Article |
Address correspondence to Kozo Kaibuchi, Dept. of Cell Pharmacology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2074. Fax: 81-52-744-2083. E-mail: kaibuchi{at}med.nagoya-u.ac.jp
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Abstract |
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Key Words: IGF-1; Rho; LARG; PDZ domain; GEF
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Introduction |
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The Rho family GTPases play roles in cytoskeletal rearrangements and in cell adhesion in response to extracellular signals. Rho participates in regulation of various cellular functions such as stress fiber and focal adhesion formation (Ridley and Hall, 1992, 1994), smooth muscle contraction (Hirata et al., 1992; Gong et al., 1996), and membrane ruffling (Nishiyama et al., 1994; Takaishi et al., 1995). Rho has GDP-bound inactive and GTP-bound active forms, which are interconvertible by GDP/GTP exchange and GTPase reactions (Nobes and Hall, 1994). The GTPase reaction is regulated by Rho GTPase-activating proteins. The GDP/GTP exchange reaction is regulated by various GEFs and by inhibitory proteins such as Rho GDI (Fukumoto et al., 1990). GTP-bound Rho exerts its biological functions through interaction with specific effectors (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998; Kaibuchi et al., 1999).
GEFs for the Rho family GTPases share a common sequence motif designated as the Dbl homology (DH) domain (Cerione and Zheng, 1996; Whitehead et al., 1997; Stam and Collard, 1999). In addition to the DH domain, GEFs for the Rho family GTPases contain a nearby pleckstrin homology (PH) domain. Both domains are essential for the GEF activity. Although several GEFs have been identified, the molecular mechanisms by which the activation of GEFs is modulated by extracellular signals are largely unknown except in the case of p115 RhoGEF, PDZ-RhoGEF, and Vav. Studies on heterotrimeric G proteins have clarified the mechanism by which p115 RhoGEF is regulated. p115 RhoGEF has the regulator of G protein signaling (RGS) domain, and the RGS domain of p115 RhoGEF specifically stimulates the intrinsic GTPase activity of G12 or G
13 (Hart et al., 1998; Kozasa et al., 1998). Activated G
13 conversely binds to p115 RhoGEF and stimulates its ability to catalyze nucleotide exchange of Rho. Thus, upon stimulation by extracellular signals the activation of G
13 is thought to be linked to the activation of Rho via p115 RhoGEF. Similarly, it has been reported that the RGS domain of PDZ-RhoGEF (KIAA0380) interacts with activated G
12 or G
13 (Fukuhara et al., 1999). Vav and Vav-2 are other GEFs for the Rho family GTPases (Crespo et al., 1997; Schuebel et al., 1998; Abe et al., 2000). The phosphorylation of Vav and Vav-2 on tyrosine residues leads to the activation of the Rho family GTPases (Crespo et al., 1997; Schuebel et al., 1998). Recently, it has been reported that Vav-2 participates in the hepatocyte growth factor (HGF)stimulated activation of Rho (Kodama et al., 2000).
It has been suggested that the Rho family GTPases participate in the IGF-1 signaling pathway (Thomson et al., 1997; Cheng et al., 2000); however, the mechanism of regulation of the Rho family GTPases by IGF-1 is unknown. In this study, we found that the IGF-1 receptor formed a complex with LARG and that IGF-1 induced the activation of Rho and its effector Rho-associated kinase (Rho-kinase)/ROK/ROCK (Leung et al., 1995; Ishizaki et al., 1996; Matsui et al., 1996) in MDCKII epithelial cells. The IGF-1induced activation of Rho-kinase was inhibited by pretreatment of the cells with Rho-kinase inhibitors and by overexpression of the PDZ and RGS domains of LARG.
