then for 5 min on ice. After centrifugation for 3 min at 6,000 rpm in a microcentrifuge, cells were lysed for 20-60 min on ice in lysis buffer HTG (20 mM Hepes (pH 7.2), 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM NaVO, 5-10 µg/ml each aprotinin and leupeptin). The lysates were cleared by a 5-min centrifugation at 13,000 g, at 4 °C.

Immunoprecipitation and Electrophoresis

For immunoprecipitation with rabbit antibodies, the lysates were precleared with normal rabbit serum (NRS) previously coupled to protein A- or protein G-Sepharose, as indicated, for 1-12 h at 4 °C; preclearings were repeated at least three times. Precleared lysates were then immunoprecipitated for periods ranging from 2 h to overnight at 4 °C with specific antibodies. Protein-antibody complexes were precipitated by incubation with protein A- or protein G-Sepharose for 90 min at 4 °C. p21 immunoprecipitation with anti-p21Y13-259 was performed by modifications of the method described previously by Downward et al.(34) . Permeabilized and labeled cells were lysed in 600 µl of 50 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM MgCl, 1% Triton X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride. The lysates were cleared by centrifugation as described above, and supernatant fractions were transferred to microcentrifuge tubes with 75 µl of 4 M NaCl, 75 µl of 4% sodium deoxycholate, and 0.4% SDS. Lysates were precleared three times with rat IgG-agarose (Sigma), before the Y13-259 antibody was added. Samples were incubated overnight at 4 °C. Y13-259-bound p21 was precipitated with anti-rat IgG-agarose (Sigma). Immunoprecipitates were washed up to six times with lysis buffer. 40 µl of 3 Laemmli sample buffer was added to the washed beads, and they were then heated at 90 °C for 5 min. Labeled proteins were separated by SDS-PAGE in a 15% acrylamide gel, or as indicated. The gels were fixed and dried. Dried gels were autoradiographed using Kodak XAR-5 film, unless otherwise indicated.

Two-dimensional Gel Electrophoresis

A combination of isolectric focusing (IEF) and SDS-polyacrylamide gel electrophoresis (PAGE) was used to resolve proteins in two dimensions essentially as described(35) . For IEF, samples were solubilized in 9.3 M urea, 50 mM dithiothreitol, 2.5% Triton X-100, 2% ampholines (80% ampholines at pH 5-7 and 20% ampholines at pH 3-10 (Bio-Rad)). Tube gels for first dimension IEF were 11-12 cm long, with 1.5-mm internal diameter. As internal markers for two-dimensional electrophoresis, a mixture of four proteins (pI range: 3.8-7.6, in a range of 17-89 kDa) (Sigma) was added. IEF gels were prerun for 15 min at 200 V, 30 min at 300 V, and 30 min at 400 V. Samples were run for 15-17 h at 800 V, followed by 1 h at 1000 V. The pH gradient after electrophoresis ranged from 4.5 to 6.5. For the second dimension, a 15% SDS-PAGE was used.

Immunoblot Analysis

GTP-labeled small G-protein-antibodies complex immunoprecipitates were eluted from the beads with Laemmli sample buffer, separated on a 5-15% gradient SDS-PAGE gel, and electrophoretically transferred to nitrocellulose (0.45 µm, Schleicher & Schuell) using a semi-dry transfer apparatus (Hoeffer Scientific, CA), in a continuous buffer system (39 mM glycine, 48 mM Tris, 0.0375% SDS, 20% methanol), for 1 h at a constant current of 0.8 mA/cm, unless otherwise indicated. Filters were blocked for 2 h at 37 °C or overnight at 4 °C, with 3% bovine serum albumin, 10 mM Tris (pH 8), 150 mM NaCl, 0.05% Tween 20. Filters were incubated with a mouse mAb against phosphotyrosine residues, 4G10 (IgG; UBI). As a secondary antibody, a biotinylated goat anti-mouse antibody (Vector Laboratories) was used, and the immunoblot was developed with an ABC amplification kit (Vector), coupled to alkaline phosphatase, following the manufacturer's protocol. A 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate premixed solution was used (Sigma).

Proteolytic Cleavage Analysis

Small G-protein immunoprecipitates derived from GTP-labeled Balb cells were washed up to six times with lysis buffer as described above. They were desalted by washing twice with distilled water and resuspended in 50 mM ammonium acetate (pH 4.4) and treated with 3-5 µg of Staphylococcus aureus V8 endoproteinase Glu-C (Promega) for 20 h at 37 °C. The reaction was stopped by addition of 40 µl of 3 X sample buffer (4% SDS, 12% glycerol, 50 mM Tris, 2% -mercaptoethanol, 0.01% Serva Blue)(36) , and incubating for 30 min at 40 °C. Labeled peptides resulting from cleavage were separated electrophoretically using a high resolution Tris-Tricine buffer SDS-PAGE system (16.5% T, 3% C, containing glycerol)(36) . The gels were either fixed for 1 h in 50% methanol, 10% acetic acid and dried, or transferred onto Immobilon-PVDF membranes in a continuous buffer system without SDS (25 mM Tris, pH 8, 192 mM glycine, in 20% methanol)(37) . Dried gels and filters were analyzed in a PhosphorImager (Molecular Dynamics) or in a InstantImager (Packard), respectively. In addition gels and filters were exposed to Kodak-XAR film or Hyperfilm-MP (Amersham).

Cellular Membrane Preparation

Balb fibroblasts were grown and rendered quiescent as described above. After PDGF-BB in vivo stimulation, cells were washed twice in cold PBS, scraped from plates, and centrifuged at 1800 g at 4 °C for 5 min. Cells were resuspended in 1.0 ml of ice-cold TMSDE buffer (50 mM Tris, pH 7.6, 75 mM sucrose, 6 mM MgCl, 1 mM dithiothreitol, 1 mM EDTA, with 10 µg/ml aprotinin, 10 µg/ml leupeptin added prior to use) and incubated at 0 °C for 10 min. To disrupt the cells, they were frozen at -70 °C and then thawed. Broken cells were homogenized by shearing through a 27-gauge needle with a tuberculin syringe, 8-10 times, on ice. The particulate suspension was centrifuged in a Sorvall Superspeed centifuge at 20,000 g at 4 °C for 30 min, and the pellet containing membranes was then resuspended in 0.1-0.2 ml of TMSDE supplemented with protease inhibitors(38) . Protein concentration was quantitated using the Bio-Rad assay standardized against a bovine serum albumin standard curve. Membranes were stored at -70 °C until use.

