Original Article

Binding of Integrin 6ß4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

Correspondence to: Arnoud Sonnenberg, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel:(31) 20-5121942 Fax:(31) 20-5121944 E-mail:asonn{at}nki.nl.

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the 6ß4 integrin and have shown that the cytoplasmic domain of the ß4 subunit associates with an NH₂-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with 6ß4 in basal hemidesmosome-like clusters or associated 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 assay to demonstrate that ß4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the ß4 cytoplasmic domain. Mapping of the ß4 and actin-binding sites 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 ß4 can compete out the plectin ABD fragment from its association with F-actin. The ability of ß4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when ß4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

Key Words: actin, epidermolysis bullosa, hemidesmosome, 6ß4 integrin, plectin

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