Binding of Integrin α6β4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

Abstract

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 α6β4 integrin and have shown that the cytoplasmic domain of the β4 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 α6β4 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 β4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the β4 cytoplasmic domain. Mapping of the β4 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 β4 can compete out the plectin ABD fragment from its association with F-actin. The ability of β4 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 β4 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.

Complete Cytolysis and Neonatal Lethality in Keratin 5 Knockout Mice Reveal Its Fundamental Role in Skin Integrity and in Epidermolysis Bullosa Simplex

“…The proximity of the two type I keratins to hemidesmosomes was in agreement with a recent report identifying K14 but not K5 as a binding partner for the hemidesmosomal plaque protein plectin (Geerts et al, 1999). In fact, staining for plectin revealed an almost complete colocalization with K14.…”

“…An additional explanation for the phenotypic differences between K5 Ϫ/Ϫ and K14 Ϫ/Ϫ mice might be related to the observation that types I and II keratins interact with distinct subsets of associated proteins. The hemidesmosomal proteins plectin and BPAG1, for example, were found to associate with type I keratins, providing the stability of the intermediate filament system in the cell (Geerts et al, 1999). In K5 Ϫ/Ϫ mice, K14 colocalized with hemidesmosomes.…”

“…The hemidesmosomal proteins plectin and BPAG1, for example, were found to associate with type I keratins, providing the stability of the intermediate filament system in the cell (Geerts et al, 1999). In K5 Ϫ/Ϫ mice, K14 colocalized with hemidesmosomes.…”

“…Transient transfection experiments and yeast 2 hybrid assays demonstrated that the amino terminal head domain of type II keratins (e.g., K5) binds to the C-terminal tail of desmoplakin I, whereas no association was seen with type I keratins (Kouklis et al, 1994;Meng et al, 1997). More recently, it was shown that the major hemidesmosomal plaque protein plectin interacts with K14 but not with K5 (Geerts et al, 1999). On the basis of these findings it is conceivable that the ablation of a type II keratin might affect a different subset of partner proteins than that of a type I keratin, leading to different phenotypes in mice.…”

A Compound Heterozygous One Amino-Acid Insertion/Nonsense Mutation in the Plectin Gene Causes Epidermolysis Bullosa Simplex with Plectin Deficiency

“…The N-terminal globular domain of plectin harbors an actin binding domain 12 and regions for integrin ␤4 binding. 9,10 The importance of this region has also been demonstrated by a three amino-acid deletion in the Nterminal globular domain of plectin in a patient with EBS with muscular dystrophy. 17 In that study the functional effects of the mutation were not analyzed.…”