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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 inhibition of cell spreading on vitronectin. (A) NIH3T3 cells were transiently transfected with myc–ICAP-1, detached with EDTA, and allowed to attach on vitronectin-coated coverslips (10 μg/ml) in serum-free medium for 1 h. Transfected cells were visualized by anti-myc monoclonal antibody, whereas actin organization was detected in the same sample with FITC phalloidin. (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means ± SD from three independent experiments in which >20 cells per condition were scored.
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fig2: ICAP-1 inhibition of cell spreading on vitronectin. (A) NIH3T3 cells were transiently transfected with myc–ICAP-1, detached with EDTA, and allowed to attach on vitronectin-coated coverslips (10 μg/ml) in serum-free medium for 1 h. Transfected cells were visualized by anti-myc monoclonal antibody, whereas actin organization was detected in the same sample with FITC phalloidin. (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means ± SD from three independent experiments in which >20 cells per condition were scored.

Mentions: Given that ICAP-1 binds to β1A but not to β3 and β5 integrin subunits (unpublished data; Chang et al., 1997), we wondered whether the ICAP-1 antispreading effect was dependent on integrin β1A binding. To this purpose, we analyzed spreading of ICAP-1–expressing NIH3T3 cells on vitronectin, a substrate for αVβ3 and αvβ5. As shown in Fig. 2 , ICAP-1–expressing cells spread poorly on vitronectin after 1 h of adhesion. This ICAP-1 inhibitory effect was comparable to that observed on fibronectin (Fig. 1, A and B, compared with Fig. 2, A and B). Taken together, these results suggest that in our experimental condition ICAP-1 effect seems not to be dependent on integrin binding.


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 inhibition of cell spreading on vitronectin. (A) NIH3T3 cells were transiently transfected with myc–ICAP-1, detached with EDTA, and allowed to attach on vitronectin-coated coverslips (10 μg/ml) in serum-free medium for 1 h. Transfected cells were visualized by anti-myc monoclonal antibody, whereas actin organization was detected in the same sample with FITC phalloidin. (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means ± SD from three independent experiments in which >20 cells per condition were scored.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: ICAP-1 inhibition of cell spreading on vitronectin. (A) NIH3T3 cells were transiently transfected with myc–ICAP-1, detached with EDTA, and allowed to attach on vitronectin-coated coverslips (10 μg/ml) in serum-free medium for 1 h. Transfected cells were visualized by anti-myc monoclonal antibody, whereas actin organization was detected in the same sample with FITC phalloidin. (B) The percentage of spread cells was calculated as described in Materials and methods. Values are means ± SD from three independent experiments in which >20 cells per condition were scored.
Mentions: Given that ICAP-1 binds to β1A but not to β3 and β5 integrin subunits (unpublished data; Chang et al., 1997), we wondered whether the ICAP-1 antispreading effect was dependent on integrin β1A binding. To this purpose, we analyzed spreading of ICAP-1–expressing NIH3T3 cells on vitronectin, a substrate for αVβ3 and αvβ5. As shown in Fig. 2 , ICAP-1–expressing cells spread poorly on vitronectin after 1 h of adhesion. This ICAP-1 inhibitory effect was comparable to that observed on fibronectin (Fig. 1, A and B, compared with Fig. 2, A and B). Taken together, these results suggest that in our experimental condition ICAP-1 effect seems not to be dependent on integrin binding.

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