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 phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely 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 displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.
Cestra
G
G
Dipartimento di Biologia, Università di Roma Tor Vergata, Rome 00133, Italy.
Castagnoli
L
L
Dente
L
L
Minenkova
O
O
Petrelli
A
A
Migone
N
N
Hoffmüller
U
U
Schneider-Mergener
J
J
Cesareni
G
G
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019151:Peptide Library; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011392:Proline / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009419:Nerve Tissue Proteins; D019151:Peptide Library; C107640:SH3GL2 protein, human; C075637:amphiphysin; D011392:Proline; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases