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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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Endogenous AF-6 coimmunoprecipitates with EphB2  (a) and EphB3 (b) receptors from whole adult rat brain extracts  prepared in HO-buffer or NL-buffer, respectively but not in  RIPA-buffer. Whole adult rat brain extracts were immunoprecipitated (IP) with either monoclonal anti–AF-6 antibody, polyclonal rabbit antisera against EphB2 (EphB2), EphB3 (EphB3),  or with the monoclonal M2 antibody, which recognizes the  FLAG epitope. The immunoprecipitates were probed on a Western blot (WB) using the monoclonal anti-AF-6 antibody. AF-6 is  recognized as a double band in the rat brain. This has also been  observed by others (Yamamoto et al., 1997).
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Figure 8: Endogenous AF-6 coimmunoprecipitates with EphB2 (a) and EphB3 (b) receptors from whole adult rat brain extracts prepared in HO-buffer or NL-buffer, respectively but not in RIPA-buffer. Whole adult rat brain extracts were immunoprecipitated (IP) with either monoclonal anti–AF-6 antibody, polyclonal rabbit antisera against EphB2 (EphB2), EphB3 (EphB3), or with the monoclonal M2 antibody, which recognizes the FLAG epitope. The immunoprecipitates were probed on a Western blot (WB) using the monoclonal anti-AF-6 antibody. AF-6 is recognized as a double band in the rat brain. This has also been observed by others (Yamamoto et al., 1997).

Mentions: Finally, to confirm that these findings are also biologically relevant in the living organism, we tested whether complexes of endogenous AF-6 with Eph receptors occur in total rat brain lysates. Indeed, endogenous AF-6 coimmunoprecipitated with both EphB2 and EphB3, but not when the total rat brain extract was immunoprecipitated with an anti-FLAG M2 antibody (Fig. 8, a and b, left). The pellet, obtained after the first extraction, was reextracted with the stronger RIPA buffer, known to disrupt protein–protein interactions. This treatment destroyed the interaction even though endogenous AF-6 was still precipitable (Fig. 8, a and b, right). This further demonstrates the specificity of the interaction and suggest a physiological role for the interaction between AF-6 and Eph receptors in the brain.


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)

Endogenous AF-6 coimmunoprecipitates with EphB2  (a) and EphB3 (b) receptors from whole adult rat brain extracts  prepared in HO-buffer or NL-buffer, respectively but not in  RIPA-buffer. Whole adult rat brain extracts were immunoprecipitated (IP) with either monoclonal anti–AF-6 antibody, polyclonal rabbit antisera against EphB2 (EphB2), EphB3 (EphB3),  or with the monoclonal M2 antibody, which recognizes the  FLAG epitope. The immunoprecipitates were probed on a Western blot (WB) using the monoclonal anti-AF-6 antibody. AF-6 is  recognized as a double band in the rat brain. This has also been  observed by others (Yamamoto et al., 1997).
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Related In: Results  -  Collection

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Figure 8: Endogenous AF-6 coimmunoprecipitates with EphB2 (a) and EphB3 (b) receptors from whole adult rat brain extracts prepared in HO-buffer or NL-buffer, respectively but not in RIPA-buffer. Whole adult rat brain extracts were immunoprecipitated (IP) with either monoclonal anti–AF-6 antibody, polyclonal rabbit antisera against EphB2 (EphB2), EphB3 (EphB3), or with the monoclonal M2 antibody, which recognizes the FLAG epitope. The immunoprecipitates were probed on a Western blot (WB) using the monoclonal anti-AF-6 antibody. AF-6 is recognized as a double band in the rat brain. This has also been observed by others (Yamamoto et al., 1997).
Mentions: Finally, to confirm that these findings are also biologically relevant in the living organism, we tested whether complexes of endogenous AF-6 with Eph receptors occur in total rat brain lysates. Indeed, endogenous AF-6 coimmunoprecipitated with both EphB2 and EphB3, but not when the total rat brain extract was immunoprecipitated with an anti-FLAG M2 antibody (Fig. 8, a and b, left). The pellet, obtained after the first extraction, was reextracted with the stronger RIPA buffer, known to disrupt protein–protein interactions. This treatment destroyed the interaction even though endogenous AF-6 was still precipitable (Fig. 8, a and b, right). This further demonstrates the specificity of the interaction and suggest a physiological role for the interaction between AF-6 and Eph receptors in the brain.

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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