ALPHA-3A-BETA-1 INTEGRIN LOCALIZES TO FOCAL CONTACTS IN RESPONSE TO DIVERSE EXTRACELLULAR-MATRIX PROTEINS

In vitro binding assays and inhibition of cell adhesion with monoclona l antibodies have implicated the integrin alpha 3 beta 1 as a receptor for a variety of extracellular ligands. However, reports of alpha 3 b eta 1-ligand interactions are inconsistent, and transfection studies h ave suggested that alpha 3 beta 1 is not sufficient for cell attachmen t to ligands other than kalinin/laminin 5. We used immunofluorescence to study subcellular localization of the alpha 3A cytoplasmic domain v ariant in different cultured cell types. Using standard fixation and p ermeabilization methods, antibodies specific for alpha 3A stained most cell types in a diffuse pattern, consistent with previous reports. Su rprisingly, however, chemical cross-linking of integrins to the extrac ellular matrix and extraction of the cytoskeleton prior to immunofluor escence revealed alpha 3A in focal contacts of most cells tested, sugg esting that the cytoplasmic domain was concealed in intact focal conta cts by cytoskeletal or other cytoplasmic proteins. The alpha 3A subuni t localized to focal contacts in several cell types cultured on fibron ectin, kalinin/laminin 5, EHS-laminin/laminin 1, type IV collagen, or vitronectin. In contrast, alpha 5 and alpha V integrins were detected in focal contacts only in cells grown on their known ligands (fibronec tin, and fibronectin or vitronectin, respectively). Therefore, our res ults show that alpha 3A beta 1 responds to a broad spectrum of extrace llular ligands. Time course comparisons of the recruitment of alpha su bunits from different fibronectin receptors suggested that localizatio n of alpha 3A beta 1 to fibronectin-induced focal contacts was indepen dent of the recruitment of alpha 5 and alpha 4 integrins. However, oth er studies have shown that alpha 3A beta 1 does not mediate initial ce ll adhesion to many of the ligands that induced its focal contact loca lization, including fibronectin. Therefore, we suggest that alpha 3A b eta 1 may be a secondary receptor with post-cell-adhesion functions fo r a broad spectrum of extracellular matrices.