Integrin α6β4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes

José A Manso, María Gómez-Hernández, Arturo Carabias, Noelia Alonso-García, Inés García-Rubio, Maaike Kreft, Arnoud Sonnenberg, Jose M. de Pereda
2019 Social Science Research Network  
Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6β4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of β4. The crystal structure of the complex and mutagenesis
more » ... x and mutagenesis analysis revealed that BP230 binds between the two domains of β4. BP230 induces closing of the two FnIII domains that are locked in place by an inter-domain ionic clasp required for binding. Disruption of BP230-β4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in β4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation. Keywords Cell adhesion, Epithelia, Keratinocytes, Plakins, Protein-protein interactions epidermolysis bullosa simplex, a blistering disease characterized by skin fragility (Groves et al., 2010) . Ablation of BP230 in mice resulted in a similar phenotype of blistering caused by mechanical stress (Guo et al., 1995) . In the absence of BP230, HDs lack an intracellular substructure called the inner plaque, and the bundles of keratin filaments do not attach to the HDs. The role of BP230 in the attachment of intermediate filaments is further supported by the absence of the inner plaque in type II HDs and the less robust connection of intermediate filaments to type II than to type I HDs (Uematsu et al., 1994) . HDs are dynamic complexes that disassemble when epithelial cells migrate, for example during wound healing (Gipson et al., 1993) and in invasive carcinoma cells (Herold-Mende et al., 2001) . In keratinocytes, epidermal growth factor and phorbol myristate acetate promote HD disassembly by inducing phosphorylation of the β4 subunit through the activation of the Ras/ERK-1/2 and protein kinase C (PKC) pathway (Frijns et al., 2010; Margadant et al., 2008; Rabinovitz et al., 1999) . Phosphorylation of Ser residues in the CS (Frijns et al., 2010; Rabinovitz et al., 2004; Wilhelmsen et al., 2007) and in the Ctail (Frijns et al., 2012) of β4 disrupts the interaction with plectin and is a major mechanism for HD disassembly. Phosphorylation of β4 at S1424 correlates with a loss of co-localization with BP230 and BP180 (Germain et al., 2009); yet, little is known about the mechanisms that regulate the interaction of α6β4 with BP proteins during HD disassembly. The structural understanding of protein-protein interactions in HDs is limited to the primary contact between α6β4 and plectin Song et al., 2015) . The isolated region FnIII-3,4 of β4 is reluctant to crystallize, thus, we had solved its structure using hybrid methods (Alonso-Garcia et al., 2015) . Nonetheless, how the FnIII-3,4 engages with BP230 or other proteins remained unknown. Here, we present a detailed mapping of the mutual binding sites in β4 and BP230, the 3D structure of the β4-BP230 complex, and the conformational changes that binding causes in β4. Finally, we have identified potentially phosphorylatable residues in BP230 and β4 that play key roles in their interaction. RESULTS Integrin β4 binds to a segment of the N-terminal tail of BP230. Residues 1-56 of BP230 interact with the FnIII-3,4 of β4 in yeast two-hybrid assays; and the longer segment 1-92 associated more efficiently with β4 (Koster et al., 2003) . The region 1-92 includes the N-tail (residues 1-55) and the first -helix of the SR2 (BP230 does not have the SR1). To investigate the binding to β4 in vitro, we created constructs of BP230 that include the complete SR2 (56-162) to maintain the integrity of the SR fold. The fragment 1-162 of BP230 had extremely low solubility that hampered its characterization. The longer construct of this region suitable for analysis was the 10-162. Using size exclusion chromatography (SEC), interaction was detected between β4 1436-1666 (4-CS-FnIII-3,4) and
doi:10.2139/ssrn.3330561 fatcat:ipjvhstr7nayza7aiw6tkh5jze