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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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Transient transfection of the EphA7 receptor in MDCK cells causes a clustering of endogenous AF-6. Subconfluent MDCK  cells were transfected with a full-length EphA7 receptor construct and doubly stained with anti-EphA7 (green) and anti-AF-6 (red) antibodies. AF-6 clusters with the EphA7 receptor at sites of cell–cell contact (a–d). Endogenous AF-6 is recruited to a more apical position in EphA7 transfected cells. EphA7 transient expression signal (e, green) with endogenous AF-6 expression (f, red) are shown for  comparison. Bars, 10 μm.
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Figure 4: Transient transfection of the EphA7 receptor in MDCK cells causes a clustering of endogenous AF-6. Subconfluent MDCK cells were transfected with a full-length EphA7 receptor construct and doubly stained with anti-EphA7 (green) and anti-AF-6 (red) antibodies. AF-6 clusters with the EphA7 receptor at sites of cell–cell contact (a–d). Endogenous AF-6 is recruited to a more apical position in EphA7 transfected cells. EphA7 transient expression signal (e, green) with endogenous AF-6 expression (f, red) are shown for comparison. Bars, 10 μm.

Mentions: It has recently been reported that AF-6 is localized at the plasma membrane at specialized sites of cell–cell contact. In the polarized epithelial cell line MDCKII, AF-6 has been shown to colocalize with the tight junction-associated protein ZO-1 at apical lateral membrane sites of cell–cell contact (Yamamoto et al., 1997). In fibroblasts, AF-6 has also been shown to colocalize with ZO-1 at cadherin-based, spot-like cell–cell adherens junction (Mandai et al., 1997). To determine the effect of exogenous Eph receptors on the subcellular localization of endogenous AF-6, EphA7 was transfected into MDCK cells. This caused an enrichment of the endogenous AF-6 protein at sites of cell–cell contact (Fig. 4, a–f), indicating that Eph receptors can be a target for the endogenous AF-6 protein. The colocalization and enrichment of endogenous AF-6 with EphA7 seems to occur only at their extreme apical positions (Fig. 4, compare e with f). This could mean that endogenous AF-6 can not freely diffuse along the lateral membrane, whereas the transfected and overexpressed receptor can do so. Alternatively, the interaction may require additional factors only present at extreme apical positions.


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)

Transient transfection of the EphA7 receptor in MDCK cells causes a clustering of endogenous AF-6. Subconfluent MDCK  cells were transfected with a full-length EphA7 receptor construct and doubly stained with anti-EphA7 (green) and anti-AF-6 (red) antibodies. AF-6 clusters with the EphA7 receptor at sites of cell–cell contact (a–d). Endogenous AF-6 is recruited to a more apical position in EphA7 transfected cells. EphA7 transient expression signal (e, green) with endogenous AF-6 expression (f, red) are shown for  comparison. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Transient transfection of the EphA7 receptor in MDCK cells causes a clustering of endogenous AF-6. Subconfluent MDCK cells were transfected with a full-length EphA7 receptor construct and doubly stained with anti-EphA7 (green) and anti-AF-6 (red) antibodies. AF-6 clusters with the EphA7 receptor at sites of cell–cell contact (a–d). Endogenous AF-6 is recruited to a more apical position in EphA7 transfected cells. EphA7 transient expression signal (e, green) with endogenous AF-6 expression (f, red) are shown for comparison. Bars, 10 μm.
Mentions: It has recently been reported that AF-6 is localized at the plasma membrane at specialized sites of cell–cell contact. In the polarized epithelial cell line MDCKII, AF-6 has been shown to colocalize with the tight junction-associated protein ZO-1 at apical lateral membrane sites of cell–cell contact (Yamamoto et al., 1997). In fibroblasts, AF-6 has also been shown to colocalize with ZO-1 at cadherin-based, spot-like cell–cell adherens junction (Mandai et al., 1997). To determine the effect of exogenous Eph receptors on the subcellular localization of endogenous AF-6, EphA7 was transfected into MDCK cells. This caused an enrichment of the endogenous AF-6 protein at sites of cell–cell contact (Fig. 4, a–f), indicating that Eph receptors can be a target for the endogenous AF-6 protein. The colocalization and enrichment of endogenous AF-6 with EphA7 seems to occur only at their extreme apical positions (Fig. 4, compare e with f). This could mean that endogenous AF-6 can not freely diffuse along the lateral membrane, whereas the transfected and overexpressed receptor can do so. Alternatively, the interaction may require additional factors only present at extreme apical positions.

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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