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Results |
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Distribution of LARG
To understand the functions of LARG, we produced anti-LARG antibody against 249 amino acids of the COOH-terminal site and performed immunoblot analysis of several rat tissues. LARG was expressed in rat adult brain, lung, spleen, thymus, testis, and ovary but not detectable in heart, kidney, liver, prostate, small intestine, and colon (Fig. 2 A). Interestingly, LARG was expressed most abundantly in the developing brains (Fig. 2 A). Moreover, LARG was highly expressed in various cell lines such as MDCKII, C6 glioma, human gastric cancer TMK1, and human intestinal cancer HT29 cells (Fig. 2 B). LARG appeared as a doublet in rat tissues and cells. When we isolated the LARG cDNA from the human fetal brain cDNA library, we obtained the shorter form of the LARG cDNA, which lacked the middle region (235261 amino acids). The higher band of 172 kD may be an intact LARG, judging from the molecular mass of intact LARG (173,230 D), and the lower band of
170 kD may be an alternatively spliced form of LARG.
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Interaction of LARG with the IGF-1 receptor in vivo
To determine whether LARG interacts with the IGF-1 receptor in vivo, we performed coimmunoprecipitation assay. When the IGF-1 receptor was immunoprecipitated from lysates of MDCKII cells with antiIGF-1 receptor antibody, the molecule corresponding to LARG was coimmunoprecipitated with the IGF-1 receptor (Fig. 3 A, top). LARG was not coimmunoprecipitated with control rabbit IgG. The IGF-1 receptor was also coimmunoprecipitated with LARG (Fig. 3 A, bottom) when LARG was immunoprecipitated with anti-LARG antibody. Similar results were obtained when C6 glioma cells were used instead of MDCKII cells (unpublished data). These results suggest that LARG interacts and forms a complex with the IGF-1 receptor in vivo.
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IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to interact with various signaling molecules. We examined whether the treatment of serum-deprived MDCKII cells with IGF-1 affected the binding state of LARG and the IGF-1 receptor. The amounts of LARG that were coimmunoprecipitated with the IGF-1 receptor were not changed under the conditions in which the phosphorylation of IGF-1 receptor was induced (Fig. 3 C). Under the same conditions, the tyrosine phosphorylated LARG was not detected (Fig. 3 D). Taken together, these results suggest that the interaction of LARG and the IGF-1 receptor is constitutive and does not require the phosphorylation of the IGF-1 receptor.
GEF activity of LARG for the Rho family GTPases
We examined the GEF activity for the Rho family GTPases in vitro. In this assay, we used a GST fusion protein carrying the DH/PH domain of LARG for monitoring the exchange activity toward the Rho family GTPases. The DH/PH domain of LARG enhanced the dissociation of [3H]-labeled GDP from RhoA in a time-dependent manner under the conditions where Dbl stimulated the dissociation of GDP from RhoA (Fig. 4 A). To investigate whether LARG is a specific GEF for Rho, we further examined the effect of the DH/PH domain of LARG on other small GTPases, Rac1, Cdc42, and Ras. The DH/PH domain of LARG showed an exchange activity for RhoA but not for Rac1, Cdc42, or Ras (Fig. 4 B). These results indicate that LARG has the exchange activity for RhoA in vitro but not for Rac1, Cdc42, or Ras.
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Roles of LARG in the IGF-1/Rho signaling pathway
It has been reported that the RGS domain of PDZ-RhoGEF (KIAA0380) inhibits stress fibers formation induced by the heterotrimeric G proteincoupled lysophosphatidic acid receptor (Rümenapp et al., 1999). The RGS domain is thought to be a functional interface for interaction with heterotrimeric G proteins. Here, we showed that the PDZ domain of LARG interacted with the IGF-1 receptor and that IGF-1 activated the Rho signaling pathway. We speculated that the PDZ domain of LARG serves as the dominant negative mutant for the IGF-1induced Rho/Rho-kinase activation. We examined whether the PDZ domain of LARG inhibits the complex formation of LARG with the IGF-1 receptor. The beads, which were coated with GSTIGF-1 receptor ß-subunit, were mixed with endogenous LARG from lysates of MDCKII cells and MBP alone, MBP-LARG-PDZ domain, or AF-6-PDZ domain, and then the amounts of endogenous LARG bound to GSTIGF-1 receptor ß-subunit were measured. Endogenous LARG interacted with GSTIGF-1 receptor ß-subunit but not with GST alone (Fig. 7 A). The amounts of endogenous LARG bound to GSTIGF-1 receptor ß-subunit were reduced by the addition of MBP-LARG-PDZ domain but not by the addition of MBP alone or the PDZ domain of AF-6 (Fig. 7 A). Thus, it is probable that the PDZ domain of LARG can dissociate endogenous LARG from the IGF-1 receptor and serve as the dominant negative form.
