Mapping of the molecular determinants involved in the interaction between eps15 and AP-2.
eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans approximately 80 amino acids. At least three molecular determinants, located at residues 650-660, 680-690, and 720-730, are involved in the binding. AP-2 binds to eps15 through its alpha subunit (alpha-adaptin); in particular, the COOH-terminal region of alpha-adaptin, the so-called alpha-ear, contains the eps15 binding region.
Iannolo
G
G
Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
Salcini
A E
AE
Gaidarov
I
I
Goodman
O B
OB
Baulida
J
J
Carpenter
G
G
Pelicci
P G
PG
Di Fiore
P P
PP
Keen
J H
JH
D016475:3T3 Cells; D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics
D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins