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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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Transfected AF-6 and Eph receptors cocluster at sites  of cell–cell contact in 293T cells. Subconfluent cells were transiently transfected with the indicated Eph receptor and a GFP-tagged AF-6 expression vector and stained with Eph receptor-specific antibodies. Red, Eph receptor localization; green, GFP  AF-6 expression. GFP AF-6 is clustered with the Eph receptors  at sites of cell–cell contact (a1–d3). Ephrin-B1 ligand, which does  not bind to the GFP AF-6 protein at the membrane, was used as  a negative control. This leads to a diffuse staining of GFP AF-6 in  these cells (e1–e3). Bars, 10 μm.
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Figure 3: Transfected AF-6 and Eph receptors cocluster at sites of cell–cell contact in 293T cells. Subconfluent cells were transiently transfected with the indicated Eph receptor and a GFP-tagged AF-6 expression vector and stained with Eph receptor-specific antibodies. Red, Eph receptor localization; green, GFP AF-6 expression. GFP AF-6 is clustered with the Eph receptors at sites of cell–cell contact (a1–d3). Ephrin-B1 ligand, which does not bind to the GFP AF-6 protein at the membrane, was used as a negative control. This leads to a diffuse staining of GFP AF-6 in these cells (e1–e3). Bars, 10 μm.

Mentions: To observe a possible direct interaction between AF-6 and these RTK proteins, a plasmid was constructed coding for a full-length AF-6 fused to GFP at its NH2 terminus, and cotransfected with various full-length receptors in 293T cells. The GFP-tagged AF-6 protein gave a generally diffuse fluorescent signal throughout the cytoplasm and nucleus when transfected alone (data not shown). However, when cotransfected with full-length EphB2, EphA7, or EphB3 RTKs (Fig. 3, a1–d1), a dramatic shift in the subcellular localization of the GFP-tagged AF-6 protein occurred with a strong signal enriched at the cellular membrane, often at sites of cell–cell contact (Fig. 3, a2–d2, compare with e2). Costaining for the transfected receptors in these cells with a rhodamine-coupled antibody specific for the primary anti-receptor antibodies revealed that the GFP AF-6 signal colocalized with the membrane-associated receptor signals. As a control Ephrin-B1 and GFP AF-6 were transfected. Even though the Ephrin-B1 signal was enriched at the plasma membrane there was no colocalization of the GFP AF-6 signal and Ephrin-B1, with the GFP AF-6 signal diffusely spread throughout the cotransfected cells (Fig. 3, e1 and e2). This is consistent with the result that AF-6 is unable to associate with the COOH-terminal peptide of Ephrin-B1 in a mammalian two-hybrid experiment (Fig. 1).


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)

Transfected AF-6 and Eph receptors cocluster at sites  of cell–cell contact in 293T cells. Subconfluent cells were transiently transfected with the indicated Eph receptor and a GFP-tagged AF-6 expression vector and stained with Eph receptor-specific antibodies. Red, Eph receptor localization; green, GFP  AF-6 expression. GFP AF-6 is clustered with the Eph receptors  at sites of cell–cell contact (a1–d3). Ephrin-B1 ligand, which does  not bind to the GFP AF-6 protein at the membrane, was used as  a negative control. This leads to a diffuse staining of GFP AF-6 in  these cells (e1–e3). Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Transfected AF-6 and Eph receptors cocluster at sites of cell–cell contact in 293T cells. Subconfluent cells were transiently transfected with the indicated Eph receptor and a GFP-tagged AF-6 expression vector and stained with Eph receptor-specific antibodies. Red, Eph receptor localization; green, GFP AF-6 expression. GFP AF-6 is clustered with the Eph receptors at sites of cell–cell contact (a1–d3). Ephrin-B1 ligand, which does not bind to the GFP AF-6 protein at the membrane, was used as a negative control. This leads to a diffuse staining of GFP AF-6 in these cells (e1–e3). Bars, 10 μm.
Mentions: To observe a possible direct interaction between AF-6 and these RTK proteins, a plasmid was constructed coding for a full-length AF-6 fused to GFP at its NH2 terminus, and cotransfected with various full-length receptors in 293T cells. The GFP-tagged AF-6 protein gave a generally diffuse fluorescent signal throughout the cytoplasm and nucleus when transfected alone (data not shown). However, when cotransfected with full-length EphB2, EphA7, or EphB3 RTKs (Fig. 3, a1–d1), a dramatic shift in the subcellular localization of the GFP-tagged AF-6 protein occurred with a strong signal enriched at the cellular membrane, often at sites of cell–cell contact (Fig. 3, a2–d2, compare with e2). Costaining for the transfected receptors in these cells with a rhodamine-coupled antibody specific for the primary anti-receptor antibodies revealed that the GFP AF-6 signal colocalized with the membrane-associated receptor signals. As a control Ephrin-B1 and GFP AF-6 were transfected. Even though the Ephrin-B1 signal was enriched at the plasma membrane there was no colocalization of the GFP AF-6 signal and Ephrin-B1, with the GFP AF-6 signal diffusely spread throughout the cotransfected cells (Fig. 3, e1 and e2). This is consistent with the result that AF-6 is unable to associate with the COOH-terminal peptide of Ephrin-B1 in a mammalian two-hybrid experiment (Fig. 1).

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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