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It has been reported that IGF-1 affects actin organization in mammalian cells (Kadowaki et al., 1986; Leventhal et al., 1997). These observations raise the possibility that the Rho/Rho-kinase pathway participates in the IGF-1induced rearrangements of the cytoskeleton. To examine whether IGF-1 affects actin organization in MDCKII cells, MDCKII cells were serum starved and then stimulated with 10 nM IGF-1 for various minutes. Under the serum-starved conditions, 10% of the cells showed thick stress fibers, and the rest of the cells showed thin and weak stress fibers (Fig. 8 A). The stimulation by IGF-1 induced thick stress fibers in
50% of cells within 2060 min (Fig. 8, A and C). The IGF-1induced enhancement of stress fibers appeared 2060 min after stimulation and then returned to basal levels later than
120 min. The PDZ domain of LARG partially inhibited the IGF-1induced enhancement of stress fibers, whereas GST (negative control) or the PDZ domain of AF-6 had no effects (Fig. 8, B and C). The RGS domain of LARG partially inhibited the IGF-1induced enhancement of stress fibers, though the inhibitory effect was weaker than that of the PDZ domain of LARG. Taken together, these results suggest that IGF-1 activates the Rho/Rho-kinase pathway via LARG.
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Discussion |
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It has been reported that RhoGEFs interact with transmembrane proteins or receptor-associated proteins. "Trio" was identified originally as the binding protein of the intracellular domain of leukocyte antigenrelated protein, which is a transmembrane protein tyrosine phosphatase (Debant et al., 1996). The NH2-terminal GEF domain of Trio has exchange activity for Rac1 and RhoG, and the COOH-terminal GEF domain of Trio has exchange activity for RhoA in vitro (Debant et al., 1996; Blangy et al., 2000). To our knowledge, the present results are the first demonstration, indicating the direct binding of RhoGEF to a growth factor receptor.
Roles of LARG in the Rho signaling pathway
LARG has the PDZ, RGS, DH, and PH domains and shows a high degree of sequence similarity to PDZ-RhoGEF (KIAA0380). LARG had the exchange activity for RhoA but not for Rac1, Cdc42, or Ras. The RGS domain of PDZ-RhoGEF interacts with activated G12 or G
13 of heterotrimeric G proteins, and extracellular signals activate Rho via this interaction (Fukuhara et al., 1999). A recent report suggests that the RGS domain of LARG interacts with G
12 or G
13 (Fukuhara et al., 2000). Thus, PDZ-RhoGEF and LARG may be structurally and functionally similar to each other. It is possible that PDZ-RhoGEF also interacts with the IGF-1 receptor, since the PDZ domain of PDZ-RhoGEF shows a high sequence similarity (75% identical) with that of LARG (Kourlas et al., 2000). Indeed, our preliminary experiments suggest that recombinant PDZ-RhoGEF interacts with the IGF-1 receptor in L fibroblasts (unpublished data). Further studies are required for evaluating the involvement of PDZ-RhoGEF in the IGF-1 signaling pathway. It has been reported that PDZ-RhoGEF (KIAA0380) translocates to the plasma membrane via its proline-rich motif COOH-terminally adjacent to its DH/PH domain and induces cortical actin reorganization in Swiss 3T3 fibroblasts (Togashi et al., 2000). The proline-rich motif truncated PDZ-RhoGEF induced the formation of stress fibers but not the cortical actin reorganization. We showed that LARG, which did not contain a proline-rich motif, induced the formation of stress fibers in NIH 3T3 fibroblasts. Our results together with the previous observations suggest that LARG is partly different from PDZ-RhoGEF in terms of the localization site on the membrane and as a result shows a different phenotype of actin reorganization.
