Distinct functions for integrins α3β1 in focal adhesions and α6β4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: Relation to hemidesmosomes

Abstract

Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin α3β1, in focal adhesions (FAs), of cultured human keratinocytes (HFKs) as a mediator of HFK adhesion to secreted BMZ-like extracellular matrix (ECM; Carter, W. G., E. A. Wayner, T. S. Bouchard, and P. Kaur. 1990. J. Cell Biol. 110: 1387-1404). Here, we have examined the relation of integrins α6β4 and α3β1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that α6β4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with α3β1-FAs to mediate adhesion to ECM, based on the following. (a) Comparison of secreted ECM, with exogenous laminin, fibronectin and collagen identified ECM as the preferred ligand for HFK adhesion and spreading and for formation of both α6β4-SACs and α3β1-FAs. (b) Inhibition of HFK adhesion with combined anti-α3β1 (P1B5) and anti-α6β4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while α3β1 played a dominant role in spreading. (c) α6β4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both α6β4-SACs and α3β1-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to α3β1-FAs, α6β4-SACs were present only in nonmotile cells, not associated with stress fibers, and were relatively stable to detergents and urea, suggesting a nonmotile, or anchoring function for SACs and motility functions for α3β1-FAs. (e) α6β4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with α6β4. (f) We suggest that α6β4/BPA-SACs in culture restrict migration of HFKs on ECM while α3β1-FAs form dynamic adhesions in spreading and migrating cells. α6β4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.