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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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Inhibitory effect of ICAP-1 on the dissociation of [α-32P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α-32P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α-32P]GDP dissociation from Cdc42 induced by chelating Mg2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α-32P]GDP remaining is expressed as the percentage of [α-32P]GDP bound to Cdc42 after loading.
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fig6: Inhibitory effect of ICAP-1 on the dissociation of [α-32P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α-32P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α-32P]GDP dissociation from Cdc42 induced by chelating Mg2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α-32P]GDP remaining is expressed as the percentage of [α-32P]GDP bound to Cdc42 after loading.

Mentions: GAPs and GDIs are important negative regulators of monomeric GTPases. Comparative sequence analysis indicated a partial degree of homology between ICAP-1 and known GDI molecules (see Fig. 8 and Discussion). To test whether ICAP-1 can exert GDI activity, Cdc42 was expressed in COS cells as a GST fusion protein, and the membrane-bound fraction was affinity purified. After loading of Cdc42 with radioactive GTP, the release of the nucleotide induced either by chelating Mg2+ ions or by the Dbl GEF was measured in the presence of purified ICAP-1, unrelated molecule, such as the carrier protein MBP, or a known GDI such as RhoGDI. As shown in Fig. 6 , ICAP-1 inhibits the dissociation of GDP induced either by EDTA (see Fig. 6 A) or by Dbl GEF (see Fig. 6 B). MBP control protein does not alter the release of GDP with respect to buffer control (see Fig. 6 C).


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)

Inhibitory effect of ICAP-1 on the dissociation of [α-32P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α-32P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α-32P]GDP dissociation from Cdc42 induced by chelating Mg2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α-32P]GDP remaining is expressed as the percentage of [α-32P]GDP bound to Cdc42 after loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199222&req=5

fig6: Inhibitory effect of ICAP-1 on the dissociation of [α-32P]GDP from Cdc42. GST-Cdc42 was expressed in COS cells, and the protein was purified from the membranous fraction and captured on glutathione-Sepharose. The Cdc42 immobilized on the beads was loaded with guanosine nucleotide by incubation with [α-32P]GTP 25 min at room temperature. The dissociation of the GDP induced by chelating Mg2+ with EDTA (A) or by the GEF activity of Dbl (B) was measured in the presence of either the buffer alone, MBP–ICAP-1(10 μg), or the same amount of GST-Rho GDI fusion proteins after 10 min at room temperature. (C) Time course for [α-32P]GDP dissociation from Cdc42 induced by chelating Mg2+ with EDTA in the presence of either 10 μg of MBP–ICAP-1, GST-Rho GDI, MBP carrier protein, or buffer alone. Results shown are means ± SD of values from three experiments. [α-32P]GDP remaining is expressed as the percentage of [α-32P]GDP bound to Cdc42 after loading.
Mentions: GAPs and GDIs are important negative regulators of monomeric GTPases. Comparative sequence analysis indicated a partial degree of homology between ICAP-1 and known GDI molecules (see Fig. 8 and Discussion). To test whether ICAP-1 can exert GDI activity, Cdc42 was expressed in COS cells as a GST fusion protein, and the membrane-bound fraction was affinity purified. After loading of Cdc42 with radioactive GTP, the release of the nucleotide induced either by chelating Mg2+ ions or by the Dbl GEF was measured in the presence of purified ICAP-1, unrelated molecule, such as the carrier protein MBP, or a known GDI such as RhoGDI. As shown in Fig. 6 , ICAP-1 inhibits the dissociation of GDP induced either by EDTA (see Fig. 6 A) or by Dbl GEF (see Fig. 6 B). MBP control protein does not alter the release of GDP with respect to buffer control (see Fig. 6 C).

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