Limits...
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.

Show MeSH

Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139800&req=5

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).

Mentions: Northern blot analysis using a sequence specific to the l-afadin cDNA as a probe detected ∼7.5-kb mRNA in all the rat tissues examined, including the heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Fig. 4 a). A similar result was obtained when a sequence common between the cDNAs of l- and s-afadins was used as a probe (data not shown). Western blot analysis using an antibody specific to l-afadin detected an ∼205-kD protein in all the rat tissues examined (Fig. 4 b1). When an antibody recognizing both l- and s-afadins was used, however, an ∼190-kD protein was detected only in the brain (Fig. 4 b2). These results indicate that l-afadin is expressed ubiquitously while s-afadin is specifically expressed in brain. The reason why the Northern blot analysis did not detect the two bands in the brain may simply be because of the similar length of the mRNAs of l- and s-afadins.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

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).
Mentions: Northern blot analysis using a sequence specific to the l-afadin cDNA as a probe detected ∼7.5-kb mRNA in all the rat tissues examined, including the heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Fig. 4 a). A similar result was obtained when a sequence common between the cDNAs of l- and s-afadins was used as a probe (data not shown). Western blot analysis using an antibody specific to l-afadin detected an ∼205-kD protein in all the rat tissues examined (Fig. 4 b1). When an antibody recognizing both l- and s-afadins was used, however, an ∼190-kD protein was detected only in the brain (Fig. 4 b2). These results indicate that l-afadin is expressed ubiquitously while s-afadin is specifically expressed in brain. The reason why the Northern blot analysis did not detect the two bands in the brain may simply be because of the similar length of the mRNAs of l- and s-afadins.

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