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Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

Kessels MM, Engqvist-Goldstein AE, Drubin DG, Qualmann B - J. Cell Biol. (2001)

Bottom Line: While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake.The endocytosis block was rescued by cooverexpression of dynamin.Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-39008 Magdeburg, Germany. kessels@ifn-magdeburg.de

ABSTRACT
The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

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Identification of proteins interacting with the SH3 domain of Abp1. (a) Blot overlay analysis using a GST-fusion protein containing the COOH-terminal half of Abp1 onto different rat tissue homogenates identified several bands prominent in brain at 75/80, 100, 145, and 180 kD. (b) Blot overlay analysis using a GST-fusion protein of the SH3 domain revealed bands of similar molecular weights not only in brain but in part also in testis and lung. (c) Affinity purifications of proteins interacting with the SH3 domains of Abp1, endophilin I, and syndapin I. Equal amounts of fusion proteins and brain extracts (0.625 mg brain protein per pull down) were used. 12.45 μg of starting material (corresponding to 1/50) were loaded for comparison. (d) Immunoprecipitated dynamin (arrow) was detected by both antidynamin antibodies (left) and by the Abp1-SH3 domain (right). Left two lanes in each gel: IP buffer with 150 mM salt; right two lanes in each gel: IP buffer with 100 mM salt.
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Figure 1: Identification of proteins interacting with the SH3 domain of Abp1. (a) Blot overlay analysis using a GST-fusion protein containing the COOH-terminal half of Abp1 onto different rat tissue homogenates identified several bands prominent in brain at 75/80, 100, 145, and 180 kD. (b) Blot overlay analysis using a GST-fusion protein of the SH3 domain revealed bands of similar molecular weights not only in brain but in part also in testis and lung. (c) Affinity purifications of proteins interacting with the SH3 domains of Abp1, endophilin I, and syndapin I. Equal amounts of fusion proteins and brain extracts (0.625 mg brain protein per pull down) were used. 12.45 μg of starting material (corresponding to 1/50) were loaded for comparison. (d) Immunoprecipitated dynamin (arrow) was detected by both antidynamin antibodies (left) and by the Abp1-SH3 domain (right). Left two lanes in each gel: IP buffer with 150 mM salt; right two lanes in each gel: IP buffer with 100 mM salt.

Mentions: To gain further insights into the functions of mammalian Abp1, which binds to F-actin via two NH2-terminal domains, we attempted to identify interaction partners of the two COOH-terminal domains of this protein. We searched for direct binding partners in a variety of rat tissues using a blot overlay technique (Fig. 1, a and b). Several bands were detected using the COOH-terminal half of Abp1 as a probe (Fig. 1 a). The most prominent bands were detected in brain (a weak band at ∼60 kD, a double band at 75/80 kD, and bands at 100, 145, and 180 kD). Proteins were also detected in testis (a weak band at 60 kD and a doublet of 100 and 105 kD) (Fig. 1 a). In the other tissues, the intensities of bands were much lower. A blot overlay using the NH2-terminal half of the protein containing the two F-actin–binding modules did not reveal any bands (data not shown). Using a GST fusion protein of the SH3 domain alone, we were able to show that the interactions detected were due to the COOH-terminal SH3 domain and not the flexible domain, which was also contained in the original fusion construct (Fig. 1 b). In general, the signal intensities were slightly higher using the SH3 domain alone compared with the COOH-terminal half of Abp1. This may be due to the fact that the flexible domain is sensitive to proteolytic degradation (Kessels et al. 2000); such cleavages would result in loss of GST detection. These experiments also showed that the binding specificities of the SH3 domain were not affected by the presence or absence of neighboring sequences.


Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin.

Kessels MM, Engqvist-Goldstein AE, Drubin DG, Qualmann B - J. Cell Biol. (2001)

Identification of proteins interacting with the SH3 domain of Abp1. (a) Blot overlay analysis using a GST-fusion protein containing the COOH-terminal half of Abp1 onto different rat tissue homogenates identified several bands prominent in brain at 75/80, 100, 145, and 180 kD. (b) Blot overlay analysis using a GST-fusion protein of the SH3 domain revealed bands of similar molecular weights not only in brain but in part also in testis and lung. (c) Affinity purifications of proteins interacting with the SH3 domains of Abp1, endophilin I, and syndapin I. Equal amounts of fusion proteins and brain extracts (0.625 mg brain protein per pull down) were used. 12.45 μg of starting material (corresponding to 1/50) were loaded for comparison. (d) Immunoprecipitated dynamin (arrow) was detected by both antidynamin antibodies (left) and by the Abp1-SH3 domain (right). Left two lanes in each gel: IP buffer with 150 mM salt; right two lanes in each gel: IP buffer with 100 mM salt.
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Related In: Results  -  Collection

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Figure 1: Identification of proteins interacting with the SH3 domain of Abp1. (a) Blot overlay analysis using a GST-fusion protein containing the COOH-terminal half of Abp1 onto different rat tissue homogenates identified several bands prominent in brain at 75/80, 100, 145, and 180 kD. (b) Blot overlay analysis using a GST-fusion protein of the SH3 domain revealed bands of similar molecular weights not only in brain but in part also in testis and lung. (c) Affinity purifications of proteins interacting with the SH3 domains of Abp1, endophilin I, and syndapin I. Equal amounts of fusion proteins and brain extracts (0.625 mg brain protein per pull down) were used. 12.45 μg of starting material (corresponding to 1/50) were loaded for comparison. (d) Immunoprecipitated dynamin (arrow) was detected by both antidynamin antibodies (left) and by the Abp1-SH3 domain (right). Left two lanes in each gel: IP buffer with 150 mM salt; right two lanes in each gel: IP buffer with 100 mM salt.
Mentions: To gain further insights into the functions of mammalian Abp1, which binds to F-actin via two NH2-terminal domains, we attempted to identify interaction partners of the two COOH-terminal domains of this protein. We searched for direct binding partners in a variety of rat tissues using a blot overlay technique (Fig. 1, a and b). Several bands were detected using the COOH-terminal half of Abp1 as a probe (Fig. 1 a). The most prominent bands were detected in brain (a weak band at ∼60 kD, a double band at 75/80 kD, and bands at 100, 145, and 180 kD). Proteins were also detected in testis (a weak band at 60 kD and a doublet of 100 and 105 kD) (Fig. 1 a). In the other tissues, the intensities of bands were much lower. A blot overlay using the NH2-terminal half of the protein containing the two F-actin–binding modules did not reveal any bands (data not shown). Using a GST fusion protein of the SH3 domain alone, we were able to show that the interactions detected were due to the COOH-terminal SH3 domain and not the flexible domain, which was also contained in the original fusion construct (Fig. 1 b). In general, the signal intensities were slightly higher using the SH3 domain alone compared with the COOH-terminal half of Abp1. This may be due to the fact that the flexible domain is sensitive to proteolytic degradation (Kessels et al. 2000); such cleavages would result in loss of GST detection. These experiments also showed that the binding specificities of the SH3 domain were not affected by the presence or absence of neighboring sequences.

Bottom Line: While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake.The endocytosis block was rescued by cooverexpression of dynamin.Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, D-39008 Magdeburg, Germany. kessels@ifn-magdeburg.de

ABSTRACT
The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin-binding protein Abp1 (SH3P7/HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1's actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein.

Show MeSH
Related in: MedlinePlus