Multiple Amphiphysin II Splice Variants Display Differential Clathrin Binding: Identification of Two Distinct Clathrin‐Binding Sites

: Amphiphysin I and II are nerve terminal‐enriched proteins that display src homology 3 domain‐mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin‐amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44‐amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin‐binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice‐specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin‐amphiphysin interactions in protein targeting during endocytosis.