Limits...
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.

Show MeSH
Two-hybrid binding analysis of the AF-6  PDZ domain and COOH-terminal peptides of  various receptors in 293T cells. (a) Fold induction  of the receptor COOH termini and AF-6 PDZ interaction is compared with that of the empty  Gal4 DBD vector. Error bars represent the standard deviation of experiments performed in triplicates. (b) Table of COOH-terminal peptide sequences used for the analysis with corresponding  fold activation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132901&req=5

Figure 1: Two-hybrid binding analysis of the AF-6 PDZ domain and COOH-terminal peptides of various receptors in 293T cells. (a) Fold induction of the receptor COOH termini and AF-6 PDZ interaction is compared with that of the empty Gal4 DBD vector. Error bars represent the standard deviation of experiments performed in triplicates. (b) Table of COOH-terminal peptide sequences used for the analysis with corresponding fold activation.

Mentions: A partial clone of mouse AF-6 (amino acids [aa] 850–1,129) encompassing the PDZ domain was subcloned into the VP16 activation domain expression vector pSNATCH (Buchert et al., 1997). Oligonucleotide adaptors encoding the last 10 amino acids of the COOH termini of the various RTKs as listed in Fig. 1 were then cloned into the Gal4 DNA-binding domain mammalian expression vector pSNAG (Buchert et al., 1997). Human 293T cells from a 12-well tissue culture plate were transfected with 20 ng of the expression plasmids along with 50 ng of a chloramphenicol acetyltransferase (CAT) reporter gene construct and the cells were harvested 36–48 h later. A CAT enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions (Boehringer Mannheim).


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)

Two-hybrid binding analysis of the AF-6  PDZ domain and COOH-terminal peptides of  various receptors in 293T cells. (a) Fold induction  of the receptor COOH termini and AF-6 PDZ interaction is compared with that of the empty  Gal4 DBD vector. Error bars represent the standard deviation of experiments performed in triplicates. (b) Table of COOH-terminal peptide sequences used for the analysis with corresponding  fold activation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132901&req=5

Figure 1: Two-hybrid binding analysis of the AF-6 PDZ domain and COOH-terminal peptides of various receptors in 293T cells. (a) Fold induction of the receptor COOH termini and AF-6 PDZ interaction is compared with that of the empty Gal4 DBD vector. Error bars represent the standard deviation of experiments performed in triplicates. (b) Table of COOH-terminal peptide sequences used for the analysis with corresponding fold activation.
Mentions: A partial clone of mouse AF-6 (amino acids [aa] 850–1,129) encompassing the PDZ domain was subcloned into the VP16 activation domain expression vector pSNATCH (Buchert et al., 1997). Oligonucleotide adaptors encoding the last 10 amino acids of the COOH termini of the various RTKs as listed in Fig. 1 were then cloned into the Gal4 DNA-binding domain mammalian expression vector pSNAG (Buchert et al., 1997). Human 293T cells from a 12-well tissue culture plate were transfected with 20 ng of the expression plasmids along with 50 ng of a chloramphenicol acetyltransferase (CAT) reporter gene construct and the cells were harvested 36–48 h later. A CAT enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions (Boehringer Mannheim).

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.

Show MeSH