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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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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.
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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.

Mentions: A rat brain cDNA library was screened using the degenerated oligonucleotide probes designed from the partial aa sequences of the seven peptide peaks described above. We obtained several overlapping clones (Fig. 2). Among these clones, clone 20 contained a coding region with ∼4.9 kb, but it lacked a predicted initiation codon. The deduced aa sequence included all the aa sequences of the peptides of p205. This aa sequence analysis indicated that the aa sequences of the two peptides specific to p205 were both located in the COOH-terminal region. Clone 94 contained a coding region with ∼4.5 kb and an in-frame stop codon upstream of the stop codon of clone 20. This clone also lacked a predicted initiation codon. The deduced aa sequence of the coding region included all the aa sequences of the peptides of p190, but not those of the peptides specific to p205. Clone 84 contained a coding region with ∼4.2 kb and a predicted initiation codon. To confirm whether these cDNAs were derived from the same locus, we made the chromosomal assignment by FISH analysis using the cDNAs as probes. All the cDNAs were localized on rat chromosome 1q12.2 (data not shown).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: A rat brain cDNA library was screened using the degenerated oligonucleotide probes designed from the partial aa sequences of the seven peptide peaks described above. We obtained several overlapping clones (Fig. 2). Among these clones, clone 20 contained a coding region with ∼4.9 kb, but it lacked a predicted initiation codon. The deduced aa sequence included all the aa sequences of the peptides of p205. This aa sequence analysis indicated that the aa sequences of the two peptides specific to p205 were both located in the COOH-terminal region. Clone 94 contained a coding region with ∼4.5 kb and an in-frame stop codon upstream of the stop codon of clone 20. This clone also lacked a predicted initiation codon. The deduced aa sequence of the coding region included all the aa sequences of the peptides of p190, but not those of the peptides specific to p205. Clone 84 contained a coding region with ∼4.2 kb and a predicted initiation codon. To confirm whether these cDNAs were derived from the same locus, we made the chromosomal assignment by FISH analysis using the cDNAs as probes. All the cDNAs were localized on rat chromosome 1q12.2 (data not shown).

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