INTRODUCTION

The insulin-like growth factor-I (IGF-I)¹ receptor, a transmembrane heterotetrameric tyrosine kinase, is expressed by most normal and transformed cells (1). Upon ligand binding, IGF-I receptor undergoes autophosphorylation on intracellular tyrosine residues and activation of its intrinsic tyrosine kinase. Mice without the IGF-I receptor gene invariably die of respiratory failure at birth and exhibit severe intrauterine growth retardation, general organ hypoplasia, and delayed ossification, demonstrating the essential role of the IGF-I receptor in normal growth and development (1-3). Overexpression of the IGF-I receptor can transform mouse fibroblasts (4). The presence of the IGF-I receptor was found to be necessary for proteins such as SV40 large T antigen, Ha-Ras, EWS/FLI-1 (a fusion protein produced by Ewing's family of tumors), v-Src, and bovine papilloma virus to transform target cells (5-8). Furthermore, decreasing the number of IGF-I receptors by antisense RNA technology causes a reversal of the transforming phenotype, including arrest of in vivo tumor growth (7), suggesting that the IGF-I receptor is also essential for oncogene-induced cellular transformation.

Heterotrimeric (, , and  subunits) guanine nucleotide-binding proteins (G proteins) couple to hundreds of different receptors in mammals and are central to the signaling processes of multicellular organisms (9). The  subunits of G proteins belong to a large group of GTPases, which are classified into four sub-families based on amino acid homology: G_s, G_i/o, G_q/11, and G_12/13 (10). Stimulatory (G_s) and inhibitory (G_i) G protein subtypes have been implicated in the regulation of adenylyl cyclase and the gating of certain ion channels (11). The G_q family of proteins couple to the phospholipase C pathway (12). The ubiquitously expressed G_12/13 proteins are involved in mitogenesis and transformation probably through activation of c-Jun N-terminal kinase (JNK) and activation or inhibition of mitogen-activated protein kinase (MAPK) pathways (13-18). Overexpression of wild type G_12/13 and the expression of GTPase-deficient active mutants of G_12/13 transform fibroblast cell lines (15, 18-22).

To test the hypothesis that IGF-I receptor is essential for cellular transformation, we stably transfected a GTPase-deficient mutant human G₁₃ (Q226L) into R fibroblasts derived from IGF-I receptor-deficient mice. These cells cannot be transformed by SV40 large T antigen and other oncoproteins (5-8). In contrast, we found that activated human G₁₃ (hG₁₃₎ is able to transform R cells independent of the IGF-I receptor and that overexpression of both G₁₃ and IGF-I receptor caused synergistic enhancement in cellular transformation.

EXPERIMENTAL PROCEDURES

Mutant Human G₁₃ Expression Vector

A GTPase-deficient mutation was introduced into hG₁₃ cDNA (Ref. 14; GenBank^TM accession number L22075) by polymerase chain reactions. The codon corresponding to glutamine (Q) at residue 226 was mutated to express leucine (L) and thereby generated a new restriction site Bsu361. The mutant cDNA (BglII-EcoRI) was then subcloned into the vector pcDNAIIIHA (Invitrogen, Carlsbad, CA). The resulting construct pcDNAIIIHAG13QL(A) (276) contains the mutant hG₁₃ cDNA (1.5 kb) downstream of a HA tag (derived from influenza protein hemagglutinin) sequence, driven by the human cytomegalovirus promoter, and genes whose products confer resistance to neomycin and ampicillin.

R cells are fibroblasts prepared from IGF-I receptor-deficient mice (5, 23). W6+ cells are R cells overexpressing IGF-I receptors (5, 23). All cells have been cultured in complete medium consists of Dulbecco's modified Essential medium (DMEM; Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and penicillin/streptomycin/fungizone (Biofluids, Rockville, MD).

R cells were transfected using electroporation (6). Geneticin (G418 sulfate, 567 mg/ml, Life Technologies, Inc.)-resistant colonies were selected, expanded, and tested for expression of the mutant hG₁₃ expression.

W6+ cells were transfected using a calcium phosphate mammalian transfection kit (Stratagene, La Jolla, CA) (24) with G₁₃ and pZeoSV (Invitrogen). Transfected cells were selected with complete medium containing zeocin (150 µg/ml, Invitrogen) for 3 weeks.

The expression of the mutant hG₁₃ was evaluated by Northern blot analysis (25). The specific bands of expected sizes were illustrated, together with ethidium bromide staining of ribosomal RNAs, demonstrating equal loading of total RNA on the gel.

