HIGH-AFFINITY BINDING OF THE DROSOPHILA NUMB PHOSPHOTYROSINE-BINDING DOMAIN TO PEPTIDES CONTAINING A GLY-PRO-(P)TYR MOTIF

The phosphotyrosine-binding (PTB) domain is a recently identified prot ein module that has been characterized as binding to phosphopeptides c ontaining an NPXpY motif (X = any amino acid). We describe here a nove l peptide sequence recognized by the PTB domain from Drosophila Numb ( dNumb), a protein involved in cell fate determination and asymmetric c ell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGP Y phi motif (phi represents a hydrophobic residue). A synthetic peptid e containing this sequence bound specifically to the isolated dNumb PT B domain in solution with a dissociation constant (K-d) of 5.78 +/- 0. 74 mu M. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (K-d = 1.41 +/- 0.10 mu M) when the second tyros ine in the sequence was phosphorylated. Amino acid substitution studie s of the phosphopeptide demonstrated that a core motif of sequence GP( p)Y is required for high-affinity binding to the dNumb PTB domain. Nuc lear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the pept ide. The in vitro selectivity of the dNumb PTB domain is therefore mar kedly different from those of the She and IRS-1 PTB domains, in that i t interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our r esults suggest that the PTB domain is a versatile protein module, capa ble of exhibiting varied binding specificities.