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Comparative Study
, 141 (1), 199-208

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts With ZO-1 and Occludin

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Comparative Study

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts With ZO-1 and Occludin

J Haskins et al. J Cell Biol.

Abstract

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.

Figures

Figure 4
Figure 4
Northern blots of ZO-3 and ZO-2. 5 μg of MDCK cell poly(A)+ RNA probed for ZO-3 shows a ∼3.4-kb transcript. Blot stripped and reprobed for ZO-2 shows the expected 5.2-kb transcript (21). RNA standards at right (kb).
Figure 2
Figure 2
Schematic representation of the full-length ZO-3 cDNA. (a) The 3,072-bp cDNA, with EcoRI (E) and XhoI (X) restriction sites at the 5′ and 3′ ends. (Solid lines) Untranslated regions. Open reading frame indicated by the box, with XcmI, EagI, PstI (unique to the A1 cDNA), and internal XhoI restriction sites. (b) Individual clones obtained from library screening. All partial cDNAs were subjected to double-stranded sequencing. (c) Scale bar in 1-kb segments.
Figure 3
Figure 3
Full-length nucleotide and amino acid sequence of ZO-3. ZO-3 contains 3 PDZ domains (thin underlines), an SH3 domain (thick underline), and a GUK domain (box), demonstrating that it is another member of the MAGUK family of proteins. ZO-3 also contains a basic domain between PDZ1 and PDZ2, a proline-rich domain between PDZ2 and PDZ3 and an acidic domain (dashed underline). The double underline within the GUK box indicates the original peptide obtained by amino acid sequencing. The ZO-3 sequence is available from the GenBank database under accession number AF023617.
Figure 9
Figure 9
Binding of recombinant ZO-3 to tight junction proteins. (a) ZO-3 binds ZO-1 and ZO-2 from MDCK cell extracts. Affinity resin containing either full-length ZO-3 (lanes 1) or negative control peptide (lanes 2) were incubated with high salt extracts of MDCK cell membranes, washed, solubilized, and immunoblotted for either ZO-1 (left) or ZO-2 (right). The resin containing ZO-3 specifically retains both ZO-1 and ZO-2. (b) ZO-3 binds ZO-1 directly. Radioactively labeled ZO-1 generated by in vitro transcription/translation (lane 1) was incubated with affinity resin containing either full-length ZO-3 (lane 2) or negative control peptide (lane 3). Resin was washed, solubilized, and subjected to SDS-PAGE. The resin containing ZO-3 specifically retains a band which runs at 215 kD. This band was confirmed as ZO-1 by immunoblotting an identical aliquot of the bound material in lane 2 with anti-human ZO-1 antisera (lane 4). Lanes 1–3, autoradiograms; lane 4, ECL. (c) Partial characterization of an anti–ZO-3 antisera. Guinea pig antisera generated against a portion of ZO-3 reacts with a 130-kD band present in whole MDCK cell lysate (lane 1). It also shows faint reactivity with a band that comigrates with ZO-1 (arrowhead). No reaction with MDCK cell proteins was detected with preimmune sera (lane 2). (d) ZO-3 binds directly to the cytoplasmic tail of occludin. Recombinant ZO-3 was incubated with affinity resin containing either the COOH-terminal 148 aa of occludin (lane 1) or negative control peptide (lane 2). Bound material was eluted from washed resin and immunoblotted with anti–ZO-3 antisera. The resin containing occludin specifically retains ZO-3. (e) ZO-3 does not bind to ZO-2. Recombinant ZO-2 (lane 1) was incubated with affinity resin containing ZO-3 (lane 2) or negative control peptide (lane 3). Bound material was eluted from washed resin and immunoblotted with anti–ZO-2 antisera. No binding was detected in either case. Unbound fractions collected from resin containing ZO-3 (lane 4) or negative control peptide (lane 5) were also immunoblotted with anti–ZO-2 antisera. The presence of ZO-3 on the resin of lane 2 was verified by stripping the blot and reprobing with anti–ZO-3 antisera (lane 6).
Figure 1
Figure 1
Immunoprecipitation from metabolically labeled MDCK cell extracts with an anti–ZO-1 mAb (+) or a nonspecific but identical subclass mAb (−) under conditions that preserve protein–protein interactions shows that both ZO-2 (160 kD) and ZO-3 (130 kD) specifically coprecipitate with ZO-1 (215 kD). Molecular mass markers at right (kD).
Figure 5
Figure 5
Multiple sequence alignments of the MAGUK domains of canine ZO-3, human ZO-1 (44), canine ZO-2 (7), Drosophila TamA (41), and dlg-A (46), constructed with GCG PILEUP. The GUK domain is also compared with the yeast guanylate kinase sequence (8). Dark shaded regions indicate identity to the ZO-3 sequence, lighter shades are conserved aa substitutions. Amino acids in the PDZ domains believed to be important in protein–protein interactions (32) are marked (+), as are the aa involved in ATP (bar), GMP (*) and Mg2+ (o) binding in the GUK domain (26). # indicates the aa of the putative leucine zipper motif.
Figure 6
Figure 6
An exogenous protein of ∼130 kD is expressed by the MDCK/Z3 cell line. (a) Immunoblot of whole cell lysates from the MDCK/Z3 line (lane 1) or parental MDCK cells (lane 2) probed with anti–VSV-G to detect the epitope-tagged ZO-3 construct. A reactive band, running at ∼130 kD, appears only in the MDCK/Z3 cell line. (b) Low-stringency immunoprecipitation of ZO-1 from metabolically labeled MDCK cells (LS) shows coprecipitation of ZO-2 and ZO-3 (see also Fig. 1). High-stringency immunoprecipitation from metabolically labeled MDCK/Z3 or parental (MDCK/P) cells using anti–VSV-G (lanes 1), anti–ZO-2 (lanes 2), or anti–ZO-1 (lanes 3) demonstrates the presence of a band only in the MDCK/Z3 cells that runs a fraction higher than the endogenous 130-kD band. This slight difference in molecular mass can be accounted for by the additional 1,338 D of the epitope tag.
Figure 7
Figure 7
Immunofluorescent costaining of MDCK/Z3 cells for ZO-3 (a) and ZO-1 (b). ZO-3 and ZO-1 are identically distributed at cell borders. Additional staining of the cell cytoplasm is visible for ZO-3. Bar, 5 μm.
Figure 8
Figure 8
ImmunoEM colocalization of ZO-1 (10 nm gold) and ZO-3 (5 nm gold) on membranes isolated from MDCK/Z3 cells. (a–c) Three different images of labeling are displayed, showing that ZO-3 and ZO-1 are colocalized at sites of tight junction membrane contact, while all other membranes, including a desmosome (D) in b, are free of label. Bars, 100 nm.
Figure 10
Figure 10
Schematic diagram showing the domain arrangement of the three MAGUK family members found at the tight junction. PDZ, diagonal stripes; SH3, horizontal stripes; GUK, black; basic domain, dots (+); acidic domain, horizontal dashes (−); proline-rich, wavy horizontal lines, alternative splices, α (ZO-1) and β (ZO-2).

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