Molecular characterization of the tight junction protein ZO-1 in MDCK cells

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino a cids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respe ctively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not be en reported in human and mouse counterparts, and gamma, which was previousl y sequenced in human and mouse ZO-1, is now identified as a splicing region . The expression of different beta and gamma isoforms varies according to t he tissue tested. With the information provided by the sequence, Southern b lot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expressi on is regulated by calcium, while the expression of MDCK-ZO-1 protein is no t. (C) 1999 Academic Press.