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Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

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.

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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.
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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.

Mentions: The localization of l-afadin was first examined by immunofluorescence microscopy of the frozen sections of various rat or mouse tissues using the anti–l-afadin antibody. In the liver, l-afadin was localized at the beltlike junctional complex region along the bile canaliculi (Fig. 6 a). When the frozen sections of the small intestine were doubly stained with the monoclonal anti–E-cadherin, l-afadin was concentrated with E-cadherin at the junctional complex region of intestine absorptive epithelia, but l-afadin was more highly concentrated at the junctional complex region than E-cadherin (Fig. 6, b1–3 and c1–3). The frozen sections of the heart were doubly stained with the monoclonal antivinculin antibody. Vinculin is known to be a marker, not only for cell-to-cell AJ, but also for cell-to-matrix AJ (Geiger, 1979, 1983). l-Afadin was colocalized with vinculin at the intercalated disc (Fig. 6 d1–3). Vinculin was also periodically located along the lateral borders of cardiac muscle cells (costamere) while l-afadin was not. When the cultured EL cells expressing E-cadherin (Nagafuchi et al., 1987) were doubly stained with the anti–ZO-1 antibody, l-afadin showed localization similar to that of ZO-1 (Fig. 7, a and b). ZO-1 is known to be concentrated at cadherin-based, spotlike cell-to-cell AJ in fibroblasts (Itoh et al., 1991, 1993). These results suggest that l-afadin is localized at cadherin-based cell-to-cell AJ.


Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139800&req=5

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.
Mentions: The localization of l-afadin was first examined by immunofluorescence microscopy of the frozen sections of various rat or mouse tissues using the anti–l-afadin antibody. In the liver, l-afadin was localized at the beltlike junctional complex region along the bile canaliculi (Fig. 6 a). When the frozen sections of the small intestine were doubly stained with the monoclonal anti–E-cadherin, l-afadin was concentrated with E-cadherin at the junctional complex region of intestine absorptive epithelia, but l-afadin was more highly concentrated at the junctional complex region than E-cadherin (Fig. 6, b1–3 and c1–3). The frozen sections of the heart were doubly stained with the monoclonal antivinculin antibody. Vinculin is known to be a marker, not only for cell-to-cell AJ, but also for cell-to-matrix AJ (Geiger, 1979, 1983). l-Afadin was colocalized with vinculin at the intercalated disc (Fig. 6 d1–3). Vinculin was also periodically located along the lateral borders of cardiac muscle cells (costamere) while l-afadin was not. When the cultured EL cells expressing E-cadherin (Nagafuchi et al., 1987) were doubly stained with the anti–ZO-1 antibody, l-afadin showed localization similar to that of ZO-1 (Fig. 7, a and b). ZO-1 is known to be concentrated at cadherin-based, spotlike cell-to-cell AJ in fibroblasts (Itoh et al., 1991, 1993). These results suggest that l-afadin is localized at cadherin-based cell-to-cell AJ.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

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.

Show MeSH
Related in: MedlinePlus