Signal transduction by β1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-I receptor

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-β1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including α-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with β1, α1 and α5 integrins, but not with α3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

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RACK1, an Insulin-Like Growth Factor I (IGF-I) Receptor-Interacting Protein, Modulates IGF-I-Dependent Integrin Signaling and Promotes Cell Spreading and Contact with Extracellular Matrix

Molecular and Cellular Biology ◽

10.1128/mcb.22.7.2345-2365.2002 ◽

2002 ◽

Vol 22 (7) ◽

pp. 2345-2365 ◽

Cited By ~ 129

Author(s):

Ulrich Hermanto ◽

Cong S. Zong ◽

Weiqun Li ◽

Lu-Hai Wang

Keyword(s):

Extracellular Matrix ◽

Growth Factor ◽

Protein Kinase ◽

Focal Adhesion ◽

Cell Spreading ◽

Β1 Integrin ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I

ABSTRACT The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gβ homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and β1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible β1 integrin-associated kinase activity and association of Crk with p130CAS were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G1 or G1/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21Cip1/WAF1 and p27Kip1, and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.

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The B domain of Insulin-like Growth Factor I (IGF-I) is recognized by IGF carrier proteins but not by type II IGF receptors

Pediatric Research ◽

10.1203/00006450-198411000-00058 ◽

1984 ◽

Vol 18 (11) ◽

pp. 1210-1210

Author(s):

M A De Vroede ◽

S Joshi ◽

P G Katsoyannis ◽

S P Nissley ◽

M M Rechler

Keyword(s):

Growth Factor ◽

Insulin Like Growth Factor ◽

Type Ii ◽

Carrier Proteins ◽

Factor I ◽

Igf Receptors ◽

Igf I ◽

Growth Factor I

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Insulin-Like Growth Factor I Controls a Mutually Exclusive Association of RACK1 with Protein Phosphatase 2A and β1 Integrin To Promote Cell Migration

Molecular and Cellular Biology ◽

10.1128/mcb.01868-05 ◽

2006 ◽

Vol 26 (11) ◽

pp. 4041-4051 ◽

Cited By ~ 67

Author(s):

Patrick A. Kiely ◽

Denise O'Gorman ◽

Ken Luong ◽

Dorit Ron ◽

Rosemary O'Connor

Keyword(s):

Cell Migration ◽

Growth Factor ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Β1 Integrin ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Phosphatase 2A ◽

Growth Factor I

ABSTRACT The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit β1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and β1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either β1 integrin or PP2A in a complex at the IGF-IR.

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