Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.
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.
Yamabhai
M
M
Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.
Hoffman
N G
NG
Hardison
N L
NL
McPherson
P S
PS
Castagnoli
L
L
Cesareni
G
G
Kay
B K
BK
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D034281:Dynamins; D004705:Endocytosis; D020558:GTP Phosphohydrolases / Q000378:metabolism; D015723:Gene Library; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009842:Oligopeptides; D009865:Oocytes; D019151:Peptide Library; D010750:Phosphoproteins / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010940:Plant Proteins; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C486206:EPS15 protein, human; C082729:Eps15 protein, mouse; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494372:Hspa8 protein, mouse; C088757:IBP1 protein, Zea mays; C088758:IBP2 protein, Zea mays; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D019151:Peptide Library; D010750:Phosphoproteins; D010940:Plant Proteins; D011506:Proteins; C115919:intersectin 1; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D034281:Dynamins