[P]NAD-ribosylation of Balb Cell Membrane Proteins

[P]ADP-ribosylation of Balb cell membranes (80-200 µg of protein) by botulinum C3 ADP-ribosyltransferase was performed in a reaction buffer containing 50 mM triethanolamine-HCl, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM ATP, 2.5 mM MgCl, 10 mM thymidine, 0.2% Triton X-100, [P]NAD (1 µCi/tube), and 6-7 µg/ml purified recombinant C3 exoenzyme, in a total volume of 80-200 µl, for 60 min at 37 °C(39) . Reaction was terminated by addition of 3 Laemmli sample buffer and boiling for 5 min. In other cases, the reaction was terminated by addition of 900 µl of HTG lysis buffer. In this case, the membranes were incubated for 60 min at 4 °C. The lysates were cleared by a 5-min centrifugation at 13,000 g at 4 °C prior to the immunoprecipitation, as described above. Proteins were resolved in a 12.5% SDS-PAGE gel; the gel was transferred to Immobilon-PVDF following the procedure described above. The radioactivity in the gels and filters were quantified in an InstantImager (Packard). Dried gels and filters were autoradiographed using Hyperfilm-MP (Amersham).


RESULTS

Affinity Labeling of GTP-binding Proteins in Permeabilized Balb Cells

To identify potential GTP-binding proteins involved in signal transduction through the PDGF receptor, a recently developed technique for labeling of nucleotide binding proteins was utilized. This technique involves the introduction of [-P]GTP into permeabilized cells, followed by ``in situ'' periodate oxidation(30, 31) . This method has recently established that the T cell receptor chain has the capacity to bind GTP and GDP(31) . This technique permits specific labeling of such different types of G-proteins as the -subunits of the heterotrimeric G and G proteins, as well as small G-proteins such as p21 protein(27) . In the present study, this technique was used to label GTP-binding proteins in Balb cells and Balb cells expressing activated (v-K)-p21. Confluent cells were serum-starved and in vivo stimulated with 30 ng/ml PDGF-BBh for 5 min at 37 °C or left untreated. The cells were then permeabilized with 50 µg/ml LPC for 5 min on ice. The cells were scraped from the plates into microcentrifuge tubes, where they were GTP-labeled after incubation with 1 µM [-P]GTP for 30 min at 37 °C. Using this in situ cross-linking method, we were able to selectively label several G-proteins in the range of 21-27 kDa with [-P]GTP, as seen in whole lysates of Balb or v-ras-expressing Balb cells, resolved on a 15% SDS-PAGE gel (Fig. 1A).


Figure 1: Affinity labeling of nucleotide-binding proteins in permeabilized Balb/c-3T3 and KBalb fibroblasts. A, [-P]GTPlabeling of cellular GTP-binding proteins. Confluent Balb (lanes1 and 2) and v-ras-containing Balb (Kbalb) cells (lanes3 and 4) were serum-starved (0.5%) for 24-48 h, and in vivo stimulated with 30 ng of PDGF-BBh/ml for 5 min at 37 °C (+), or left untreated (-). Cells were then permeabilized with 50 µg/ml LPC for 5 min on ice. Cells were then scraped from the plates into tubes, where they were GTP-labeled after incubation with 1 µM [-P]GTP for 30 min at 37 °C. Whole cell lysates were then resolved by 15% SDS-PAGE. An autoradiogram of the dried gel is shown here. The position of molecular mass markers is indicated to the left of the figure. Using this method, several G-proteins in the range of 21-27 kDa could be selectively labeled with [-P]GTP. B, competition of insitu affinity labeling of GTP-binding proteins. Dog kidney epithelial cells (TRMP) expressing wild type human PDGF type- receptor were serum-starved for 48 h, then in vivo stimulated with 30 ng of PDGF-BBh/ml for 5 min at 37 °C. The cells were then permeabilized with LPC, and labeled with [-P]GTP after 30 min at 37 °C, as described, in the absence or presence of 100 µM cold deoxynucleotides as competitors. The labeling reaction was inhibited by the addition of cold dGDP or dGTP, but not dCTP or dATP. The [-P]GTP-labeled proteins were separated by 15% SDS-PAGE after lysis of the cells. An autoradiogram of the dried gel is shown here. The position of molecular mass markers is indicated to the left of the figure.



To evaluate the specificity of the in situ labeling technique, competition experiments were carried out using unoxidizable deoxyribonucleotides. TRMP cells transfected with the human PDGF type- receptor gene were used(33) . Cross-linking with [-P]GTP in permeabilized TRMP cells was completely abolished in the presence of 100 µM cold dGTP or 100 µM cold dGDP, whereas addition of cold dATP or dCTP did not affect labeling (Fig. 1B). Thus, the observed 21-27-kDa GTP-labeled bands were the result of a specific affinity labeling, and only those proteins that bound guanine nucleotides were labeled (Fig. 1B).

To demonstrate that known low molecular weight GTP-binding proteins could be specifically labeled in Balb fibroblasts with this in situ [-P]GTP-labeling method, Balb cells were stimulated with PDGF-BB for 2 min at 37 °C and then permeabilized and labeled as described above (Fig. 2A). Cell lysates were precleared up to three times with NRS coupled to protein G-Sepharose (lane1). The precleared lysates were independently immunoprecipitated with one of several different specific anti-small G-protein antibodies. Lanes 3-5 show the immunoprecipitation of GTP-labeled RhoA, RhoB, and Rap1A/B, precipitated with affinity-purified anti-RhoA, anti-RhoB, and anti-Rap1A/B, respectively. The GTP-labeled p21 protein was immunoprecipitated from Balb cells with mAb Y13-259 (lane6). Immunoprecipitated [-P]GTP-labeled proteins migrated in a slightly retarded fashion during SDS-PAGE separation, due to the cross-linking, presenting apparent molecular masses greater than 21 kDa.


