Parallel Dimers and Anti-parallel Tetramers Formed by Epidermal Growth Factor Receptor Pathway Substrate Clone 15 (EPS15)

The recently discovered localization of epidermal growth factor receptor pathway substrate clone 15 (Eps15) to plasma membrane clathrin-coated pits and its constitutive association with the endocytic clathrin adaptor protein complex, AP-2, strongly suggest that Eps15 has an important role in the pathway of clathrin-dependent endocytic traffic. We report here that Eps15 forms dimers and tetramers of distinct shape. The Eps15 dimer is an elongated molecule, 32 nm in length. There is a globular “head” at one end of the molecule and an extended “stalk” of 25 nm which is kinked at about 17 nm away from the head. In the Eps15 dimer, two subunits are arranged parallel to each other, so that the head corresponds to two side by side copies of the N-terminal region I, which contains the three Eps15 homology domains. The proximal part of the stalk is the coiled-coil central region II containing 20 heptad repeats. The kink is at the boundary between region II and the C-terminal region III, which contains the AP-2 binding site, 15 aspartic-proline-phenylalanine repeats, and proline-rich Src homology domain ligand sites. The Eps15 tetramer has a “dumbbell” shape, ∼31 nm in length; it is formed by the anti-parallel association of two Eps15 dimers. Formation of these Eps15 tetramers appears to require contacts between regions I of one dimer and regions III of a second apposing dimer. The extended shapes of the Eps15 dimers and tetramers suggest how Eps15 oligomers are located in the clathrin coat. We discuss the implications for accessibility to partners and for proposed functions of Eps15.