ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin.
A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19-amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of approximately 130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.
Haskins
J
J
Department of Cell Biology and Anatomy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Gu
L
L
Wittchen
E S
ES
Hibbard
J
J
Stevenson
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004285:Dogs; D004330:Drosophila; D007668:Kidney; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D062793:Occludin; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019108:Tight Junctions / Q000737:chemistry / Q000502:physiology*; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D018909:src Homology Domains
D002352:Carrier Proteins; D018076:DNA, Complementary; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein