Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.
The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain 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 Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-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 receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.
Mesh Terms:
3T3 Cells, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Amino Acid Sequence, Animals, Antigens, Polyomavirus Transforming, Binding Sites, CHO Cells, Cricetinae, Ligands, Mice, Models, Structural, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Phosphotyrosine, Protein Biosynthesis, Proteins, Receptor, Insulin, Receptors, Nerve Growth Factor, Recombinant Fusion Proteins, Shc Signaling Adaptor Proteins, Src Homology 2 Domain-Containing, Transforming Protein 1, Transfection
3T3 Cells, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport, Amino Acid Sequence, Animals, Antigens, Polyomavirus Transforming, Binding Sites, CHO Cells, Cricetinae, Ligands, Mice, Models, Structural, Molecular Sequence Data, Phosphopeptides, Phosphorylation, Phosphotyrosine, Protein Biosynthesis, Proteins, Receptor, Insulin, Receptors, Nerve Growth Factor, Recombinant Fusion Proteins, Shc Signaling Adaptor Proteins, Src Homology 2 Domain-Containing, Transforming Protein 1, Transfection
Proc. Natl. Acad. Sci. U.S.A.
Date: Feb. 06, 1996
PubMed ID: 8577769
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