Figure 2: Identification of known small GTP-binding proteins labeled with [-P]GTP. A, Balb cells were stimulated with PDGF-BBh for 2 min at 37 °C, permeabilized and labeled with [-P]GTP as described in Fig. 1. The lysates were precleared with NRS-protein G-Sepharose (lane1) and immunoprecipitated with antibodies specific for small G-proteins: affinity-purified anti-RhoA (lane3), affinity-purified anti-RhoB (lane4), affinity-purified anti-Rap1A/B (lane5), and anti-p21 mAb Y13-259 (lane6). Lane2 shows a small G-protein co-immunoprecipitated with a specific anti-PDGF type- receptor antibody. B, anti-RhoA-specific immunoprecipitation of TRMP cell lysates labeled with [-P]GTP, without (lane1) or with (lanes 2-4) 100 µM competing cold deoxynucleotides. The immunoprecipitated proteins were separated in a 15% SDS-PAGE after elution from the Sepharose. An autoradiogram of the dried gel is shown here. The position of molecular mass markers is indicated to the left of the figure.



To further document the specificity of the labeling method for known small G-proteins, the RhoA small G-protein was labeled in the presence or absence of competing deoxynucleotides, and immunoprecipitated by anti-RhoA antiserum. Labeling of RhoA in TRMP cells (Fig. 2B, lane 1) was completely abrogated by the addition of 100 µM cold dGDP (lane2) or 100 µM cold dGTP (lane3), whereas 100 µM cold dCTP (lane4) had no effect.

A Small G-protein Associates with the PDGF Type- Receptor

A specific rabbit anti-peptide serum directed against the external region of the PDGF type- receptor co-precipitated a 23-27-kDa [-P]GTP-labeled protein along with the PDGF type- receptor from a labeled Balb cell lysate (Fig. 2A, lane2).

To further study the PDGF type- receptor/small G-protein interaction, the amount of PDGF type- receptor-associated G-proteins was compared before and after stimulation with PDGF-BB. In cells stimulated for 2 and 5 min with PDGF-BB, there was a significant increase in the amount of labeled G-proteins co-immunoprecipitated with the PDGF type- receptor (Fig. 3A, compare lanes5 and 6 with lane4). In four independent experiments, the binding of the small G-protein to the PDGF type- receptor peaked at 2 min after PDGF-BB stimulation. Thus, there was a rapid agonist-dependent induction of G-protein association to the PDGF type- receptor.


Figure 3: Small [-P]GTP-labeled protein associated with PDGF type- receptor. A, stimulation with PDGF-BB increases the association of the small G-protein with the PDGF type- receptor in a time-dependent manner. Confluent Balb cells were serum-starved, and in vivo stimulated with 30 ng of PDGF-BBh/ml for 2 min (lanes2 and 5) or 5 min (lanes3 and 6) at 37 °C (+), or left untreated(-). Cells were permeabilized with LPC for 5 min on ice and labeled with [-P]GTP after 30 min at 37 °C. Lysates were precleared three times with NRS. The (nonspecific) proteins bound to the NRS-protein A beads after the final preclearing were eluted and the proteins were separated by 15% SDS-PAGE (lanes 1-3). Precleared lysates were immunoprecipitated with a specific anti-PDGF type--receptor antiserum, eluted, and separated in the same gel (lanes 4-6). An autoradiogram of the dried gel is shown here. The position of molecular mass markers are indicated to the left of the figure. Co-precipitation of labeled small G-proteins with the PDGF type- receptor was observed, and an increase in the amount of the GTP-labeled small G-binding protein associated with the receptor at 2 min of stimulation with PDGF-BB (lane5) was apparent. This is a representative result from at least four separate experiments. B, association of the small G-protein with the PDGF type- receptor requires a functional PDGF type- Receptor. Dog kidney epithelial cell lines (TRMP), untransfected (WT, lane2), transfected with human PDGF type- receptor gene (lanes1 and 3), or transfected with a kinase-negative mutant PDGF type- receptor gene (L635R) (lane4) were used. Cells were treated as described in Fig. 2. Lane1 is the eluate from the last preclearing by NRS-protein A-Sepharose from the TRMP transfected with human PDGF type- receptor gene. An autoradiogram of the dried gel is shown here. The position of molecular size markers is indicated to the left of the figure. A GTP-labeled small G-protein co-immunoprecipitated with the transfected human wild type PDGF type- receptor (lane3). In contrast, only minimal amounts of labeled small G-proteins co-immunoprecipitate from lysates of the parental cell line (lane2) or from the cell line expressing the mutant kinase-negative receptor, L635R (lane4).



To determine whether the G-protein(s)/PDGF type- receptor interaction was dependent on the expression of a functionally-active PDGF type- receptor, co-immmunoprecipitation experiments were carried out in TRMP cells transfected with human wild type or with a kinase-negative mutant PDGF type- receptor gene. In the mutant receptor, a single point mutation in the ATP-binding site (L635R) inhibits the ligand-induced autophosphorylation of the PDGF type- receptor(33) . In cells stimulated with PDGF-BB, much lower levels of a labeled small G-protein were associated with the kinase-negative PDGF type- receptor, as compared with the wild type human PDGF type- receptor (Fig. 3B, compare lane4 with lane3). Note that in wild type TRMP cells, which lack PDGF type- receptors (but express low levels of PDGF type- receptors),() the anti-PDGF type- receptor antibody co-immunoprecipitated a similar amount of small G-protein as in the PDGF type- receptor kinase mutant-containing cells (Fig. 3B, compare lane2 with lane4). Taken together, these results support the hypothesis that a functionally-active PDGF type- receptor is required for ligand-stimulated G-protein binding to the receptor.

Efficient detection of small G-proteins by the in situ GTP-labeling technique depends largely on the ability to exchange radiolabeled nucleotide for endogenous nucleotide under physiological conditions in semi-intact cells. This process requires an extended incubation time with radiolabeled GTP prior to chemical cross-linking. It was therefore necessary to determine the functional status of the stimulated PDGF type- receptor at the time when the cross-linking occurred. The autophosphorylation capability of the PDGF type- receptor was assessed in cells that were stimulated with PDGF-BB in vivo for 2 min, washed, and kept permeabilized at 37 °C for 30 min before performing the GTP labeling. As shown in Fig. 4(panelA), the PDGF type- receptor was tyrosine-phosphorylated after PDGF-BB-stimulated cells were GTP-labeled, whereas in unstimulated cells the PDGF type- receptor remained largely unphosphorylated on tyrosine residues. As expected, there was an increase in the amount of labeled small G-protein associated with the PDGF type- receptor upon PDGF-BB stimulation (Fig. 4, panelB), which closely correlated with the PDGF-BB-induced autophosphorylation of the PDGF type- receptor.


Figure 4: Ligand-stimulated autophosphorylation of PDGF type- receptor in GTP-labeled cells. Serum-starved Balb cells were stimulated in vivo with PDGF-BB (lane3), or left untreated (lanes1 and 2), permeabilized with LPC, and labeled with [-P]GTP, as described in Fig. 1A. Precleared lysates were immunoprecipitated with an anti-PDGF type- receptor antibody (lanes2 and 3), as described in Fig. 3. Lane1 shows the third preclearing with NRS. The eluted samples were separated on a 5-15% gradient SDS-PAGE gel. The gel was transferred to a nitrocellulose filter. PanelA, the upper part of the nitrocellulose filter was immunoblotted with 4G10 anti-phosphotyrosine antibody (Wb:-pTyr). The PDGF type- receptor was phosphorylated on tyrosine upon ligand binding. PanelB, the lower part of the nitrocellulose filter was exposed to XAR5 Kodak film. An increase in the amount of the labeled G-protein associated with the PDGF type- receptor was observed after PDGF-BB stimulation. Molecular mass markers are shown at right.



Identification of the G-protein Associated with the PDGF Type- Receptor

Analysis by Two-dimensional Protein Separation

Members of the family of Ras-like small G-proteins possess a high degree of homology (40, 41) . A combination of the [-P]GTP labeling technique and two-dimensional gel electrophoresis has been used to identify small G-proteins in different tissues(29) . This technique was used here to identify the small G-protein associated with the PDGF type- receptor. Serum-starved Balb cells that had been stimulated for 2 min with PDGF-BB were permeabilized and GTP-labeled as described. The electrophoretic mobilities of the immunoprecipitated GTP-labeled proteins indicated that they migrated at a pI range between 5 and 5.6, which appears to be a slightly more acidic range than for native forms of these proteins (Fig. 5). This finding has also been observed by Huber and Peter(29) . A comparison of relative migration patterns (Fig. 5, panels A-D), indicated that the PDGF type- receptor-associated small G-protein had a two-dimensional profile (panelA) strikingly similar to that of the Rho family of small G-proteins (panelC). Only some spots of immunoprecipitated Rap1A/B (panelB) migrated at similar positions as those of the small G-protein co-immunoprecipitated by the anti-PDGF receptor antibody. In contrast, two-dimensional electrophoretic profile of the p21 protein (panelD) was significantly different from that of the PDGF type- receptor-associated small G-protein. These results suggest that the PDGF receptor-associated small G-protein is closely related to the Rho family of proteins.


Figure 5: 2-Dimensional gel analysis of the GTP-labeled small G-proteins immunoprecipitating with the PDGF type- receptor. Balb cells were treated as described in Fig. 1. The cells were stimulated in vivo with PDGF-BBh (30 ng/ml) for 2 min at 37 °C. Permeabilization and GTP labeling were carried out as described. Postnuclear lysis proteins were precleared three times with NRS conjugated to protein G-Sepharose before the antibody-specific immunoprecipitations. Aliquots of the same lysates were immunoprecipitated with: anti-PDGF type- receptor (A), anti-Rap1A/B (B), anti-RhoB (C), and anti-p21 Y13-259 (D). Immunoprecipitated proteins were subjected to two-dimensional IEF/followed by a 15% SDS-PAGE. Autoradiograms of the dried gels are shown here. The positions of molecular mass markers are indicated to the left of the figures. The dottedcircles in the figure represent the migration of the ovalbumin two-dimensional-IEF marker (45 kDa and a pI of 5.1). The pH gradient after electrophoresis was linear from 4.5 to 6.5.



Proteolytic Cleavage Analysis

To further identify the small G-protein associated with the PDGF type- receptor, [-P]GTP-labeled proteins co-immunoprecipitating with the receptor were digested with S. aureus endoproteinase Glu-C (V8 protease), and the resulting peptides were separated by a high resolution Tris-Tricine SDS-PAGE system and then transferred to an Immobilon-PVDF membrane, as described above (Fig. 6A). Three [P]GTP-labeled, well separated, and PDGF type- receptor-associated peptides were detected (Fig. 6A, lane2). For comparison, a number of known, affinity-purified small [-P]GTP-labeled G-proteins were also subjected to V8 protease digestion, and the digestion products analyzed. The digestion patterns of GTP-labeled RhoA (lane4), and of PDGF-type- receptor-associated small G-protein (lane2) were almost identical. The digestion pattern of RhoB protein (lane5) was also very similar to RhoA (lane4). Upon V8 protease digestion, labeled Rap1A/B protein (lane3) was converted to three peptides with similar mobilities to the ones observed in the digestion of the PDGF type- receptor-associated G-protein, plus an extra lower band that was unique and was not present in digests of any of the other proteins. V8 protease digestion of the p21 protein (lane6) gave a fragment with a quite distinct mobility. Quantitative analysis of the radioactivity on the Immobilon filter is shown in Fig. 6B. The analysis of these proteolytic cleavage studies further demonstrated that the small G-protein associated with the PDGF type- receptor belongs to the Rho family of small G-proteins.


Figure 6: Proteolytic cleavage of immunoprecipitated [P] GTP--labeled small G-proteins. A, [-P]GTP-labeled small G-proteins from Balb cells were immunoprecipitated with anti-PDGF type- receptor antibody or with specific anti-small G-protein antibodies, as described. Immunoprecipitates were digested with S. aureus proteinase Glu-C V8. A small aliquot of uncleaved immunoprecipitated PDGF receptor-associated small G-protein is shown in lane1. The V8-cleaved G proteins immunoprecipitating with anti-PDGF receptor, anti-Rap1A/B, anti-RhoA, anti-RhoB, and anti-Ras are shown in lanes 2-6, respectively. The samples were separated in a high resolution Tris-Tricine gel (16.5% T, 3% C). The gel was transferred to an Immobilon-PVDF membrane. The membrane was exposed for 1 day with an enhancing screen at -70 °C. The positions of low molecular mass markers are indicated to the left of the figures. B, InstantImager analysis of the membrane shown in panelA. The membrane was counted for 18 h in an InstantImager. The figure shows a profile with the quantitation of the radioactivity throughout each lane containing the V8-digested peptides. The analysis was done by counting the radioactivity through a rectangle comprising each whole lane of the gel. The width of each rectangle was kept constant for all the lanes. The profile is presented in counts, in arbitrary units. These experiments were repeated at least four times.



The Small G-protein Co-immunoprecipitated by the Anti-PDGF Type- Receptor Antibody Is a Substrate for the Exoenzyme C3 Transferase

The association of a Rho small G-protein with the PDGF type- receptor was confirmed by an alternative strategy. In these experiments, purified recombinant C3 transferase was utilized to ADP-ribosylate Rho proteins in Balb cell membranes. The exoenzyme C3 transferase from C. botulinum specifically ADP-ribosylates Rho proteins on amino acid Asn-41, which is located in the putative effector domain(42, 43) . The members of Rho subfamily are the major substrates for the ADP-ribosylation by C. botulinum ADP-ribosyltransferase C3 exoenzyme. In contrast, the members of the Rac subfamily are very poor substrates for the C3 transferase(44, 45, 46) .

As expected, Balb cell membranes were readily [P]ADP-ribosylated, demonstrating the presence of Rho proteins in Balb cells (Fig. 7, panel A, lane2; and panel B, lane1). This was confirmed through direct immunoprecipitation of RhoA and RhoB by specific antibodies (Fig. 7B, lanes3 and 4). As suggested by the amount of radioactivity incorporated, RhoA was the predominant C3 substrate in Balb membranes (lane3). An ADP-ribosylated p21-protein was co-immunoprecipitated by the anti-PDGF type- receptor antibody. Quantitative analysis indicated 3-fold more radioactivity in anti-PDGF type- receptor immunoprecipitates than in the anti-p21 or NRS immunoprecipitates used as negative controls (Fig. 7B, compare lane2 with lanes5 and 6). Moreover, the amount of [P]-ADP-ribosylated protein in PDGF type- receptor immunoprecipitates represented 10-25% of the [P]ADP-ribosylated proteins immunoprecipitated by the anti-RhoA and anti-RhoB antibodies, respectively.


Figure 7: C3-ADP-ribosylation of Balb cell membrane proteins. Purified recombinant C3 transferase was utilized to ADP-ribosylate Rho G-proteins in Balb cell membranes. PanelA shows the [P]ADP-ribosylated protein in presence (+) or in absence(-) of 6-7 µg of C3 transferase/ml. In panelB, membranes (100 µg of protein/reaction) were prepared from serum-starved Balb cells that had been stimulated for 2 min with PDGF-BB. The ribosylation reactions were stopped by lysing the membranes with HTG buffer, as described under ``Materials and Methods.'' The precleared lysates were independently immunoprecipitated with anti-PDGF type- receptor or with several specific anti-small G-protein antibodies: anti-PDGF type- receptor (lane2), anti-RhoA (lane3), anti-RhoB (lane4), anti-Ras (lane5), and NRS (lane6). Lane1 shows the ribosylated material in 20 µg of membrane protein. An ADP-ribosylated small G-protein was co-immunoprecipitated by the anti-PDGF type- receptor antibody (lane2). ADP-ribosylated RhoA is the predominant C3 substrate in Balb cell membranes (lane3), followed by RhoB (lane4), which migrated as a double band. p21 protein was not ribosylated (lane5).



These results, together with the data on V8 digestion and two-dimensional IEF/SDS-PAGE analysis, confirmed that a small G protein of the Rho family is associated with the PDGF-BB-stimulated PDGF type- receptor in Balb fibroblasts.


DISCUSSION

Ras proteins and related small GTPases play critical roles in the control of normal and transformed cell growth(47, 48, 49) . The Ras superfamily of GTPases contains over 60 small (20-27-kDa) guanine-nucleotide binding proteins that are involved in the control of a wide variety of cellular functions(50) . Based on their sequences, Ras-like proteins fall into five main families: Ras, Rho, Rab, Ran, and Arf(40, 41) , each of whose members have similar structural characteristics, with very distinct functions(46, 48, 50, 51) .

We report here that small GTP-binding proteins bind specifically to the murine and human PDGF type- receptor in response to PDGF-BB stimulation. Two-dimensional electrophoresis and proteolytic cleavage analysis indicated that the small G-protein associated with the PDGF type- receptor is a member of the Rho family of G-proteins. These results were confirmed by demonstrating that the small G-protein co-immunoprecipitated by the anti-PDGF receptor antibody was a substrate for the ADP-ribosyltransferase C3 exoenzyme, which specifically ADP-ribosylates Rho proteins(45) . Thus, the PDGF type- receptor may form a complex with small G-proteins upon binding PDGF-BB, and the Rho G-protein is likely to be an important component of the proteins making up the multimeric signaling complex of the PDGF type- receptor.

Our results suggest that the association of the Rho small G-protein with the PDGF type- receptor is of sufficient affinity as to allow co-immunoprecipitation with an unstimulated receptor. Thus, the association appears to differ substantially from the chain of adaptor proteins involved in the transmission of signals from the PDGF type- receptor to p21. There was a significant increase in the amount of GTP-labeled Rho small G-protein associated with the PDGF type- receptor upon ligand binding, and this increase correlated with PDGF type- receptor tyrosine autophosphorylation. This agonist-induced association also appeared to require a ``functional'' receptor. The Rho protein did not undergo ligand-induced association with a mutant receptor protein that was unable to bind ATP. This mutant was tyrosine kinase-negative, but this result does not necessarily imply that receptor tyrosine kinase activity is required for Rho association. In addition to being kinase-deficient, this same mutant receptor does not undergo conformation changes or dimerization in response to PDGF-BB binding, and either of these two events, rather than tyrosine kinase activity, may instead be required for Rho association.

