Regulation of integrin affinity and clustering plays a key role in the control of cell adhesion and migration. The protein ICAP-1␣ (integrin cytoplasmic domain-associated protein-1␣) binds to the cytoplasmic domain of the ␤ 1A integrin and controls cell spreading on fibronectin. Here, we demonstrate that, despite its ability to interact with ␤ 1A integrin, ICAP-1␣ is not recruited in focal adhesions, whereas it is colocalized with the integrin at the ruffling edges of the cells. ICAP-1␣ induced a rapid disruption of focal adhesions, which may result from the ability of ICAP-1␣ to inhibit the association of ␤ 1A integrin with talin, which is crucial for the assembly of these structures. ICAP-1␣-mediated dispersion of ␤ 1A integrins is not observed with ␤ 1D integrins that do not bind ICAP. This strongly suggests that ICAP-1␣ action depends on a direct interaction between ICAP-1␣ and the cytoplasmic domain of the ␤ 1 chains. Altogether, these results suggest that ICAP-1␣ plays a key role in cell adhesion by acting as a negative regulator of ␤ 1 integrin avidity.

Cell adhesion-dependent signaling implicates cytoplasmic proteins interacting with the intracellular tails of integrins. Among those, the integrin cytoplasmic domain-associated protein 1␣ (ICAP-1␣) has been shown to interact specifically with the ␤ 1 integrin cytoplasmic domain. Although it is likely that this protein plays an important role in controlling cell adhesion and migration, little is known about its actual function. To search for potential ICAP-1␣-binding proteins, we used a yeast two-hybrid screen and identified the human metastatic suppressor protein nm23-H2 as a new partner of ICAP-1␣. This direct interaction was confirmed in vitro, using purified recombinant ICAP-1␣ and nm23-H2, and by coimmunoprecipitation from CHO cell lysates over-expressing ICAP-1␣. The physiological relevance of this interaction is provided by confocal fluorescence microscopy, which shows that ICAP-1␣ and nm23-H2 are colocalized in lamellipodia during the early stages of cell spreading. These adhesion sites are enriched in occupied ␤ 1 integrins and precede the formation of focal adhesions devoid of ICAP-1␣ and nm23-H2, indicating the dynamic segregation of components of matrix adhesions. This peripheral staining of ICAP-1␣ and nm23-H2 is only observed in cells spreading on fibronectin and collagen and is absent in cells spreading on poly-L-lysine, vitronectin, or laminin. This is consistent with the fact that targeting of both ICAP-1␣ and nm23-H2 to the cell periphery is dependent on ␤ 1 integrin engagement rather than being a consequence of cell adhesion. This finding represents the first evidence that the tumor suppressor nm23-H2 could act on ␤ 1 integrin-mediated cell adhesion by interacting with one of the integrin partners, ICAP-1␣.Cell adhesion to the extracellular matrix is mediated mainly by integrin clusters organized in transient focal complexes and more stable focal adhesions (1-3). These structures are linked physically to the actin cytoskeleton. Besides being the mechanical anchors of the cells, focal adhesions participate in outside-in and inside-out signaling. Integrin cytoplasmic domains have no known catalytic function, but they play a key role in the control of cytoskeleton organization and in signal transduction by recruiting many structural and signaling proteins (4). Although it is well documented that the adhesive function of most members of the integrin family can be activated in a phenotypically similar fashion, it is unclear whether common or independent cellular pathways underlie this apparent uniformity. Several proteins interacting with specific integrin cytoplasmic tails have been identified recently, suggesting that although the cytoplasmic domains of integrin ␤ subunits are quite similar, they are coupled to distinct functional pathways. For instance, ␤ 3 -endonexin binds specifically to the ␤ 3 integrin cytoplasmic tail (5) and increases the affinity of the integrin ␣ IIb ␤ 3 (6). A ␤ 2 integrin cytoplasmic domain-binding protein, cytohesin-1, has been found to increase ␣ L ␤ 2 -mediated cell ...