Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction - PubMed (original) (raw)
. 1997 Oct 20;139(2):517-28.
doi: 10.1083/jcb.139.2.517.
H Nakanishi, A Satoh, H Obaishi, M Wada, H Nishioka, M Itoh, A Mizoguchi, T Aoki, T Fujimoto, Y Matsuda, S Tsukita, Y Takai
Affiliations
- PMID: 9334353
- PMCID: PMC2139800
- DOI: 10.1083/jcb.139.2.517
Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction
K Mandai et al. J Cell Biol. 1997.
Erratum in
- J Cell Biol 1997 Nov 17;139(4):1060
Abstract
A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.
Figures
Figure 1
Mono Q column chromatography. (a) Absorbance at 280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3 μl) of each fraction was subjected to 125I-labeled F-actin blot overlay. (c) Protein staining with Coomassie brilliant blue. An aliquot (10 μl) of each fraction was subjected to SDS-PAGE (8% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue.
Figure 2
Schematic drawings of l-afadin (p205) and s-afadin (p190) cDNAs. (1) alternative insertion of 21 bp; (2) alternative insertion of 36 bp. The sequence data of the l- and s-afadin genes are available from GenBank/EMBL/DDBJ under accession numbers U83230 and U83231, respectively.
Figure 3
Domain organization of l-afadin (p205) and s-afadin (p190). (a) F-actin–binding activity of various fragments of recombinant l-afadin. Full-length l-afadin (aa 1–1829) (myc–l-afadin) and its deletion mutant (aa 1–1673) (myc–l-afadinΔC) were expressed as myc-tagged proteins in COS7 cells. The COOH-terminal fragment (aa 1631–1829) (GST–l-afadin-C) was expressed as a GST fusion protein in E. coli and was purified with glutathione-Sepharose. The extracts from the COS7 cells and the protein purified from the bacteria were subjected to a 125I-labeled F-actin blot overlay. The extracts from the COS7 cells were subjected to Western blot analysis using the myc epitope antibody to confirm the expression amounts. (Left) 125I-labeled F-actin blot overlay; (right) Western blot analysis using the anti-myc antibody. (Asterisks) endogenous proteins of COS7 cells with 125I-labeled F-actin–binding activity. (b) Schematic drawings of l- and s-afadin structures.
Figure 4
Tissue distribution of l-afadin. (a) Northern blot analysis. A RNA blot membrane (CLONTECH, Palo Alto, CA) was hybridized with the 32P-labeled fragment (bp 5327–5812) of the l-afadin cDNA according to the manufacturer's protocol. In addition to the ∼7.5-kb mRNA of l-afadin, a smaller mRNA of ∼4.2 kb was detected in several tissues, but significance of this is unknown. (b1 and b2) Western blot analyses. Various rat tissue homogenates (10 μg of protein each) were subjected to SDS-PAGE (8% polyacrylamide gel), followed by immunoblot using the anti–l-afadin (b1) or the anti–l- and –s-afadin antibody (b2). The lower band in the heart lane with the anti–l-afadin antibody was nonspecific, since the treatment with the peptide that was used to raise the antibody did not quench this lower band, while it quenched the band with a molecular mass of ∼205 kD (l-afadin).
Figure 5
Biochemical properties of l-afadin. (a) Inhibition by myosin S1 of the binding of l-afadin to 125I-labeled F-actin. The Mono Q sample of l-afadin (0.1 μg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by the blot overlay with 125I-labeled F-actin pretreated with myosin S1 in the presence or absence of ATP. (b) Increase by l-afadin of the viscosity of F-actin. The viscosity of F-actin was measured by low shear viscometry. •, With l-afadin; ○, with α-actinin. (c) Binding of His6–l-afadin-C to F-actin. The binding curve was generated by the cosedimentation of His6–l-afadin-C with F-actin.
Figure 6
Localization of l-afadin, E-cadherin, and vinculin in various rat or mouse tissues. (a) The staining of rat liver with the anti– l-afadin antibody. Arrow, longitudinal-section view of bile canaliculi; arrowhead, cross-section view of bile canaliculi. (b1–b3) The double staining of mouse small intestinal epithelial cells with the anti–l-afadin and anti–E-cadherin antibodies: (b1) l-afadin; (b2) E-cadherin; (b3) l-afadin and E-cadherin. Arrows, basal level; arrowheads, apical level. (c1–c3) The double staining of the tangential section of mouse small intestinal cells at the level of the apical surface with the anti–l-afadin and anti–E-cadherin antibodies: (c1) l-afadin; (c2) E-cadherin; (c3) l-afadin and E-cadherin. (d1–d3) The double staining of mouse heart with the anti–l-afadin and antivinculin antibodies. It should be noted that the staining of l-afadin is also associated with the blood vessels: (d1) l-afadin; (d2) vinculin; (d3) l-afadin and vinculin. arrows, intercalated disc; arrowhead, costamere; double arrowheads, blood vessel. Bars: (a and b) 10 μm; (c) 5 μm; (d) 50 μm.
Figure 7
Localization of l-afadin and ZO-1 in the EL cells expressing E-cadherin. The EL cells expressing E-cadherin were doubly stained with the anti–l-afadin and anti–ZO-1 antibodies. l-Afadin was concentrated at cell-to-cell AJ. There were perinuclear and nuclear stainings with this anti–l-afadin antibody, but its significance is not clear. (a) l-afadin; (b) ZO-1. arrows, cell-to-cell AJ. Bar, 25 μm.
Figure 8
Different localization of l-afadin, ZO-1, and desmoplakin. (a1–a3) Localization of l-afadin and ZO-1 in mouse small intestine: (a1) l-afadin; (a2) ZO-1; (a3) l-afadin and ZO-1. arrows, the sites where green (ZO-1), yellow (the mixture of l-afadin and ZO-1), and red (l-afadin) signals occurred in this order from the apical side to the basal side. (b1–b3) Localization of l-afadin and desmoplakin in the isolated bile canaliculi: (b1) l-afadin; (b2) desmoplakin; (b3) l-afadin and desmoplakin. Asterisk, the inner space of the bile canaliculi. Bars: (a) 15 μm; (b) 5 μm.
Figure 9
Ultrastructural localization of l-afadin in rat small intestine. Intestinal epithelial cells were labeled singly with the anti–l-afadin antibody. (a) silver enhancement technique; (b) ultrathin cryosection technique. Open arrow, tight junction; closed arrow, cell-to-cell AJ; mv, microvilli. Bars, 0.2 μm.
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