The Rho family of small G-proteins consists of RhoA, RhoB, RhoC, Rac, Cdc42Hs, and TC10. Proteins of this subgroup are involved in organization of the cytoskeleton. Rac is required for membrane ruffling induced by growth factors (a result of actin reorganization at the plasma membrane)(51) , and the subsequent formation of actin stress fibers requires Rho(46, 52) . PDGF and epidermal growth factors have been shown to induce cytoskeletal changes in several cell types. Studies carried out by microinjection of purified Rho and Rac proteins indicated that there is a sequential involvement of the Rac followed by the Rho small G-proteins in these cellular responses(51) .

PDGF and other growth factors presumably induce Rho-dependent responses by increasing the level of GTP-bound Rho in cells, and this could be achieved either by activating proteins that stimulate nucleotide exchange on Rho, or by inhibiting proteins that enhance GTP hydrolysis, such as RhoGAP (GTPase-Activating Proteins)(53, 54) . Several proteins have been identified as candidates for GAPs, among them the cellular protein p190, which forms a stable complex with Ras-GAP (the upstream/downstream effector of Ras)(55) . This is of particular interest, raising the possibility that the Ras and Rho signaling pathways are coordinately controlled(56) . One of the exchange factors for the Rho family is the human oncoprotein Dbl, which catalyzes nucleotide exchange on Cdc42Hs (57) and on Rho(53) . A family of proteins with a Dbl-like domain has been identified(53) . Although some of them form in vitro complexes with several members of the Rho family of proteins, many of them showed no exchange activity on Rho(58) . Whether these proteins provide a link between growth factors receptors and Rho-like GTPase remains to be determined. Another potential guanine dissociation stimulator (GDS) has been identified for Rho proteins, smgGDS (small GTP-binding protein GDS)(59) , but smgGDS appears not to be completely specific for the Rho subfamily, since it is also active on Ki-Ras and Rap1(60) .

The activation of the Rho subfamily of G-proteins differs significantly with respect to rates of GTP exchange and hydrolysis. Rho, like Ras, has a slow intrinsic rate of hydrolysis(61) , but Rac and Cdc42 have much shorter half-lives for bound GTP. Conversely, nucleotide exchange is much slower for Rac than Ras(44, 62) . The increase in GTP labeling of the receptor-associated Rho protein that we observe after ligand binding to the PDGF type- receptor may reflect a change in the rate of GTP-GDP exchange rate, with a resulting change in the ability of the Rho to be cross-linked by the GTP-labeling technique, as well as a change in the amount of Rho protein bound. This alternative possibility for PDGF-induced signaling through Rho is under investigation.

What downstream events might be activated through Rho association with the PDGF type- receptor? Biochemical and genetic data implicate p21 in growth factor-stimulated pathways involving tyrosine and serine/threonine kinases. One of the targets or mediators of p21 in these cascades appears to be Raf-1(63, 64, 65, 66, 67, 68, 69, 70, 71, 72) . The biochemical targets of the Rho family of small G-proteins, however, remain largely uncharacterized. There is now evidence that a genistein-sensitive tyrosine kinase is required in the Rho-mediated signal transduction pathways involved in thrombin-induced changes in cytoskeleton(45) . It has been shown in Swiss-3T3 cells and in human platelet cytosolic extracts that activation of phosphatidylinositol 3-kinase is dependent on Rho(73, 74) . In addition, in rat liver membranes, RhoA protein has a role in the activation of the membrane-associated phospholipase D(75) , although no evidence exists for a direct interaction between phospholipase D and Rho proteins. Recently, others have identified a serine/threonine kinase, STE 20, as a target of Cdc42 and Rac1(76) . This in vitro study demonstrated that this kinase complexes specifically with the activated (or GTP-bound) p21, inhibiting p21GTPase activity and leading to kinase autophosphorylation and activation. The resulting autophosphorylated kinase had a decreased activity for Cdc42/Rac1, freeing the p21 for further stimulatory activities or for down-regulation by GTPase-activating proteins. Thus, there may be as-yet-unexplored regulatory interactions between the PDGF type- receptor and the Rho small G-protein family, analogous to the link between the PDGF type- receptor and p21.


FOOTNOTES

*
This work was supported by National Cancer Institute Grant R01-CA50459 and a grant from the Council for Tobacco Research (to D. V. F.) and by Grant PB92-0123 from the Dirección General de Investigatión Cientifica y Técnica (Spain) (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Cancer Research Center, E-124, Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-4173; Fax: 617-638-4176.

The abbreviations used are: PDGF, platelet-derived growth factor; PBS, phosphate-buffered saline; IEF, isolectric focusing; PAGE, polyacrylamide gel electrophoresis; PDGF-BBh, human recombinant platelet-derived growth factor-BB; mAb, monoclonal antibody; NRS, normal rabbit serum; LPC, L--lysophosphatidylcholine; PVDF, polyvinylidene difluoride; GDS, guanine dissociation stimulator; Tricine, N-tris(hydroxymethyl)methylglycine.

A. Kazlauskas, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. T. Daniels for generously providing antisera and Drs. A. Kazlauskas and J. Cooper for generously providing the TRMP cells and the PDGF-receptor mutants.