The expression of the G₁₃ protein was further confirmed by Western blot analysis using standard procedures (26) and rabbit anti-G₁₃ antiserum (1:1000, Calbiochem, San Diego, CA), which is specific to an 11-amino acid (LHDNLKQLMLQ) C-terminal region of hG₁₃. The level of MAPK activity was determined by using phospho-specific MAPK and p44/42 MAPK antibodies (New England Biolabs, Beverly, MA). Rabbit polyclonal Crk-L (C20) and mouse monoclonal JNK1 (F3) antibodies (Santa Cruz Biotech, Santa Cruz, CA) were also employed in Western blots.

For assays of anchorage-independent growth, suspensions of 1000 cells in 1 ml of DMEM with 10% FBS, 20 mM HEPES, pH 7.5, and 0.2% agarose were lain onto 1.5 ml of 0.4% agarose-containing medium in 35-mm tissue culture dishes (27). Colonies >0.05 mm in diameter were scored after 3 weeks of incubation at 37 C, 5% CO₂ and humidified (90%) atmosphere. Three dishes were plated each time for each clone.

To determine the level of IGF-I receptor expression, displacement binding of ¹²⁵I-IGF-I was done as described previously (28). The Scatchard analysis was performed using Scapre v4.42 and MacScafit v4.94 computer programs (National Institutes of Health, Bethesda, MD).

Confluent cells in 100-mm diameter dishes was incubated with serum-free medium for 20 h and stimulated with 10% FBS for 10 min. Whole cell lysate (1 mg of protein) was immunoprecipitated with 1 µg of JNK1 (C17) antibody and 2.5% protein A-Sepharose (Santa Cruz) overnight at 4 °C, washed twice in ice-cold RIPA buffer (phosphate-buffered saline containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaV₃O₄, 3% aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and washed twice with kinase buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1% Tween 20, 0.1% bovine serum albumin, 0.1 mM NaV₃O₄, and 0.1 mM phenylmethylsulfonyl fluoride). The beads were resuspended in 100 µl of kinase buffer containing 3 µg of GST-Jun (Santa Cruz), 5 µM ATP, and 5 µCi of [-³²P]ATP (NEN Life Science Products). The reactions were terminated after 15 min at room temperature by adding 5 × Laemmli buffer (29). Samples were separated by 9% SDS-PAGE and blotted onto Protran membrane (Scheicher & Schuell). After blocking with 3% bovine serum albumin, the blot was first exposed to a Kodak X-Omat film and then blotted with mouse monoclonal JNK1 antibody (Santa Cruz) to determine the amount of JNK1 present in the reaction.

Unpaired t test was performed with InStat 2.03 computer program (GraphPad Software, San Diago, CA)

RESULTS

Three cell lines were used in transformation studies. Previously, IGF-I receptor gene-deficient R cells have been demonstrated to not express IGF-I receptor using immunoprecipitation, IGF-I-cross-linking, and RNase protection assay, in contrast to wild type mouse fibroblasts (5, 6). Indeed, R cells demonstrate a total lack of binding to ¹²⁵I-IGF-I as determined by Scatchard analysis (data not shown). W6+ cells that overexpress IGF-I receptors demonstrate a level of IGF-I receptor expression (730,000 receptors/cell, K_d of 0.16 nM) comparable with that of NWTb3 (550,000 receptors/cell, K_d of 0.55 nM), which are NIH 3T3 fibroblasts overexpressing the IGF-I receptors (30).

Expression of Human Mutant G₁₃ in IGF-I Receptor-deficient R Cells

To study the role of the IGF-I receptor in G₁₃-induced cellular transformation, we transfected a constitutive active mutant hG₁₃ into R cells. All G418 resistance transfected R clones express a 1.8-kb mRNA hybridized to an antisense hG₁₃ riboprobe (Fig. 1, upper panel). This band is absent in untransfected cells, despite equal amounts of total RNA loaded as shown by staining of the rRNA bands (middle panel). A 6-kb endogenous mouse G₁₃ mRNA has been observed in all clones (data not shown). At the protein level, G₁₃ (wild type and mutant forms) are present mainly as a 40-kDa band (lower panel), which is increased 2-fold upon transfection in all the clones selected (clones 5-26), as analyzed by densitometry.

Expression of G₁₃ mRNA and protein in R cells following electroporation.