Activating mechanism of LARG in the IGF-1 signaling pathway
In this study, we showed that the IGF-1 receptor forms a complex with LARG, IGF-1 induced the activation of Rho and Rho-kinase, and overexpression of the PDZ or RGS domain of LARG partially inhibited the IGF-1induced activation of Rho-kinase and the enhancement of stress fibers in a dominant negative fashion. The IGF-1induced Rho activation peaked at 2.55 min after stimulation and then returned to basal levels at 2030 min. The IGF-1induced MBS phosphorylation peaked at
10 min and then returned to basal levels at
60 min. The IGF-1induced enhancement of stress fibers appeared 2060 min after stimulation and then returned to basal levels later than
120 min. We think that the time courses of IGF-1/Rho/Rho-kinase signaling pathways are reasonable. These results suggest that IGF-1 activates the Rho/Rho-kinase pathway via a complex of the IGF-1 receptor and LARG. As to the molecular mechanism in which the activation of GEFs can be modulated by extracellular signals, Vav and Vav-2 are thought to be phosphorylated on tyrosine residues, and then this leads to activation of the Rho family GTPases (Crespo et al., 1997; Schuebel et al., 1998). Thus, we speculated that the phosphorylation of LARG on tyrosine residues might be necessary for the activation of LARG. We examined whether IGF-1 induced the tyrosine phosphorylation of LARG in MDCKII cells and found that the tyrosine phosphorylation of LARG was not detected under our conditions in which the IGF-1 receptor was coimmunoprecipitated with LARG and tyrosine phosphorylated (Fig. 3, C and D). These results suggest that the interaction of the IGF-1 receptor with LARG is constitutive and the tyrosine phosphorylation of LARG is not necessary for its activation. Since Rho/Rho-kinase is activated by the addition of IGF-1, the phosphorylation of the IGF-1 receptor and/or some other unidentified mechanism may account for the activation of LARG. In this context, molecules, which interact with the RGS domain such as heterotrimeric G proteins, may participate in the activation of LARG. Alternatively, the binding of IGF-1 to the IGF-1 receptor may induce the conformational change of the receptor, leading to the activation of LARG. Further studies are necessary for understanding the mode of activation of LARG by IGF-1.
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Materials and methods |
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Plasmid constructions
The human IGF-1 receptor cDNA was amplified by PCR from the human fetal liver library. The pLexA- or pBTM116IGF-1 receptor plasmid, the fragments of the IGF-1 receptor (13481367 amino acids) inserted into pLexA or pBTM116, were provided by Dr. Hollenberg (Fred Hutchinson Cancer Research Center, Seattle, WA), respectively. The 9.5- and 10.5-d mouse embryo cDNA-VP16 fusion libraries and pLexA-Lam were also provided by Dr. Hollenberg (Fred Hutchinson Cancer Research Center). The pLexA-Lam plasmid expressed human lamin C (66230 amino acids) as a fusion product between it and the DNA-binding domain of LexA. For the mammalian expression plasmid pEF-BOSHAIGF-1 receptor ß-subunit or the Escherichia coli expression plasmid pGEXIGF-1 receptor ß-subunit, the fragment of the IGF-1 receptor (8991367 amino acids) was inserted into pEF-BOS-HA or pGEX-4T-1 (Amersham Pharmacia Biotech), respectively. The human LARG cDNA was amplified by PCR from the human fetal brain MATCHMAKER cDNA library (CLONTECH Laboratories, Inc.) and then cloned into pGEM-T-Easy (Promega). The fragment of LARG (321544 amino acids), LARG-PDZ (1531544 amino acids), LARG-
RGS (the deletion of 262685 amino acids), LARG-DH/PH (6861544 amino acids), LARG-PDZ domain (32295 amino acids), or LARG-RGS domain (153670 amino acids) was cloned into pEF-BOS-Myc or -HA, respectively. The fragment of the LARG-PDZ domain (32190 amino acids) or LARG-DH/PH (6861544 amino acids) was inserted into pMAL-c2 (New England Biolabs, Inc.) or pGEX-4T-2, respectively.