REFERENCES
  1. Bowen-Pope, D. F., Hart, C. F., and Seifert, R. A.(1989)J. Biol. Chem. 264, 2502-2508 [Abstract/Free Full Text]
  2. Claesson-Welch, L., Eriksson, A., Moren, A., Severinsson, L., Ek, B., Ostman, A., Betcholtz, C., and Heldin, C. H.(1988)Mol. Cell. Biol. 8, 3476-3486 [Medline] [Order article via Infotrieve]
  3. Escobedo, J. A., Keating, M. T., Ives, H. E., and Williams, L. T.(1988)J. Biol. Chem. 263, 1482-1487 [Abstract/Free Full Text]
  4. Seifert, R. A., Hart, C. E., Phillips, P. E., Forstrom, J. W., Ross, R., Murray, M. J., and Bowen-Pope, D. F.(1989)J. Biol. Chem. 264, 8771-8778 [Abstract/Free Full Text]
  5. Matsui, T., Pierce, J. H., Fleming, T. P., Greenberger, J. S., Larochelle, W. J., Ruggiero, M., and Aaronson, S. T.(1989)Proc. Natl. Acad. Sci. U. S. A. 89, 8314-8318
  6. Yarden, Y., Escobedo, J. A., Kuang, W., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried, V. A., Ullrich, M. A., and Williams, L. T. (1986) Nature323, 226-232 [Medline] [Order article via Infotrieve]
  7. Williams, L. T. (1989)Science 243, 1564-1570 [Medline] [Order article via Infotrieve]
  8. Heldin, C.-H., Ernlund, A., Rorsman, C., and Ronnstrand, L.(1989)J. Biol. Chem. 264, 8905-8912 [Abstract/Free Full Text]
  9. Koch, C. A., Anderson, D., Moran, M. F., Ellis, C., and Pawson, T.(1991) Science 252, 668-674 [Medline] [Order article via Infotrieve]
  10. Mayer, B. J., Ron, R., Clark, K. L., and Baltimore, D.(1993)Cell 73, 629-630 [Medline] [Order article via Infotrieve]
  11. Pawson, T., and Gish, G. D.(1992)Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  12. Li, W., Nishimura, R., Kashishian, A., Batzer, A. G., Kim, W. J. H., Cooper, J. A., and Schlessinger, J.(1994)Mol. Cell. Biol. 14, 509-517 [Abstract]
  13. Case, R. D., Piccione, E., Wolf, G., Benett, A. M., Lechleider, R. J., Neel, B. G., and Shoelson, S. E.(1994)J. Biol. Chem. 269, 10467-10474 [Abstract/Free Full Text]
  14. Meisenhelder, J., Suh, P.-G., Rhee, S. G., and Hunter, T.(1989)Cell 57, 1109-1122 [Medline] [Order article via Infotrieve]
  15. Morrison, D. K., Kaplan, D. R., Escobedo, J. A., Rapp, U. R., Roberts, T. M., and Williams, L. T.(1989)Cell 58, 649-657 [Medline] [Order article via Infotrieve]
  16. Kumjian, D. A., Wahl, M. I., Rhee, S. G., and Daniel, T. O.(1989)Proc. Natl. Acad. Sci. U. S. A. 89, 8232-8236
  17. Wahl, M. I., Olashaw, N. E., Nishibe, S., Rhee, S. G., Pledger, W. J., and Carpenter, G. (1989)Mol. Cell. Biol. 9, 2934-2943 [Medline] [Order article via Infotrieve]
  18. Mundschau, L. J., Forman, L. W., Weng, H., and Faller, D. V.(1994)J. Biol. Chem. 269, 16137-16142 [Abstract/Free Full Text]
  19. Quinones, M. A., Mundschau, L. J., Rake, J. B., and Faller, D. V.(1991) J. Biol. Chem. 266, 14055-14063 [Abstract/Free Full Text]
  20. Rake, J. B., Quinones, M. A., and Faller, D. V.(1991)J. Biol. Chem. 266, 5348-5352 [Abstract/Free Full Text]
  21. Olivier, J. P., Raabe, T., Henkemeyer, M., Dickson, B., Mbamalu, G., Margolis, B., Schlessinger, J., Hafen, E., and Pawson, T.(1993) Cell 73, 179-191 [Medline] [Order article via Infotrieve]
  22. Buday, L., and Downward, J.(1993)Cell 73, 611-620 [Medline] [Order article via Infotrieve]
  23. Li, B. Q., Subleski, M., Shalloway, D., Kung, H. F., and Kamata, T.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8504-8508 [Abstract/Free Full Text]
  24. Egan, S. E., Giddings, B. W., Brooks, M. W., Buday, L., Sizeland, A. M., and Weinberg, R. A. (1993)Nature 363, 15-16 [CrossRef][Medline] [Order article via Infotrieve]
  25. Wu, J., Dent, P., Jelinek, T., Wolfman, A., Weber, M. J., and Sturgill, T. W. (1993)Science 262, 1065-1069 [Medline] [Order article via Infotrieve]
  26. Baltensperger, K., Kozma, L. M., Cherniack, A. D., Klarlund, J. K., Chawla, A., Banerjee, U., and Czech, M. P.(1993)Science 260, 1950-1952 [Medline] [Order article via Infotrieve]
  27. Peter, M. E., She, J., Huber, L. A., and Terhorst, C.(1993)Anal. Biochem. 210, 77-85 [CrossRef][Medline] [Order article via Infotrieve]
  28. Low, A., Sprinzl, M., and Faulhammer, H. G.(1993)Eur. J. Biochem. 215, 473-479 [Abstract]
  29. Huber, L. A., and Peter, M. E.(1994)Electrophoresis 15, 283-288 [Medline] [Order article via Infotrieve]
  30. Peter, M. E., Wileman, T., and Terhorst, C.(1993)Eur. J. Biochem. 23, 461-466
  31. Peter, M. E., Hall, C., Ruhlmann, A., Sancho, J., and Terhorst, C.(1992)EMBO J. 11, 933-941 [Abstract]
  32. Zullo, J. N., and Faller, D. V.(1988)Mol. Cell. Biol. 8, 5080-5085 [Medline] [Order article via Infotrieve]
  33. Kazlauskas, A., and Cooper, J. A.(1989)Cell 58, 1121-1133 [Medline] [Order article via Infotrieve]
  34. Downward, J., Graves, J. D., Warne, P. H., Rayter, S., and Cantrell, D. A.(1990) Nature 346, 719-723 [CrossRef][Medline] [Order article via Infotrieve]
  35. Jones, P. P. (1984)Methods Enzymol. 108, 452-466 [Medline] [Order article via Infotrieve]
  36. Schagger, H., and von Jagow, G.(1987)Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  37. Patterson, S. D., Hess, D., Yungwirth, T., and Aebersold, R.(1992)Anal. Biochem. 202, 193-203 [Medline] [Order article via Infotrieve]
