Binding of integrin alpha 6 beta 4 to plectin prevents plectin associationwith F-actin but does not interfere with intermediate filament binding

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracell ular matrix. We have investigated the recruitment of plectin into hemidesmo somes by the alpha 6 beta 4 integrin and have shown that the cytoplasmic do main of the beta 4 subunit associates with an NH2-terminal fragment of plec tin that contains the actin-binding domain (ABD). When expressed in immorta lized plectin-deficient keratinocytes from human patients with epidermolysi s bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is c olocalized with alpha 6 beta 4 in basal hemidesmosome-like clusters or asso ciated with F-actin in stress fibers or focal contacts. We used a yeast two -hybrid binding assay in combination with an in vitro dot blot overlay assa y to demonstrate that beta 4 interacts directly with plectin, and identifie d a major plectin-binding site on the second fibronectin type III repeat of the beta 4 cytoplasmic domain. Mapping of the beta 4 and actin-binding sit es on plectin showed that the binding sites overlap and are both located in the plectin ABD, Using an in vitro competition assay, we could show that b eta 4 can compete out the plectin ABD fragment from its association with F- actin. The ability of beta 4 to prevent binding of F-actin to plectin expla ins why F-actin has never been found in association with hemidesmosomes, an d provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes whe n beta 4 is expressed. Finally, by mapping of the COOH-terminally located b inding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EB S keratinocytes, we confirm that plectin interacts with different cytoskele tal networks.