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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 stimulated release of Cdc42 from the membranes of COS cells. Membranes from COS cells transfected with myc-Cdc42 were prepared as described in Materials and methods. The membranes were incubated with the indicated amounts of purified MBP–ICAP-1 or MBP alone as a control for 25 min at room temperature. After centrifugation, the supernatant fractions were examined for the presence of Cdc42 using SDS-PAGE and Western blotting with a specific antibody (A). The relative amount of Cdc42 released into supernatant fraction was determined by densitometric scanning analysis (B).
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fig7: ICAP-1 stimulated release of Cdc42 from the membranes of COS cells. Membranes from COS cells transfected with myc-Cdc42 were prepared as described in Materials and methods. The membranes were incubated with the indicated amounts of purified MBP–ICAP-1 or MBP alone as a control for 25 min at room temperature. After centrifugation, the supernatant fractions were examined for the presence of Cdc42 using SDS-PAGE and Western blotting with a specific antibody (A). The relative amount of Cdc42 released into supernatant fraction was determined by densitometric scanning analysis (B).

Mentions: A known activity of GDIs is their ability to induce the release of the GTPase from cellular membranes (Leonard et al., 1992). To test for this activity, membrane fractions from COS cells transfected with Cdc42 constructs were incubated with purified MBP–ICAP-1 fusion protein or MBP alone. As shown in Fig. 7 , increasing amounts of purified ICAP-1 caused an increasing level of Cdc42 to be released into a soluble supernatant fraction. The result demonstrates that ICAP-1, besides being able to inhibit GDP dissociation, is also able to specifically elicit the release of Cdc42 from the membrane.


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 stimulated release of Cdc42 from the membranes of COS cells. Membranes from COS cells transfected with myc-Cdc42 were prepared as described in Materials and methods. The membranes were incubated with the indicated amounts of purified MBP–ICAP-1 or MBP alone as a control for 25 min at room temperature. After centrifugation, the supernatant fractions were examined for the presence of Cdc42 using SDS-PAGE and Western blotting with a specific antibody (A). The relative amount of Cdc42 released into supernatant fraction was determined by densitometric scanning analysis (B).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: ICAP-1 stimulated release of Cdc42 from the membranes of COS cells. Membranes from COS cells transfected with myc-Cdc42 were prepared as described in Materials and methods. The membranes were incubated with the indicated amounts of purified MBP–ICAP-1 or MBP alone as a control for 25 min at room temperature. After centrifugation, the supernatant fractions were examined for the presence of Cdc42 using SDS-PAGE and Western blotting with a specific antibody (A). The relative amount of Cdc42 released into supernatant fraction was determined by densitometric scanning analysis (B).
Mentions: A known activity of GDIs is their ability to induce the release of the GTPase from cellular membranes (Leonard et al., 1992). To test for this activity, membrane fractions from COS cells transfected with Cdc42 constructs were incubated with purified MBP–ICAP-1 fusion protein or MBP alone. As shown in Fig. 7 , increasing amounts of purified ICAP-1 caused an increasing level of Cdc42 to be released into a soluble supernatant fraction. The result demonstrates that ICAP-1, besides being able to inhibit GDP dissociation, is also able to specifically elicit the release of Cdc42 from the membrane.

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