Tight-junction protein ZO-1 isoforms (alpha(+) and alpha(-)) show differential extractability and epidermal-growth-factor-induced tyrosine phosphorylation in A431 cells

ZO-1, a cytoplasmic plaque protein of tight junctions, exists in vivo as tw o major isoforms which are defined by the presence or absence of an 80 amin o acid domain termed alpha. The ZO-1 alpha(+) isoform is expressed in most epithelial cells while ZO-1 alpha(-) isoform expression is restricted to en dothelial cells and some highly specialized epithelial cells, suggesting th at the isoforms serve different functions. We had previously demonstrated t hat both ZO-1 isoforms are expressed in A431 cells and are tyrosine phospho rylated in response to epidermal-growth-factor treatment. In the present st udy, we found that the alpha(-) isoform of ZO-1 was more tightly associated with the cytoskeleton than was the alpha(+) isoform, based on extraction i n nonionic detergent. In addition, the ZO-1 alpha(-) was preferentially tyr osine phosphorylated in response to epidermal-growth-factor treatment. Howe ver, both isoforms became more tightly associated with the cytoskeleton aft er A431 cells were exposed to epidermal growth factor. Immunofluorescence a nalysis of A431 cells with isoform-specific antibodies demonstrated that fu nctional differences in ZO-1 isoform behavior were not due to differences i n their subcellular locations. The coincident localization of these isoform s does not rule out different affinities for interacting proteins related t o the presence or absence of the alpha domain, and it is these interactions that are likely to explain functional differences between the isoforms.