Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains

doi:10.1074/jbc.273.47.31401

Intersectin, a Novel Adaptor Protein with Two Eps15 Homology and Five Src Homology 3 Domains*

From the ^‡Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, the ^§University of North Carolina, Chapel Hill, North Carolina 27599-3280, the ^¶Duke University Medical Center, Durham, North Carolina 27710, the ^‖Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4, Canada, and the ^‡Department of Biology, Enrico Calef, University of Rome Tor Vergata, Via Della Ricerca Scientifica, 00173 Rome, Italy

Abstract

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX _1–2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF032118, AF057287, AF057285, and AF057286.
↵** Financial support provided by the Natural Sciences and Engineering Research Council (Grant 197685). Scholar of the Medical Research Council of Canada and an Alfred P. Sloan Research Fellow.
↵§§ Support provided by the Italian Association for Cancer Research and the CNR.
↵¶¶ Support provided by the Cytogen Corp. (Princeton, NJ), the University of North Carolina Honors Program, the Muscular Dystrophy Association, and the University of Wisconsin-Madison Graduate School. To whom correspondence should be addressed. Tel.: 608-265-5218; Fax: 608-262-1257; E-mail: bkkay{at}facstaff.wisc.edu.
↵2 B. K. Kay, M. Yamabhai, B. Wendland, and S. D. Emr, submitted for publication.
↵3 B. K. Kay, N. G. Hoffman, and N. L. Hardison, unpublished results.
↵4 Guipponi, M., Scott, H. S., Chen, H., Schebesta, A., Rossier, C., and Antonarakis, S. E. (1998) Genomics, in press.
Abbreviations:

EH

Eps15 homology

AP

alkaline phosphatase

COLT

cloning of ligand targets

Dap

dynamin-associated protein

GST

glutathione S-transferase

Ibp

Intersectin-binding protein

MP90

mitotic phosphoprotein of 90 kDa

RAB

Rev-associated binding protein

Rip

Rev interacting protein

SH3

Src homology 3

PAGE

polyacrylamide gel electrophoresis.
- Received May 14, 1998.
- Revision received September 2, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.