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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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Immunogold labeling for AF-6 and Eph receptors in  cryo-ultrathin sections of CA1 stratum radiatum. Immunogold  particles for AF-6 (5-nm immunogold in a, arrows), EphB3 (15-nm  immunogold in b) and EphB2 (15-nm immunogold in c) are  shown at postsynaptic densities of axo-spinous synapses. (d–f)  AF-6 (8-nm immunogold particles, arrows) EphB3 receptor (d  and f, 15 nm immunogold particles, arrowheads), and EphB2 receptor (e, arrowhead) are shown at postsynaptic membranes. ax,  axonal terminal; s, spine. Bar, 0.2 μm.
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Figure 6: Immunogold labeling for AF-6 and Eph receptors in cryo-ultrathin sections of CA1 stratum radiatum. Immunogold particles for AF-6 (5-nm immunogold in a, arrows), EphB3 (15-nm immunogold in b) and EphB2 (15-nm immunogold in c) are shown at postsynaptic densities of axo-spinous synapses. (d–f) AF-6 (8-nm immunogold particles, arrows) EphB3 receptor (d and f, 15 nm immunogold particles, arrowheads), and EphB2 receptor (e, arrowhead) are shown at postsynaptic membranes. ax, axonal terminal; s, spine. Bar, 0.2 μm.

Mentions: Immunocytochemical techniques with light and electron microscopic analyses were combined in order to determine whether AF-6 colocalizes with Eph receptors in rat brain. In addition, the localization of ZO-1, a protein which is found together with AF-6 at tight junctions in polarized epithelial cells and at cadherin-based cell–cell junctions in fibroblasts (Mandai et al., 1997; Yamamoto et al., 1997), was examined. At low magnification, we observed overlapping spatial distributions in the immunolabeling patterns of AF-6, ZO-1, EphB2, EphA7, and EphB3. Strong immunoreactivity occurred in a variety of regions with high synaptic density, specifically the hippocampus, caudate putamen, cerebellum, superficial gray layer of superior colliculus, external cortex of inferior colliculus, medial mammillary nucleus, sensory nuclei of thalamus, and neocortex (data not shown). Because the architecture and function of hippocampal circuitry is well known (Johnston and Amaral, 1998), we focused our examination on the subcellular localization of AF-6, ZO-1, and Eph receptors in hippocampal regions. The strong immunolabeling of AF-6, ZO-1, and Eph receptors was found in all dendritic layers, strata oriens and radiatum of CA1 and CA3 regions, and the dentate moleculare of the hippocampus (data not shown). Electron microscopic analysis revealed the presence of immunoperoxidase reaction products at the majority of asymmetric axo-spinous and axo-dendritic synapses in stratum radiatum of the CA1 region (Fig. 5 a, c–e). Immunogold labeling of cryo-ultrathin sections of CA1 have shown that AF-6 (Fig. 6 a) and Eph receptors (Fig. 6, b and c) are localized at postsynaptic densities. Further examination of double-immunogold staining revealed colocalization of AF-6 and Eph receptors over postsynaptic specializations of excitatory synapses in CA1 (Fig. 6, d–f).


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)

Immunogold labeling for AF-6 and Eph receptors in  cryo-ultrathin sections of CA1 stratum radiatum. Immunogold  particles for AF-6 (5-nm immunogold in a, arrows), EphB3 (15-nm  immunogold in b) and EphB2 (15-nm immunogold in c) are  shown at postsynaptic densities of axo-spinous synapses. (d–f)  AF-6 (8-nm immunogold particles, arrows) EphB3 receptor (d  and f, 15 nm immunogold particles, arrowheads), and EphB2 receptor (e, arrowhead) are shown at postsynaptic membranes. ax,  axonal terminal; s, spine. Bar, 0.2 μm.
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Related In: Results  -  Collection

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Figure 6: Immunogold labeling for AF-6 and Eph receptors in cryo-ultrathin sections of CA1 stratum radiatum. Immunogold particles for AF-6 (5-nm immunogold in a, arrows), EphB3 (15-nm immunogold in b) and EphB2 (15-nm immunogold in c) are shown at postsynaptic densities of axo-spinous synapses. (d–f) AF-6 (8-nm immunogold particles, arrows) EphB3 receptor (d and f, 15 nm immunogold particles, arrowheads), and EphB2 receptor (e, arrowhead) are shown at postsynaptic membranes. ax, axonal terminal; s, spine. Bar, 0.2 μm.
Mentions: Immunocytochemical techniques with light and electron microscopic analyses were combined in order to determine whether AF-6 colocalizes with Eph receptors in rat brain. In addition, the localization of ZO-1, a protein which is found together with AF-6 at tight junctions in polarized epithelial cells and at cadherin-based cell–cell junctions in fibroblasts (Mandai et al., 1997; Yamamoto et al., 1997), was examined. At low magnification, we observed overlapping spatial distributions in the immunolabeling patterns of AF-6, ZO-1, EphB2, EphA7, and EphB3. Strong immunoreactivity occurred in a variety of regions with high synaptic density, specifically the hippocampus, caudate putamen, cerebellum, superficial gray layer of superior colliculus, external cortex of inferior colliculus, medial mammillary nucleus, sensory nuclei of thalamus, and neocortex (data not shown). Because the architecture and function of hippocampal circuitry is well known (Johnston and Amaral, 1998), we focused our examination on the subcellular localization of AF-6, ZO-1, and Eph receptors in hippocampal regions. The strong immunolabeling of AF-6, ZO-1, and Eph receptors was found in all dendritic layers, strata oriens and radiatum of CA1 and CA3 regions, and the dentate moleculare of the hippocampus (data not shown). Electron microscopic analysis revealed the presence of immunoperoxidase reaction products at the majority of asymmetric axo-spinous and axo-dendritic synapses in stratum radiatum of the CA1 region (Fig. 5 a, c–e). Immunogold labeling of cryo-ultrathin sections of CA1 have shown that AF-6 (Fig. 6 a) and Eph receptors (Fig. 6, b and c) are localized at postsynaptic densities. Further examination of double-immunogold staining revealed colocalization of AF-6 and Eph receptors over postsynaptic specializations of excitatory synapses in CA1 (Fig. 6, d–f).

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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