LIGAND-DEPENDENT AND LIGAND-INDEPENDENT INTEGRIN FOCAL CONTACT LOCALIZATION - THE ROLE OF THE ALPHA-CHAIN CYTOPLASMIC DOMAIN

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha1, wild-type subunit (HA1 cells) or the cytoplasmic domain deleted al subunit (CYT cells). Both cell lines express similar levels of the human al subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha1beta1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha5beta1 but not for alpha1beta1, there is a striking difference in the cellular localization of alpha1beta1. The HA1 cells show only alpha5 in focal contacts, without alpha1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha1, appears in focal contacts along with the alpha5/beta1. Thus, there is both ligand-dependent (alpha5/beta1) and ligand-independent (alpha1/beta1) focal contact localization in these cells. The truncated alpha1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.