INTERSECTIN, A NOVEL ADAPTER PROTEIN WITH 2 EPS15 HOMOLOGY AND 5 SRC HOMOLOGY 3 DOMAINS

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class LT. peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a centr al coiled-coil region, and five SH3 domains. We named this protein Int ersectin, because it potentially brings together EH and SH3 domain-bin ding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NP F) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displ ayed combinatorial peptide library used. Screens of a mouse embryo cDN A library with the EH domains of Intersectin yielded clones for the Re v-associated binding/Rev-interacting protein (RAB/Rip) and two novel p roteins, which we named Intersectin-binding proteins (lbps) 1 and 2. A ll three proteins contain internal and C-terminal NPF peptide sequence s, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Del etion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated E H domain binding, whereas fusion of the same peptide sequence to gluta thione S-transferase generated strong binding to the EH domains of Int ersectin, Several experiments support the conclusion that the free car boxylate group contributes to binding of the NPFL motif at the C termi nus of RAB/Rip to the EH domains of Intersectin, Finally, affinity sel ection experiments with the SR3 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.