The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphat idylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the en dophilin family. These interactions are mediated by SH3 domains and are bel ieved to play a regulatory role in synaptic vesicle recycling, We have prec isely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage disp layed peptide libraries, substitution analyses of peptides synthesized on c ellulose membranes, and a peptide scan spanning a 252-residue long synaptoj anin fragment, we have concluded that amphiphysin binds to two sites, PIRPS R and PTIPPR, whereas endophilin has a distinct preferred binding site, PKR PPPPR, The comparison of the results obtained by phage display and substitu tion analysis permitted the identification of proline and arginine at posit ions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determin ants of the recognition specificity mediated by the SH3 domain of amphiphys in I. More complex is the structural rationalization of the preferred endop hilin ligands where SH3 binding cannot be easily interpreted in the framewo rk of the "classical" type I or type II SH3 binding models. Our results sug gest that the binding repertoire of SH3 domains may be more complex than or iginally predicted.