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Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Taya S, Inagaki N, Sengiku H, Makino H, Iwamatsu A, Urakawa I, Nagao K, Kataoka S, Kaibuchi K - J. Cell Biol. (2001)

Bottom Line: Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule.When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced.Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya 466-8550, Japan.

ABSTRACT
Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. 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 initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

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Identification of the IGF-1 receptor-interacting protein. (A) Amino acid sequences of the IGF-1 receptor-interacting peptide and human LARG. The numbers denote amino acid positions. Asterisks denote identical amino acids. The black bars show amino acid positions of PDZ domain. (B) Schematic representation of the IGF-1 receptor-interacting peptide and human LARG. The numbers indicate the amino acid sequence identity in the PDZ domains or amino acid numbers. (C) Interaction of MBP-LARG-PDZ domain with GST–IGF-1 receptor β-subunit in vitro. MBP, MBP-LARG-PDZ domain, or the AF-6–PDZ domain was mixed with GST- or GST–IGF-1 receptor-coated beads. The interacting proteins were coeluted with GST fusion proteins. The eluates were subjected to SDS-PAGE and subjected to immunoblot analysis with anti-MBP or anti–AF-6 antibody. The results shown are representative of three independent experiments.
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fig1: Identification of the IGF-1 receptor-interacting protein. (A) Amino acid sequences of the IGF-1 receptor-interacting peptide and human LARG. The numbers denote amino acid positions. Asterisks denote identical amino acids. The black bars show amino acid positions of PDZ domain. (B) Schematic representation of the IGF-1 receptor-interacting peptide and human LARG. The numbers indicate the amino acid sequence identity in the PDZ domains or amino acid numbers. (C) Interaction of MBP-LARG-PDZ domain with GST–IGF-1 receptor β-subunit in vitro. MBP, MBP-LARG-PDZ domain, or the AF-6–PDZ domain was mixed with GST- or GST–IGF-1 receptor-coated beads. The interacting proteins were coeluted with GST fusion proteins. The eluates were subjected to SDS-PAGE and subjected to immunoblot analysis with anti-MBP or anti–AF-6 antibody. The results shown are representative of three independent experiments.

Mentions: To clarify the mechanism underlying the IGF-1/IGF-1 receptor signaling pathway, we tried to identify IGF-1 receptor-interacting molecules by the yeast two-hybrid screening method. We used the COOH-terminal 20 amino acids of the IGF-1 receptor β-subunit (His-Met-Asn-Gly-Gly-Arg-Lys-Asn-Glu-Arg-Ala-Leu-Pro-Leu-Pro-Gln-Ser-Ser-Thr-Cys-COOH) as a bait and identified a cDNA encoding 111 amino acids from the mouse embryo cDNA library. The cDNA product was almost identical to the PDZ domain of human LARG except for Lys-135 and Ile-166 (Fig. 1, A and B). LARG had DH and PH domains (Kourlas et al., 2000) and showed a high degree of sequence similarity to PDZ-RhoGEF (KIAA0380) except for a proline-rich motif COOH-terminally adjacent to the DH/PH domain (Fukuhara et al., 1999; Rümenapp et al., 1999; Togashi et al., 2000). Thus, we regarded the cDNA encoding the 111 amino acids as a part of murine LARG. However, we can not rule out the possibility that the identified fragment by yeast two-hybrid screening is a LARG-like molecule.


Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Taya S, Inagaki N, Sengiku H, Makino H, Iwamatsu A, Urakawa I, Nagao K, Kataoka S, Kaibuchi K - J. Cell Biol. (2001)

Identification of the IGF-1 receptor-interacting protein. (A) Amino acid sequences of the IGF-1 receptor-interacting peptide and human LARG. The numbers denote amino acid positions. Asterisks denote identical amino acids. The black bars show amino acid positions of PDZ domain. (B) Schematic representation of the IGF-1 receptor-interacting peptide and human LARG. The numbers indicate the amino acid sequence identity in the PDZ domains or amino acid numbers. (C) Interaction of MBP-LARG-PDZ domain with GST–IGF-1 receptor β-subunit in vitro. MBP, MBP-LARG-PDZ domain, or the AF-6–PDZ domain was mixed with GST- or GST–IGF-1 receptor-coated beads. The interacting proteins were coeluted with GST fusion proteins. The eluates were subjected to SDS-PAGE and subjected to immunoblot analysis with anti-MBP or anti–AF-6 antibody. The results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150867&req=5

fig1: Identification of the IGF-1 receptor-interacting protein. (A) Amino acid sequences of the IGF-1 receptor-interacting peptide and human LARG. The numbers denote amino acid positions. Asterisks denote identical amino acids. The black bars show amino acid positions of PDZ domain. (B) Schematic representation of the IGF-1 receptor-interacting peptide and human LARG. The numbers indicate the amino acid sequence identity in the PDZ domains or amino acid numbers. (C) Interaction of MBP-LARG-PDZ domain with GST–IGF-1 receptor β-subunit in vitro. MBP, MBP-LARG-PDZ domain, or the AF-6–PDZ domain was mixed with GST- or GST–IGF-1 receptor-coated beads. The interacting proteins were coeluted with GST fusion proteins. The eluates were subjected to SDS-PAGE and subjected to immunoblot analysis with anti-MBP or anti–AF-6 antibody. The results shown are representative of three independent experiments.
Mentions: To clarify the mechanism underlying the IGF-1/IGF-1 receptor signaling pathway, we tried to identify IGF-1 receptor-interacting molecules by the yeast two-hybrid screening method. We used the COOH-terminal 20 amino acids of the IGF-1 receptor β-subunit (His-Met-Asn-Gly-Gly-Arg-Lys-Asn-Glu-Arg-Ala-Leu-Pro-Leu-Pro-Gln-Ser-Ser-Thr-Cys-COOH) as a bait and identified a cDNA encoding 111 amino acids from the mouse embryo cDNA library. The cDNA product was almost identical to the PDZ domain of human LARG except for Lys-135 and Ile-166 (Fig. 1, A and B). LARG had DH and PH domains (Kourlas et al., 2000) and showed a high degree of sequence similarity to PDZ-RhoGEF (KIAA0380) except for a proline-rich motif COOH-terminally adjacent to the DH/PH domain (Fukuhara et al., 1999; Rümenapp et al., 1999; Togashi et al., 2000). Thus, we regarded the cDNA encoding the 111 amino acids as a part of murine LARG. However, we can not rule out the possibility that the identified fragment by yeast two-hybrid screening is a LARG-like molecule.

Bottom Line: Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule.When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced.Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya 466-8550, Japan.

ABSTRACT
Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. 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 initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

Show MeSH
Related in: MedlinePlus