IDENTIFICATION OF RESIDUES THAT CONTROL SPECIFIC BINDING OF THE SHC PHOSPHOTYROSINE-BINDING DOMAIN TO PHOSPHOTYROSINE SITES

The She adaptor protein contains two phosphotyrosine [Tyr(P)]binding m odules-an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain, We have compared the ability of the She PTB d omain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding, The She PTB domain binds with high affinity to a phospho peptide corresponding to the nerve growth factor receptor Tyr-490 auto phosphorylation site, Analysis of individual residues within this moti f indicates that the Asn at position -3 [with respect to Tyr(P)], in a ddition to Tyr(P), is critical for PTB binding, while the Pro at posit ion -2 plays a less significant role. A hydrophobic amino acid 5 resid ues N-terminal to the Tyr(P) is also essential for high-affinity bindi ng, In contrast, the She PTB domain does not bind stably to the Asn-Pr o-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin recepto r Tyr-960 phosphopeptide to the PTB domain, These results suggest that while the She PTB domain recognizes a core sequence of Asn-Pro-Xaa-Ty r(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-rece ptor interactions, An analysis of residues in the She PTB domain requi red for binding to Tyr(P) sites identified a specific and evolutionari ly conserved Arg (Arg-175) that is uniquely important for ligand bindi ng and is potentially involved in Tyr(P) recognition.