Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

J Cell Biol. 1994 Mar;124(6):949-61. doi: 10.1083/jcb.124.6.949.

Abstract

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar 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 from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double-label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, 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 epithelial tight junction.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Dogs
  • Drosophila Proteins*
  • Endothelium / chemistry
  • Epithelium / chemistry
  • Fluorescent Antibody Technique
  • Genes, Tumor Suppressor
  • Insect Hormones / chemistry*
  • Intercellular Junctions / chemistry*
  • Membrane Proteins / analysis*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / isolation & purification
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Myocardium / chemistry
  • Phosphoproteins / chemistry*
  • Tumor Suppressor Proteins*
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein

Substances

  • Drosophila Proteins
  • Insect Hormones
  • Membrane Proteins
  • Phosphoproteins
  • TJP1 protein, human
  • TJP2 protein, human
  • Tjp1 protein, mouse
  • Tjp1 protein, rat
  • Tjp2 protein, mouse
  • Tumor Suppressor Proteins
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein
  • dlg1 protein, Drosophila

Associated data

  • GENBANK/L27152