  38. Cui, Z., Zubiaur, M., Bloch, D. B., Michel, T., Seidman, J. G., and Neer, E.(1991) J. Biol. Chem. 266, 20276-20282 [Abstract/Free Full Text]
  39. Wieland, T., Ulibarri, I., Aktories, K., Gierschik, P., and Jakobs, K. H.(1990) FEBS Lett. 263, 195-198 [CrossRef][Medline] [Order article via Infotrieve]
  40. Valencia, A., Chardin, P., Wittinghofer, A., and Sander, C.(1991) Biochemistry 30, 4637-4347 [Medline] [Order article via Infotrieve]
  41. Kahn, R. A., Der, C. J., and Bokoch, G. M.(1992)FASEB J. 6, 2512-2513 [Free Full Text]
  42. Aktories, K., Braun, U., Rosener, S., Just, I., and Hall, A.(1989)Biochem. Biophys. Res. Commun. 158, 209-213 [Medline] [Order article via Infotrieve]
  43. Sekine, A., Fujiwara, M., and Narumiya, S.(1989)J. Biol. Chem. 264, 8602-8605 [Abstract/Free Full Text]
  44. Menard, L., Tomhave, E., Casey, P., Uhing, R. J., Snyderman, R., and Didsbury, J. R. (1992)Eur. J. Biochem. 206, 537-546 [Abstract]
  45. Ridley, A. J., and Hall, A.(1994)EMBO J. 13, 2600-2310 [Abstract]
  46. Ridley, A. J., and Hall, A.(1992)Cell 70, 389-399 [Medline] [Order article via Infotrieve]
  47. Barbacid, M. (1987)Annu. Rev. Biochem. 56, 779-827 [CrossRef][Medline] [Order article via Infotrieve]
  48. Bourne, H. R., Sanders, D. A., and McCormick, F.(1990)Nature 348, 125-131 [CrossRef][Medline] [Order article via Infotrieve]
  49. Bourne, H. R., Wrischnik, L., and Kenyon, C.(1990)Nature 348, 678-679 [Medline] [Order article via Infotrieve]
  50. Lacal, J. C., and McCormick, F. (1993) The Ras Superfamily of GTPases, CRC Press, London
  51. Ridley, A. J., Paterson, H. F., Johnston, C. L., Dickmann, D., and Hall, A. (1992)Cell 70, 401-410 [Medline] [Order article via Infotrieve]
  52. Hall, A.(1994) Annu. Rev. Cell Biol. 10, 31-54 [CrossRef]
  53. Boguski, M. S., and McCormick, F.(1993)Nature 366, 643-654 [CrossRef][Medline] [Order article via Infotrieve]
  54. Homma, Y., and Emori, Y. (1995)EMBO J. 14, 286-291 [Abstract]
  55. Settleman, J., Albright, C. F., Foster, L. C., and Weinberg, R. A.(1992) Nature 359, 153-154 [CrossRef][Medline] [Order article via Infotrieve]
  56. McGlade, J., Brunkhorst, B., Anderson, D., Mbamalu, G., Settleman, J., Dedhar, S., Rozakis-Adcock, M., Chen, L. B., and Pawson, T.(1993) EMBO J. 12, 3073-3081 [Abstract]
  57. Hart, M. J., Eva, A., Evans, T., Aaronson, S. A., and Cerione, R. A.(1991) Nature 354, 311-314 [CrossRef][Medline] [Order article via Infotrieve]
  58. Gulbins, E., Coggeshall, K. M., Baier, G., Katzav, S., Burn, P., and Altman, A.(1993) Science 260, 822-825 [Medline] [Order article via Infotrieve]
  59. Ueda, T., Kikuchi, A., Ohga, N., Yamamoto, J., and Takai, Y.(1990)J. Biol. Chem. 265, 9373-9380 [Abstract/Free Full Text]
  60. Kikuchi, A., Kuroda, S., Sasaki, T., Kotani, K., and Hirata, K.(1992)J. Biol. Chem. 267, 14611-14615 [Abstract/Free Full Text]
  61. Tsai, M., Hall, A., and Stacey, D. W.(1989)Mol. Cel. Biol. 9, 5260-5264 [Medline] [Order article via Infotrieve]
  62. Hart, M. J., Maru, Y., Leonard, D., Witte, O. N., Evans, T., and Cerione, R. A.(1992) Science 258, 812-815 [Medline] [Order article via Infotrieve]
  63. Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A.(1993) Science 260, 1658-1661 [Medline] [Order article via Infotrieve]
  64. Zhang, X. F., Settleman, J., Kyriakis, J. M., Takeuchisuzuki, E., Elledge, S. J., Marshall, M. S., Bruder, J. T., Rapp, U. R., and Avruch, J.(1993) Nature 364, 308-313 [CrossRef][Medline] [Order article via Infotrieve]
  65. Warne, P. H., Viciana, P. R., and Downward, J.(1993)Nature 364, 352-355 [CrossRef][Medline] [Order article via Infotrieve]
  66. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A.(1993)Cell 74, 205-214 [Medline] [Order article via Infotrieve]
  67. Koide, H., Satoh, T., Nakafuku, M., and Kaziro, Y.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 8683-8686 [Abstract/Free Full Text]
  68. Cai, H., Erhardt, P., Troppmair, J., Diaz-Meco, M. T., Sithanandam, G., Rapp, U. R., Moscat, J., and Cooper, G. M.(1993)Mol. Cell. Biol. 13, 7645-7651 [Abstract]
  69. Hallberg, B., Rayter, S. I., and Downward, J.(1994)J. Biol. Chem. 269, 3913-3916 [Abstract/Free Full Text]
  70. Moodie, S. A., and Wolfman, A.(1994)Trends Genet. 10, 44-48 [CrossRef][Medline] [Order article via Infotrieve]
  71. Ahn, N. G., Seger, R., and Krebs, E. G.(1992)Curr. Opin. Cell Biol. 4, 992-999 [Medline] [Order article via Infotrieve]
  72. Ahn, N. G., Weiel, J. E., Chan, C. P., and Krebs, E. G.(1990)J. Biol. Chem. 265, 11487-11494 [Abstract/Free Full Text]
  73. Zhang, J., King, W. G., Dillon, S., Hall, A., Feig, L., and Rittenhouse, S. E.(1993) J. Biol. Chem. 268, 22251-22254 [Abstract/Free Full Text]
  74. Kumagi, N., Morii, N., Fujisawa, K., Nemoto, Y., and Narumiya, S.(1993)J. Biol. Chem. 268, 24535-24538 [Abstract/Free Full Text]
  75. Malcolm, K. C., Ross, A. H., Qiu, R., Symons, M., and Exton, J. H.(1994)J. Biol. Chem. 269, 25951-25954 [Abstract/Free Full Text]
  76. Manser, E., Leung, T., Salihuddin, H., Zhao, Z., and Lim, L.(1994)Nature 367, 40-44 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.