Yeast strains and media
The genotype of the S. cerevisiae reporter strain used, L40, was MATa trp1 leu2 his3 ade2 LYS2::lexA-HIS3 URA3::lexA-lacZ and that of NA87-11A was MAT leu2 his3 trp1 pho3 pho5. Yeast strains were grown at 30°C in rich medium (1% yeast extract, 2% bacto-peptone, 2% glucose) or in Burkfolder's minimal medium with appropriate supplements (Tohe et al., 1973).
Library screening
S. cerevisiae L40, which contained the pLexAIGF-1 receptor, was transformed with the mouse embryo cDNA library constructed in pVP16. Plasmid DNA transformations were performed by the lithium acetate method (Schiestl and Gietz, 1989). His+ colonies were grown in synthetic medium at 30°C for 3 d. Approximately 1.8 x 107 transformants were screened, and then 1,683 colonies were picked up as His+. Of these colonies, 99 were His+ and LacZ+ as confirmed by replicate plating. The 99 His+ and LacZ+ clones were cured of pLexAIGF-1 receptor by growing the cells in Trp-containing medium and then mating them to yeast strain NA87-11A that had been transformed with pLexAIGF-1 receptor or pLexA-Lam. Mated cells were selected for growth in medium that lacked Trp (pBTM116) and Leu (pVP16) and were tested for their ability to transactivate HIS3 and a lacZ reporter gene by a ß-galactosidase colorimetric filter assay. 60 diploids transactivated the reporter constructs. Of these 60 diploids, 10 did not transactivate the reporter construct in the presence of the LexAlamin fusion protein (Vojtek et al., 1993). The library plasmid was recovered from the 10 clones and sequenced by an ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems).
In vitro binding assay
GST or MBP fusion proteins were expressed in E. coli BL21(DE3) and purified according to the manufacturer's instructions. MBP or MBP-LARG PDZ domain (600 pmol) was mixed with glutathioneSepharose 4B beads coated with 100 pmol of either GST or GSTIGF-1 receptor ß-subunit in buffer A (20 mM Tris/HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2). The bound MBP-LARG PDZ domain was coeluted with GST fusion proteins by the addition of buffer A containing 10 mM glutathione. Portions of the eluates were subjected to SDS-PAGE followed by immunoblot analysis with anti-MBP antibody.
Cell culture
NIH 3T3 and MDCKII cells were grown in DME containing 10% calf serum, penicillin, and streptomycin. L cells were grown in DME containing 10% FBS. Although IGF-1 induced the enhancement of actin stress fibers in parental MDCKII cells, the background of stress fibers was relatively high. To monitor the enhancement of actin stress fibers, the cell line, which showed low background of actin stress fibers and high response to IGF-1, was subcloned and employed for whole studies. The cells showed the same characteristics as typical MDCK cells, which show characteristics of polarized epithelial cells. MDCKII cells stably expressing EGFP-RhoA were grown in DME containing 10% calf serum and 300 µg/ml of G418.
Immunofluorescence and laser scanning confocal microscopy
MDCKII cells plated on 13-mm round glass coverslips were fixed in 3.7% formaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. The fixed cells were incubated with primary antibodies for 12 h at 4°C and then were incubated for 1 h with secondary antibodies. The distributions of LARG and the IGF-1 receptor were examined with a laser scanning confocal microscope LSM510 (ZEISS).
Coimmunoprecipitation assay
For coimmunoprecipitation assay of the endogenous proteins, MDCKII cells were lysed with buffer B (50 mM Tris/HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, 0.5% [wt/vol] Triton X-100, 10 µM p-APMSF, and 10 µg/ml leupeptin). The lysate was sonicated and then clarified by centrifugation at 12,000 g for 30 min at 4°C. The soluble supernatant was incubated with anti-LARG antibody, antiIGF-1 receptor antibody, or rabbit IgG. For coimmunoprecipitation assay of the recombinant proteins, L cells were transfected with pEF-BOS-Myc-LARG and pEF-BOSHAIGF-1 receptor ß-subunit by lipofectamine (GIBCO BRL) and cultured for 16 h. The soluble supernatant was incubated with anti-HA or anti-Myc antibody. The immunocomplexes were then precipitated with protein ASepharose (Amersham Pharmacia Biotech) eluted by boiling in sample buffer for SDS-PAGE, and subjected to immunoblot analyses with the appropriate antibodies.