13

13

13

13

[View Larger Version of this Image (47K GIF file)]

G₁₃ Is Capable of Transforming IGF-I Receptor-deficient R Cells

G₁₃ transfection caused no morphological change in R cells. The effect of the constitutively active mutant hG₁₃ on cellular transformation was tested using the soft agar assay (Table I). In three experiments each with triplicate dishes, 5 of 6 mutant hG₁₃-expressing clones (except 12) formed significant numbers of colonies (11-50 colonies/1000 cells plated, p < 0.05) as compared with untransfected R cells, which formed only 1-3 tiny colonies as shown in Fig. 2. Cells expressing mutant hG₁₃ protein formed either significant number albeit small colonies (clones 17 and 20) or many colonies comparable in size with the ones seen in NWTb3 control cells (clones 7 and 26) (Table I and Fig. 2). This result suggests that hG₁₃ can transform R cells in the absence of the IGF-I receptor.

Table I. The effect of G13 expression in R and W6+ cells on cell growth in the soft agar assay One thousand cells were plated on soft agar with 10% FBS and allowed to grow for 3 weeks. Colony formation was analysed using a 10× reverse microscope. n indicates numbers of individual plates analyzed. Clones n Colonies (Mean ± SE) NWTb3 9 93  ± 18 R 9 2  ± 0.2 RG13-5 9 11  ± 3a RG13-7 9 50  ± 8a RG13-12 9 6  ± 2 RG13-17 9 12  ± 5a RG13-20 9 35  ± 13a RG13-26 9 16  ± 2a W6+ 3 27  ± 2b W6-G13-3 3 20  ± 2 W6-G13-12 3 15  ± 2 W6-G13-18 3 145  ± 38a W6-G13-19 3 117  ± 9a W6-G13-21 3 153  ± 16a a p < 0.05 versus untransfected R or W6+ cells. b p < 0.001 versus untransfected R cells.

Representative colonies formed on soft agar by R, RG₁₃ clone 7, W6+, and W6-G₁₃ clone 19 cells after 3 weeks of incubation.

[View Larger Version of this Image (86K GIF file)]

Expression of Human Mutant G₁₃ in IGF-I Receptor-overexpressing W6+ Cells

To determine if overexpression of IGF-I receptor can further enhance the transforming ability of hG₁₃, W6+ cells were transfected and analyzed for the expression of hG₁₃ using Northern and Western blot analysis (Fig. 3). Abundant hG₁₃ mRNA is expressed in all the clones transfected with the construct. In transfected W6+ cells, hG₁₃ mRNA is present in two molecular forms (2.2 and 1.8 kb, upper panel); the reason for the two bands is under investigation. Mutant hG₁₃ expression is undetectable in untransfected R and W6+ cells. At the protein level, G₁₃ is present in two molecular forms as well (44 and 40 kDa, lower panel), co-migrating with the positive control sample (lane C). Both forms of G₁₃ are increased at least 2-fold in clones 3, 12, and 19 but to a lesser degree in 18 and 21. 

Expression of G₁₃ mRNA and protein on W6+ cells following calcium phosphate co-transfection using pSV-Zeo.

13

13

13

13

13

[View Larger Version of this Image (72K GIF file)]

IGF-I Receptor and G₁₃ Synergize in Cellular Transformation

The transforming ability of the transfected IGF-I receptor and hG₁₃ was tested using the soft agar assay. As shown in Table I and Fig. 2, overexpression of the IGF-I receptor enabled R cells to form colonies (27 versus 2, p < 0.001). Transfection of W6+ cells with hG₁₃ caused no phenotypical change except a further increase in colony formation (117 to 153, p < 0.05) in 3 of 5 clones analyzed (although without further increase in the colony size formed by W6+ cells, Fig. 2). This effect is much greater than simple addition of the effects of hG₁₃ and IGF-I receptor alone, suggesting a synergism between them.

G₁₃-induced Cellular Transformation Does Not Work through MAPK or JNK

In an attempt to identify signaling pathways involved in G₁₃-activated cellular transformation, MAPK and JNK activities were analyzed. As shown in Fig. 4A, a 10-min incubation with 10% FBS dramatically induced MAPK phosphorylation in both untransfected R and W6+ cells. Although basal level of MAPK phosphorylation was elevated to various degree in clones that form colonies (RG7, RG20, WG18, and WG21), transfection of G₁₃ caused no obvious change in serum-activated MAPK activity. In Fig. 4B, JNK activity was studied by in vitro GST-Jun phosphorylation. As in the case of MAPK, serum incubation-activated JNK activity dramatically in both untransfected cells. With 10% serum, G₁₃ transfection caused no significant change in maximum JNK activation. Only in one clone (RG20, of six tested) G₁₃ increased basal JNK activity. Therefore, we conclude that G₁₃-induced cellular transformation is apparently not accompanied by changes in MAPK and JNK activities.

Effect of G₁₃ transfection on MAPK and JNK activities in R and W6+ cells.

