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The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM - J. Cell Biol. (1999)

Bottom Line: Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor.The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates.AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Virologie, Universität Zürich, Switzerland.

ABSTRACT
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

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Binding of the  AF-6 PDZ domain to recombinant Eph receptors. (a)  FLAG-tagged AF-6 PDZ  domain protein (FLAG AF-6  PDZ) coimmunoprecipitates  with full-length EphB2 and  EphA7 receptors but not  with Ephrin-B1. Lanes 1, 5,  and 9, direct cell lysate, untransfected cells; lanes 2, 6,  and 10, direct cell lysate, receptor alone transfected;  lanes 3, 7, and 11, receptors  and FLAG AF-6 PDZ  cotransfected; lanes 4, 8, and  12, receptors and empty vector (FLAG) transfected. (b)  GST-pull down experiments  of EphB3 COOH-terminal  peptide (last 10 amino acids)  or full-length EphB3 receptor. Lane 1, EphB3pep direct  cell lysate; lane 4, EphB3  full-length receptor direct  cell lysate; lanes 2 and 5,  EphB3pep or EphB3 extracts mixed with GST AF-6  PDZ coupled glutathionine  beads; lanes 3 and 6, GFP  EphB3 peptide or EphB3 extracts mixed with GST-coupled glutathionine beads.  Lanes 7–9, an EphB3ala mutant receptor (terminal  valine residue changed to  alanine) is unable to coimmunoprecipitate with FLAG  AF-6 PDZ or empty vector  (FLAG); lane 7, EphB3ala  receptor direct cell lysate;  lane 8, EphB3ala cotransfected with FLAG AF-6  PDZ; lane 9, EphB3ala  cotransfected with empty  vector (FLAG). Bands not  indicated by arrows are contaminants.
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Figure 2: Binding of the AF-6 PDZ domain to recombinant Eph receptors. (a) FLAG-tagged AF-6 PDZ domain protein (FLAG AF-6 PDZ) coimmunoprecipitates with full-length EphB2 and EphA7 receptors but not with Ephrin-B1. Lanes 1, 5, and 9, direct cell lysate, untransfected cells; lanes 2, 6, and 10, direct cell lysate, receptor alone transfected; lanes 3, 7, and 11, receptors and FLAG AF-6 PDZ cotransfected; lanes 4, 8, and 12, receptors and empty vector (FLAG) transfected. (b) GST-pull down experiments of EphB3 COOH-terminal peptide (last 10 amino acids) or full-length EphB3 receptor. Lane 1, EphB3pep direct cell lysate; lane 4, EphB3 full-length receptor direct cell lysate; lanes 2 and 5, EphB3pep or EphB3 extracts mixed with GST AF-6 PDZ coupled glutathionine beads; lanes 3 and 6, GFP EphB3 peptide or EphB3 extracts mixed with GST-coupled glutathionine beads. Lanes 7–9, an EphB3ala mutant receptor (terminal valine residue changed to alanine) is unable to coimmunoprecipitate with FLAG AF-6 PDZ or empty vector (FLAG); lane 7, EphB3ala receptor direct cell lysate; lane 8, EphB3ala cotransfected with FLAG AF-6 PDZ; lane 9, EphB3ala cotransfected with empty vector (FLAG). Bands not indicated by arrows are contaminants.

Mentions: The binding specificities observed in the mammalian cell two-hybrid assays with the PDZ domain of AF-6 and the peptide ligands were verified with recombinant receptors in vitro. cDNAs encoding full-length EphB2, EphA7, and EphB3 were expressed in 293T cells and their ability to immunoprecipitate with a FLAG epitope-tagged AF-6 PDZ domain construct (FLAG AF-6 PDZ) was analyzed. Fig. 2 a indicates that both the full-length EphB2 and EphA7 receptors, when cotransfected into 293T cells with FLAG AF-6 PDZ, specifically coimmunoprecipitate with the FLAG-tagged AF-6 PDZ domain fusion protein whereas no receptor immunoprecipitates with the FLAG-tagged vector alone (Fig. 2 a, FLAG, compare lanes 3 and 4 and lanes 7 and 8). As a further control the Ephrin-B1 (Xlerk2) (Jones et al., 1997) receptor ligand, which has an intracellular COOH terminus characteristic of PDZ-binding peptides but which our mammalian two-hybrid analysis had indicated did not interact with the PDZ domain of AF-6, was transfected. When the Ephrin-B1 cDNA expression vector was cotransfected with either FLAG AF-6 PDZ or the empty vector (FLAG), no Ephrin-B1 protein coimmunoprecipitated with the FLAG-tagged fusion proteins, indicating the specificity of the interaction of the AF-6 PDZ domain for only certain Eph receptors (Fig. 2 a, compare lanes 10–12).


The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.