In vitro GEF assays
Effects of LARG on the dissociation of [3H]GDP from the Rho family proteins were assayed as described previously (Hoshino et al., 1999). The [3H]GDP-bound form of small G proteins was obtained by incubating 10 pmol of each small G protein with 1 µM [3H]GDP (1,0002,000 cpm/pmol) for 20 min at 30°C in reaction mixture I (20 mM Tris/HCl, pH 7.5, 10 mM EDTA, 1 mM DTT, 5 mM MgCl2). To prevent the dissociation of [3H]GDP from the G proteins, we added MgCl2 to a final concentration of 20 mM and then cooled the mixtures immediately to 4°C. The dissociation of [3H]GDP was performed at 25°C by adding a 200-fold excess of unlabeled GTP and the indicated amounts of GST-DH/PH domain of LARG or GST-Dbl to reaction mixture II (50 mM Tris/HCl, pH 8.0, 2.9 mM EDTA, 1 mM DTT, 10 mM MgCl2). The diluted mixtures were filtered through nitrocellulose filters, and the radioactivity trapped on the filters was counted.
Transfection of NIH 3T3 or MDCKII cells with the expression plasmid of LARG
NIH 3T3 cells were transfected with pEF-BOS-HA-LARG or -LARG/-Myc dominant negative form of RhoA by using lipofectamine and cultured for 24 h. The transfected cells were serum starved for 24 h in DME. MDCKII cells were transfected with pEF-BOS-GST, or the -HA-LARG-PDZ, -HA-LARG-RGS, or -HAAF-6PDZ domain by using lipofectamine 2000 (GIBCO BRL) and cultured for 24 h. The transfected cells were serum starved for 48 h in DME and then stimulated with 10 nM IGF-1 for 1 h. The fixed cells were incubated with primary antibody and then incubated with secondary antibody and TRITC phalloidin. Fluorescent images were taken with a laser scanning confocal microscope LSM510.
Pull-down assay (Rho activity assay)
To obtain MDCKII cells stably expressing EGFP-RhoA, MDCKII cells were transfected with pEGFP-RhoA along with pSVIISR vector containing the neomycin resistance gene using lipofectamine 2000, and neomycin-resistant clones were selected. MDCKII cells stably expressing EGFP-RhoA were incubated for 24 h and then were deprived of serum for 48 h. The cells were next incubated in DME containing 10 nM IGF-1 at 37°C for various minutes and then lysed with buffer C (50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% NP-40, 10 µM p-APMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin). The soluble supernatant was incubated with 2 nmol of GST-rhotekin Rho-binding domain (Zondag et al., 2000). The bound EGFP-RhoA was eluted by boiling in sample buffer for SDS-PAGE and subjected to immunoblot analysis with anti-RhoA antibody.
Detection of phosphorylated MBS by immunoblot analysis
MDCKII cells were incubated for 24 h, and the cells were deprived of serum for 48 h. For inhibitor assays, serum-deprived cells were treated with various doses of HA1077 or Y-32885 for 30 min. In the experiments involving the PDZ domain of LARG, serum-deprived cells were transfected with pEF-BOS-HA, or the -LARG-PDZ, -LARG-RGS, or -AF-6-PDZ domain by using lipofectamine 2000 and then were cultured. The cells were thereafter incubated in DME containing 10 nM IGF-1 at 37°C for various minutes. The IGF-1stimulated cells were treated with 10% (wt/vol) TCA. The resulting precipitates were subjected to immunoblotting with anti-MBS or anti-pS854 antibody.
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Footnotes |
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Acknowledgments |
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This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, the Japan Society of the Promotion of Science Research for the Future, and the Human Frontier Science Program. S. Taya is a research fellow of the Japan Society for the Promotion of Science.
Submitted: 26 June 2001
Revised: 1 October 2001
Accepted: 16 October 2001
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