MOLECULAR CHARACTERIZATION AND TISSUE DISTRIBUTION OF ZO-2, A TIGHT JUNCTION PROTEIN HOMOLOGOUS TO ZO-1 AND THE DROSOPHILA DISKS-LARGE TUMOR-SUPPRESSOR PROTEIN

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytop lasmic surfaces of the zonula occludens or tight junction. A 160-kD po lypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve prot ein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Pr oc, Natl. Acad. Sci. USA, 88: 3460-3464). We have isolated ZO-2 from M DCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed b y electroelution from preparative SDS-PAGE gel slices. Amino acid sequ ence information obtained from a ZO-2 tryptic fragment was used to iso late a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very sim ilar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase- like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs -large-1 (dlg) gene of Drosophila, and a synapse-associated protein fr om rat brain, PSD-95/SAP90. The dig gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, a lthough the functions of other family members have not yet been define d. A polyclonal antiserum was raised against a unique region of ZO-2 a nd found to exclusively label the cytoplasmic surfaces of tight juncti ons in MDCK plasma membrane preparations, indicating that ZO-2 is a ti ght junction-associated protein. Immunohistochemical staining of froze n sections of whole tissue demonstrated that ZO-2 localized to the reg ion of the tight junction in a number of epithelia, including liver, i ntestine, kidney, testis, and arterial endothelium, suggesting that th is protein is a ubiquitous component of the tight junction. Double-lab el immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 stain ing at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemur a, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115: 1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epi thelial tight junction.