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- 2)NPFXXC), 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.
| Original language | English |
|---|---|
| Pages (from-to) | 31401-31407 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 273 |
| Issue number | 47 |
| DOIs | |
| Publication status | Published - Nov 20 1998 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. / Yamabhai, Montarop; Hoffman, Noah G.; Hardison, Nancy L.; McPherson, Peter S.; Castagnoli, Luisa; Cesareni, Gianni; Kay, Brian K.
In: Journal of Biological Chemistry, Vol. 273, No. 47, 20.11.1998, p. 31401-31407.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains
AU - Yamabhai, Montarop
AU - Hoffman, Noah G.
AU - Hardison, Nancy L.
AU - McPherson, Peter S.
AU - Castagnoli, Luisa
AU - Cesareni, Gianni
AU - Kay, Brian K.
PY - 1998/11/20
Y1 - 1998/11/20
N2 - 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- 2)NPFXXC), 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.
AB - 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- 2)NPFXXC), 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.
UR - http://www.scopus.com/inward/record.url?scp=0032553443&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032553443&partnerID=8YFLogxK
U2 - 10.1074/jbc.273.47.31401
DO - 10.1074/jbc.273.47.31401
M3 - Article
VL - 273
SP - 31401
EP - 31407
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 47
ER -