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 (CX1-2NPFXXC), 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF032118, AF057287, AF057285, and AF057286
Comparison of frog Intersectin to GenBank and The Institute for Genomic Research data bases has identified several cDNA entries, which are extremely similar and are likely members of a gene family. This family consists of DAP160, a protein [36] recently identified in Drosophila melanogaster, and human Intersectin, which has recently been discovered in the human genome on chromosome 21.4
As a means of understanding the cellular function of Intersectin, we have identified three mouse proteins (i.e. Rev-associated binding/Rev-interacting protein (RAB/Rip), Ibp1, Ibp2) that bind to the Eps15 homology (EH) domains of Intersectin
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF032118, AF057287, AF057285, and AF057286. Another protein interaction module is the Src homology 3 (SH3) domain. Interactions between SH3 domains and PXXP sequence-containing proteins have been shown to contribute to synaptic vesicle endocytosis. Amphiphysin I, amphiphysin II, and endophilin are SH3 domain-containing proteins that are enriched in the nerve terminal and that interact through their SH3 domains with proline-rich sequences in dynamin and synaptojanin (18 –25). We have screened cDNA expression libraries of a 16-day-old mouse embryo, human prostate, and human hematopoietic cells and identified 18 different SH3 domain containphosphatase; 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
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