Epsin binds to clathrin by associating directly with the clathrin-terminaldomain - Evidence for cooperative binding through two discrete sites

Epsin is a recently identified protein that appears to play an important ro le in clathrin-mediated endocytosis. The central region of epsin 1, the so- called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by as sociating directly with the globular appendage of the alpha subunit. We hav e found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP -2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (LMD)-L-257-LADV located within the epsin 1 DPW domain. This sequ ence, related to the known clathrin-binding sequences in the adaptor beta s ubunits, amphiphysin, and beta-arrestin, facilitates the association of eps in 1 with the terminal domain of the clathrin heavy chain, Unexpectedly, in hibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progres sively deleting DPW triplets but leaving the LMDLADV sequence intact, dimin ishes the association of clathrin in parallel with AP-2. Because the beta s ubunit of the AP-2 complex also contains a clathrin-binding site, optimal a ssociation with soluble clathrin appears to depend on the presence of at le ast two distinct clathrin-binding sites, and we show that a second clathrin -binding sequence (LVDLD)-L-480, located within the carboxyl-terminal segme nt of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting tha t they bind to different sites on the clathrin-terminal domain. The evoluti onary conservation of similar clathrin-binding sequences in several metazoa n epsin-like molecules suggests that the ability to establish multiple prot ein-protein contacts within a developing clathrin-coated bud is an importan t aspect of epsin function.