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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

The Junction-associated Protein AF-6 Interacts and Clusters with Specific Eph Receptor Tyrosine... 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. postsynaptic clustering PDZ domains receptor tyrosine kinases neuron physiology Ras-binding protein Footnotes C.M. Hovens's present address is Dept. of Surgery, Royal Melbourne Hospital, Clinical Sciences Building, Parkville VIC. 3050 Australia. Abbreviations used in this paper: aa amino acid FLAG AF-6 PDZ FLAG epitope-tagged AF-6 PDZ domain construct GFP green fluorescent protein GST glutathione-S-transferase MAGUK membrane-associated guanylate kinase NGS normal goat serum NMDA N -methyl- d -aspartate NR2 NMDA receptor 2 PB phosphate buffer RIPA radioimmunoprecipitation assay RT room temperature RTK receptor tyrosine kinase SAP synapse-associated protein Submitted: 24 June 1998 Revision received 4 November 1998 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Rockefeller University Press

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

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References (50)

Publisher
Rockefeller University Press
Copyright
© 1999 Rockefeller University Press
ISSN
0021-9525
eISSN
1540-8140
DOI
10.1083/jcb.144.2.361
Publisher site
See Article on Publisher Site

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. postsynaptic clustering PDZ domains receptor tyrosine kinases neuron physiology Ras-binding protein Footnotes C.M. Hovens's present address is Dept. of Surgery, Royal Melbourne Hospital, Clinical Sciences Building, Parkville VIC. 3050 Australia. Abbreviations used in this paper: aa amino acid FLAG AF-6 PDZ FLAG epitope-tagged AF-6 PDZ domain construct GFP green fluorescent protein GST glutathione-S-transferase MAGUK membrane-associated guanylate kinase NGS normal goat serum NMDA N -methyl- d -aspartate NR2 NMDA receptor 2 PB phosphate buffer RIPA radioimmunoprecipitation assay RT room temperature RTK receptor tyrosine kinase SAP synapse-associated protein Submitted: 24 June 1998 Revision received 4 November 1998

Journal

The Journal of Cell Biology – Rockefeller University Press

Published: Jan 25, 1999

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