Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic ve sicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain bindi ng sites for clathrin and for the clathrin adaptor AP-2, We have defined he re amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin, O verexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment t hat binds both AP-2 and clathrin resulted in a segregation of clathrin, whi ch acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15, These effects correlated with a block in clathrin -mediated endocytosis. A fragment selectively interacting with clathrin pro duced a similar effect. These results can be explained by the binding of am phiphysin to the NH2-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The inter action of amphiphysin 1 with either clathrin or AP-2 did not prevent its in teraction with dynamin, supporting the existence of tertiary complexes betw een these proteins. Together with previous evidence indicating a direct int eraction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the me mbrane to coat proteins and coat proteins to dynamin and synaptojanin.