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The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G - J. Cell Biol. (2002)

Bottom Line: Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACT
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

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ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.
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fig5: ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.

Mentions: In the experimental condition used to analyze the activation level of Cdc42 and Rac1 during cell spreading, we were not able to detect any significant activation of RhoA, as measured by pull-down assay with the Rho effector protein mDia (Kimura et al., 2000), in accordance with previous reports (Ren et al., 1999; Arthur et al., 2000). Given that the adhesive stimulus was not sufficient to induce RhoA activation, we then activated RhoA by either treating COS cells with LPA or transfecting them with pCEFL-GST-DH/PH coding for a GST fusion protein containing the DH/PH domains of the GEF protein Dbl. However, in both of these conditions ICAP-1 expression did not affect RhoA activity (unpublished data; Fig. 5 A).


The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading.

Degani S, Balzac F, Brancaccio M, Guazzone S, Retta SF, Silengo L, Eva A, Tarone G - J. Cell Biol. (2002)

ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199222&req=5

fig5: ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose coupled with MBP–ICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for the presence of Rac1 using a specific monoclonal antibody (B'). Total extract is shown as control.
Mentions: In the experimental condition used to analyze the activation level of Cdc42 and Rac1 during cell spreading, we were not able to detect any significant activation of RhoA, as measured by pull-down assay with the Rho effector protein mDia (Kimura et al., 2000), in accordance with previous reports (Ren et al., 1999; Arthur et al., 2000). Given that the adhesive stimulus was not sufficient to induce RhoA activation, we then activated RhoA by either treating COS cells with LPA or transfecting them with pCEFL-GST-DH/PH coding for a GST fusion protein containing the DH/PH domains of the GEF protein Dbl. However, in both of these conditions ICAP-1 expression did not affect RhoA activity (unpublished data; Fig. 5 A).

Bottom Line: Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain.ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix.This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126 Torino, Italy.

ABSTRACT
Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the beta1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor-induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

Show MeSH
Related in: MedlinePlus