Subunit and Plectin at Multiple Molecular Sites

Recent studies with patients suffering from epidermolysis bullosa simplex associated with muscular dystrophy and the targeted gene disruption in mice suggested that plectin, a versatile cytoskeletal linker and intermediate filament-binding protein, may play an essential role in hemidesmosome integrity and stabilization. To define plectin’s interactions with hemidesmosomal proteins on the molecular level, we studied its interaction with the uniquely long cytoplasmic tail domain of the b 4 subunit of the basement membrane laminin receptor integrin a 6 b 4 that has been implicated in connecting the transmembrane integrin complex with hemidesmosome-anchored cytokeratin filaments. In vitro binding and in vivo cotransfection assays, using recombinant mutant forms of both proteins, revealed their direct interaction via multiple molecular domains. Furthermore, we show in vitro self-interaction of integrin b 4 cytoplasmic domains, as well as disruption of intermediate filament network arrays and dislocation of hemidesmosome-associated endogenous plectin upon ectopic overexpression of this domain in PtK2 and/or 804G cells. The close association of plectin molecules with hemidesmosomal structures and their apparent random orientation was indicated by gold immunoelectron microscopy using domain-specific antibodies. Our data support a model in which plectin stabilizes hemidesmosomes, via directly interlinking integrin b 4 subunits and cytokeratin filaments. I ntegrins comprise a large family of heterodimeric receptors that mediate the adhesion of cells to extracellular matrices and other cells (Buck and Horwitz, 1987; Hynes, 1987, 1992; Ruoslahti and Pierschbacher, 1987; Ginsberg et al., 1988; Hemler, 1990; Springer, 1990; Watt et al., 1993). In addition, they are involved in transducing extracellular signals into the cell (Hynes, 1992; Juliano and Haskill, 1993; Giancotti and Mainiero, 1994). Both the a and b subunits of integrins have a large extracellular portion, a transmembrane segment, and generally a short cytoplasmic domain. The cytoplasmic domains of integrins interact with the cytoskeleton and possibly with signaling molecules, but the molecular mechanisms of these interactions are not well understood. The a 6 b 4 integrin is a basement membrane receptor for laminins (Kajiji et al., 1989; De Luca et al., 1990; Sonnenberg et al., 1990 a , b ; Lee et al., 1992) that engages in cytoplasmic interactions distinct from those of all other known integrins. Instead of being localized at adhesion plaques that serve as anchoring structures of actin filament networks, this receptor is part of hemidesmosomes (Carter et al., 1990; Stepp et al., 1990; Jones et al., 1991; Sonnenberg et al., 1991), junctional complexes that anchor cytokeratin intermediate filament (IF) 1 networks and mediate adhesion of epithelial cells to the underlying basement membrane. The intracellular interactions of integrin a 6 b 4 are mediated by the b 4 subunit, the intracellular portion of which is much larger ( z 1,000 amino acids) than that of all the other known b subunits ( z 50 amino acids) and bears no apparent sequence homology to them (Hogervorst et al., 1990; Suzuki and Naitoh, 1990; Tamura et al., 1990). It contains four regions with homology to fibronectin type III (FNIII) repeats, arranged in two pairs separated by a 143–amino acid-long connecting segment. This large cytoplasmic tail of integrin b 4 is required, and probably sufficient, for incorporation of the integrin into hemidesmosomes. Specifically, a minimal region on the integrin b 4 subunit located in the linking segment between the second and third repeat has been reported to be critical to its localization in hemidesmosomes (Niessen et al., 1997 a ). The targeted inactivation of the integrin b 4 gene in mice convincingly Address all correspondence to Gerhard Wiche, Vienna Biocenter, Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria. Tel.: 43 (1) 79515-5119. Fax: 43 (1) 79515-5121. E-mail: [email protected] 1. Abbreviations used in this paper : EBS-MD, epidermolysis bullosa simplex combined with muscular dystrophy; FNIII, fibronectin type III repeat; GFP, green fluorescent protein; IF, intermediate filament. on A uust 6, 2017 jcb.rress.org D ow nladed fom