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

The AF-6/afadin protein, which contains a single PDZ domain, forms a periph eral component of cell membranes at specialized sites of cell-cell junction s. To identify potential receptor-binding targets of AF-6 we screened the P DZ domain of AF-6 against a range of COOH-terminal peptides selected from r eceptors 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 C OOH terminus both in vitro and in vivo. Cotransfection of a green fluoresce nt protein-tagged AF-6 fusion protein with full-length Eph receptors into h eterologous cells induces a clustering of the Eph receptors and AF-6 at sit es of cell-cell contact, Immunohistochemical analysis in the adult rat brai n reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus, Furthermore, AF-6 is a sub strate for a subgroup of Eph receptors and phosphorylation of AF-6 is depen dent on a functional kinase domain of the receptor. The physical interactio n of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipit ation from whole rat brain lysates. AF-6 is a candidate for mediating the c lustering of Eph receptors at postsynaptic specializations in the adult rat brain.