[View Larger Version of this Image (35K GIF file)]

DISCUSSION

In this study, we have confirmed that IGF-I receptor-deficient R fibroblasts are incapable of forming colonies in soft agar and that active mutant G₁₃ is highly oncogenic. The unique finding is that G₁₃ is able to transform fibroblasts independent of IGF-I receptor.

The role of G_12/13, a novel class of G proteins, in signal transduction is under intense investigation. The G_12/13 proteins are known to be coupled to two receptors: thyrotropin receptors in the thyroid gland, which stimulate adenylyl cyclase and phospholipase C pathways (31), and the thrombin receptor in platelets, which stimulates platelet aggregation (32). It is relatively clear that constitutively active G_12/13 activates Ras, which recruits the small G protein Rac, and leads to activation of JNK/stress-activated protein kinase (SAPK) and enhanced transcriptional activity of c-Jun (14, 16). The effect of G_12/13 on MAPK activity is not yet fully elucidated. Although the G_12/13 proteins do not activate MAPK directly, there have been reports that these activated G proteins can enhance epidermal growth factor (EGF)-induced MAPK activation in Rat-1 cells (18) or inhibit EGF-stimulated extracellular signal-regulated kinase expression in COS-7 cells (17). In Swiss 3T3 cells, G_12/13 regulate Rho-dependent responses in actin polymerization (28). Constitutively active mutants of G_12/13 have also been known to activate the Na⁺/H⁺ exchanger (22, 33) and to potentiate serum-stimulated phospholipase A₂ activity but do not stimulate inositol phospholipid hydrolysis (19).

Recent reports by Baserga and co-workers (5, 7, 8, 23, 34-36) have demonstrated that the IGF-I receptor is required for the establishment and maintenance of the transformed phenotype in vivo and in vitro. Indeed, oncogenes including SV40 T antigen and Ha-Ras and overexpressed EGF receptors all failed to transform R cells (5, 8, 23, 34, 35); the resistance of R cells to transformation can be abolished by expressing IGF-I receptors (5, 23). Furthermore, a decrease in the number of IGF-I receptors caused a reversal of the transformed phenotype (7). C6 rat glioblastoma cells failed to grow into tumors in syngeneic rats when antisense IGF-I receptor mRNA is expressed (36). Our finding that R cells can be transformed by G₁₃ in contrast to other oncogenes suggests that IGF-I receptor is not required for all the transformation processes. Nevertheless, G₁₃ is not the only signaling protein capable of transforming R cells. When transfected with constitutively active Ras or Ras plus the SV40 T antigen, R cells were partially transformed (23). Most recently, Valentinis et al. (37) demonstrated that oncoprotein v-Src can also bypass the requirement for a functional IGF-I receptor in transforming mouse fibroblasts. However, c-Src was unable to do so. Taken together, the results of these studies suggested that G₁₃ acts independently of the IGF-I receptor in cellular transformation.

Another interesting observation from this study is that G₁₃ and the overexpressed IGF-I receptor act in concert in transforming R cells. This is a very similar scenario with the reported synergism between G₁₂ and c-Raf-1 in transforming NIH 3T3 cells (38). In that case, the synergism seemed to occur between two parallel pathways: MAPK activated by c-Raf-1 and JNK/SAPK activated by G₁₂. The IGF-I receptor is known to signal through the Ras-Raf-MAPK cascade of serine/threonine and tyrosine kinases. Therefore, our observation of similar synergism may well be explained by cooperation between a G₁₃-activated pathway and overexpressed IGF-I receptor-induced MAPK activation. Analysis of MAPK and JNK activities upon serum stimulation revealed no change caused by mutant G₁₃ expression, although in some clones their basal activities were elevated. Because the cell transformation was studied using 10% serum and the increase in basal kinase activities does not correlate with transforming ability of each clone, we propose that these two pathways are not apparently involved in G₁₃-induced cellular transformation. Investigation is underway to explore the specific targets activated by G₁₃ in R cells.

In summary, we have found that 1) IGF-I receptor-deficient R fibroblasts did not form colonies in soft agar assay; 2) oncogenically active mutant hG₁₃ was able to transform the R cells; 3) overexpression of the IGF-I receptor also enabled R cells to form colonies; 4) the effects on transformation of hG₁₃ and the overexpressed IGF-I receptor were synergistic; and 5) G₁₃-induced cellular transformation does not apparently work through MAPK or JNK pathways. Therefore, G₁₃ induces cellular transformation through pathways apparently independent of the IGF-I receptor. Furthermore, whereas IGF-I receptor is not essential for the transforming phenotype, it can enhance the effect of G₁₃-activated pathways.