The EMBO Journal Vol. 19,pp. 1263-1271, 2000, Copyright © European Molecular Biology Organization

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

Xin-Kang Tong, Natasha K. Hussain, Elaine de Heuvel, Alexei Kurakin, Elia Abi-Jaoude, Christopher C. Quinn, Michael F. Olson, Richard Marais, Danny Baranes, Brian K. Kay and Peter S. McPherson^1,3

¹ Department of Neurology and Neurosurgery, Montreal Neurological Institute and ³ Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 2B4, Canada, ² Department of Pharmacology, University of Wisconsin, Madison, WI 53706, ⁴ Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA and ⁵ CRC Center for Cell and Molecular Biology, Institute of Cancer Research, London SW3 6JB, UK
⁶   Corresponding author
   e-mail: mcpm{at}musica.mcgill.ca

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.