Buchert M, Schneider S, Meskenaite V, Adams MT, Canaani E, Baechi T, Moelling K, Hovens CM - J. Cell Biol. (1999)

Binding of the  AF-6 PDZ domain to recombinant Eph receptors. (a)  FLAG-tagged AF-6 PDZ  domain protein (FLAG AF-6  PDZ) coimmunoprecipitates  with full-length EphB2 and  EphA7 receptors but not  with Ephrin-B1. Lanes 1, 5,  and 9, direct cell lysate, untransfected cells; lanes 2, 6,  and 10, direct cell lysate, receptor alone transfected;  lanes 3, 7, and 11, receptors  and FLAG AF-6 PDZ  cotransfected; lanes 4, 8, and  12, receptors and empty vector (FLAG) transfected. (b)  GST-pull down experiments  of EphB3 COOH-terminal  peptide (last 10 amino acids)  or full-length EphB3 receptor. Lane 1, EphB3pep direct  cell lysate; lane 4, EphB3  full-length receptor direct  cell lysate; lanes 2 and 5,  EphB3pep or EphB3 extracts mixed with GST AF-6  PDZ coupled glutathionine  beads; lanes 3 and 6, GFP  EphB3 peptide or EphB3 extracts mixed with GST-coupled glutathionine beads.  Lanes 7–9, an EphB3ala mutant receptor (terminal  valine residue changed to  alanine) is unable to coimmunoprecipitate with FLAG  AF-6 PDZ or empty vector  (FLAG); lane 7, EphB3ala  receptor direct cell lysate;  lane 8, EphB3ala cotransfected with FLAG AF-6  PDZ; lane 9, EphB3ala  cotransfected with empty  vector (FLAG). Bands not  indicated by arrows are contaminants.
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Figure 2: Binding of the AF-6 PDZ domain to recombinant Eph receptors. (a) FLAG-tagged AF-6 PDZ domain protein (FLAG AF-6 PDZ) coimmunoprecipitates with full-length EphB2 and EphA7 receptors but not with Ephrin-B1. Lanes 1, 5, and 9, direct cell lysate, untransfected cells; lanes 2, 6, and 10, direct cell lysate, receptor alone transfected; lanes 3, 7, and 11, receptors and FLAG AF-6 PDZ cotransfected; lanes 4, 8, and 12, receptors and empty vector (FLAG) transfected. (b) GST-pull down experiments of EphB3 COOH-terminal peptide (last 10 amino acids) or full-length EphB3 receptor. Lane 1, EphB3pep direct cell lysate; lane 4, EphB3 full-length receptor direct cell lysate; lanes 2 and 5, EphB3pep or EphB3 extracts mixed with GST AF-6 PDZ coupled glutathionine beads; lanes 3 and 6, GFP EphB3 peptide or EphB3 extracts mixed with GST-coupled glutathionine beads. Lanes 7–9, an EphB3ala mutant receptor (terminal valine residue changed to alanine) is unable to coimmunoprecipitate with FLAG AF-6 PDZ or empty vector (FLAG); lane 7, EphB3ala receptor direct cell lysate; lane 8, EphB3ala cotransfected with FLAG AF-6 PDZ; lane 9, EphB3ala cotransfected with empty vector (FLAG). Bands not indicated by arrows are contaminants.
Mentions: The binding specificities observed in the mammalian cell two-hybrid assays with the PDZ domain of AF-6 and the peptide ligands were verified with recombinant receptors in vitro. cDNAs encoding full-length EphB2, EphA7, and EphB3 were expressed in 293T cells and their ability to immunoprecipitate with a FLAG epitope-tagged AF-6 PDZ domain construct (FLAG AF-6 PDZ) was analyzed. Fig. 2 a indicates that both the full-length EphB2 and EphA7 receptors, when cotransfected into 293T cells with FLAG AF-6 PDZ, specifically coimmunoprecipitate with the FLAG-tagged AF-6 PDZ domain fusion protein whereas no receptor immunoprecipitates with the FLAG-tagged vector alone (Fig. 2 a, FLAG, compare lanes 3 and 4 and lanes 7 and 8). As a further control the Ephrin-B1 (Xlerk2) (Jones et al., 1997) receptor ligand, which has an intracellular COOH terminus characteristic of PDZ-binding peptides but which our mammalian two-hybrid analysis had indicated did not interact with the PDZ domain of AF-6, was transfected. When the Ephrin-B1 cDNA expression vector was cotransfected with either FLAG AF-6 PDZ or the empty vector (FLAG), no Ephrin-B1 protein coimmunoprecipitated with the FLAG-tagged fusion proteins, indicating the specificity of the interaction of the AF-6 PDZ domain for only certain Eph receptors (Fig. 2 a, compare lanes 10–12).

Bottom Line: Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor.The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates.AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Medizinische Virologie, Universität Zürich, Switzerland.

ABSTRACT
The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

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