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

• Published in: The Journal of cell biology Vol. 144; no. 2; pp. 361 - 371
• Main Authors: Buchert, Michael, Schneider, Stefan, Meskenaite, Virginia, Adams, Mark T., Canaani, Eli, Baechi, Thomas, Moelling, Karin, Hovens, Christopher M.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 25-01-1999; The Rockefeller University Press
• Subjects: AF-6 protein; afadin; Animals; Antibodies; Binding Sites; Brain; Brain - metabolism; Cell Line; Cell Line, Transformed; Cell Membrane - metabolism; Cells; Dogs; Eph receptors; Epithelial cells; Hippocampus - metabolism; Humans; Intercellular Junctions; Kinesin - genetics; Kinesin - metabolism; Myosins - genetics; Myosins - metabolism; N methyl D aspartate receptors; Neurons; P branes; Precipitin Tests; Rats; Receptor Protein-Tyrosine Kinases - genetics; Receptor Protein-Tyrosine Kinases - metabolism; Receptor, EphA7; Receptor, EphB2; Receptors; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Regular; Substrate Specificity; Synapses; Tight junctions; Transfection
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.144.2.361

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.