EH domain-dependent interactions between Eps15 and clathrin-coated vesicle protein p95.
The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.
McPherson
P S
PS
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Quebec, Canada. mcpm@musica.mcgill.ca
de Heuvel
E
E
Phillie
J
J
Wang
W
W
Sengar
A
A
Egan
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002966:Clathrin; D018699:Coated Vesicles / Q000737:chemistry*; D004705:Endocytosis; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid
D002135:Calcium-Binding Proteins; D002966:Clathrin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases