Abstract
The N-terminal 200 amino acids of SHC constitute a unique phosphotyrosine (Tyr(P)) interaction (PI) domain that shows no significant sequence similarity to the other Tyr(P)-recognizing module, the SH2 domain. We describe the thermodynamic parameters characterizing PI domain binding to various tyrosyl phosphopeptides, using isothermal titration calorimetry. The PI domain forms 1:1 complexes of similar affinity with a 12-mer peptide (ISLDNPDpYQQDF) derived from Tyr-1148 of the epidermal growth factor receptor (EGFR) (KD = 28 nm) and an 18-mer (LQGHIIENPQpYFSDACVH) derived from Tyr-490 of Trk (KD = 42 nM). Binding of the EGFR-derived peptide was largely enthalpy-driven at 25 degrees C, while Trk490 peptide binding was entropy-driven. Based on the change in heat capacity upon binding, approximately 700 A2 of nonpolar surface was estimated to be buried upon interaction. Alteration of the Asn or Pro to Ala in the NPXpY motif of the EGFR Tyr-1148 peptide increased the KD of PI domain interactions to 238 and 370 nM, respectively. Alteration of a Leu at position -5 (with respect to Tyr(P)) in the EGFR peptide to Gly also reduced the binding affinity (KD = 580 nM). It is proposed that the PI domain recognizes the beta1 turn that is found in NPXpY-containing peptides and also interacts with a larger segment of the peptide than seen for SH2 domains.
Highlights
SHC, an adapter protein involved in a number of signaling pathways, consists of an N-terminal region and a C-terminal SH21 domain separated by a region rich in glycine and proline residues
Phosphopeptides corresponding to this region of epidermal growth factor receptor (EGFR) were synthesized, and their ability to compete for PI domain binding to EGFR was analyzed
From studies reported here as well as from others [21,22,23,24, 29, 31], it is clear that SHC contains two phosphotyrosine recognition regions: the PI domain at the N terminus and the SH2 domain at the C terminus
Summary
SHC, an adapter protein involved in a number of signaling pathways, consists of an N-terminal region and a C-terminal SH21 domain separated by a region rich in glycine and proline residues. Binding of Grb to activated growth factor receptors via SHC recruits Sos to the membrane surface, where it exchanges GDP for GTP on Ras, leading to its activation. Data base searches have identified a number of proteins with regions that show significant sequence similarities with the SHC PI domain [26]. The function of these conserved regions is not yet understood. The interaction of the PI domain with NPXpY-containing phosphopeptides is one of the highest affinity interactions far observed in the growth factor induced intracellular signaling process and shows an exquisite dependence on the sequence of the peptides studied
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