Abstract. Basal cells of stratified epidermis are anchored to the basement membrane zone (BMZ) of skin via hemidesmosomes. We previously identified integrin tx3B1, 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 ct6/~4 and tx3B1, to bullous pemphigoid antigen (BPA), a component of hemidesmosomes. We conclude that ot6B4 in HFKs localizes in a new stable anchoring contact (SAC) that cooperates with tx3B1-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 tx6B4-SACs and ~x3B1-FAs. (b) Inhibition of HFK adhesion with combined anti-or3/31 (P1B5) and anti-c~6B4 (GoH3) antibodies indicated that both receptors were functional in adhesion to ECM while oe3B1 played a dominant role in spreading. (c) tx6fl4 colocalized with BPA in SACs that were proximal to but excluded from FAs. Both tx6~4-SACs and ot3fll-FAs were in contact with the adhesion surface as indicated by antibody exclusion and interference reflection microscopy. (d) In contrast to ot3~l-FAs, ot6~4-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 tx3B1-FAs. (e) ct6B4 formed a detergent-insoluble complex with exogenous ECM in an affinity isolation procedure, confirming the ability of an unidentified ECM ligand to interact with ot6f14. (f) We suggest that ot6B4/BPA-SACs in culture restrict migration of HFKs on ECM while tx3fll-FAs form dynamic adhesions in spreading and migrating cells, a6fl4/BPA-SACs in culture bear functional and compositional similarities to hemidesmosomes in skin.
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