Determination of the Binding Specificity of the SH2 Domains of Protein Tyrosine Phosphatase SHP-1 through the Screening of a Combinatorial Phosphotyrosyl Peptide Library

Abstract

A method for the rapid identification of high-affinity ligands to Src homology-2 (SH2) domains is reported. A phosphotyrosyl (pY) peptide library containing completely randomized residues at positions -2 to +3 relative to the pY was synthesized on TentaGel resin, with a unique peptide sequence on each resin bead (total 2.5 x 10(6) different sequences). The library was screened against the biotinylated N- and C-terminal SH2 domains of protein tyrosine phosphatase SHP-1, and the beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay involving a streptavidin-alkaline phosphatase conjugate. Peptide ladder sequencing of the selected beads using matrix-assisted laser desorption ionization mass spectrometry revealed consensus sequences for both SH2 domains. The N-terminal SH2 domain strongly selects for peptides with a leucine at the -2 position; at the C-terminal side of the pY residue, it can recognize two distinct classes of peptides with consensus sequences of LXpY(M/F)X(F/M) and LXpYAXL (X = any amino acid), respectively. The C-terminal SH2 domain exhibits almost exclusive selectivity for peptides of the consensus sequence, (V/I/L)XpYAX(L/V). Several representative sequences selected from the library were individually synthesized and tested for binding to the SH2 domains by surface plasmon resonance and for their ability to stimulate the catalytic activity of SHP-1. Both experiments have demonstrated that the selected peptides are capable of binding to the SH2 domains with dissociation constants (K(D)) in the low micromolar range.

Gβγ-independent constitutive association of Gαswith SHP-1 and angiotensin II receptor AT2is essential in AT2-mediated ITIM-independent activation of SHP-1

“…These findings indicate that activation of SHP-1 by AT 2 receptor requires conformational transition of the receptor from inactive state to active state, in a way similar to G␣ protein activation but distinct from classical SHP-1 activation by immunoreceptors (11)(12)(13)(14)(15)(16)(17). In these non-GPCR receptors, phosphorylation of a tyrosine located in the ITIM is required for SHP-1 binding and activation (11)(12)(13)(14)(15)(16)(17).…”

“…In these non-GPCR receptors, phosphorylation of a tyrosine located in the ITIM is required for SHP-1 binding and activation (11)(12)(13)(14)(15)(16)(17).…”

“…4) derived from the last 11 residues of G ␣q , G ␣s , G 13 , and G ␣i proteins are introduced into N1E-115 cells by transfection with Lipofectamine reagent (Life Science). These peptides are able to block G protein coupling to GPCR (37).…”

“…The 78% increase in SHP-1 activation reflects a mixed effect of sequestration of G ␤␥ and modified binding to the receptor (38). SHP-1 binding to tyrosine-phosphorylated ITIM (I͞VxYxxL) of inhibitory receptors represents a general paradigm for SHP-1 activation (11)(12)(13)(14)(15)(16)(17). This finding is further supported by evidence that has identified sequence YxxL as a previously uncharacterized ITIM in the death domain of death receptor of the tumor necrosis factor and nerve growth factor superfamily (39).…”

“…Removal of this self-constraint imposed by the SH2 domains thus activates the SHP-1. The inhibitory receptors of the immune system such as killer cell inhibitory receptor, Fc␥ receptor type IIb1, and paired Ig-like receptors of inhibitory types that initiate an inhibitory signal of SHP-1 activation bear an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail (11)(12)(13). Stimulation of the inhibitory receptors result in tyrosine phosphorylation of the ITIM and subsequently activates SHP-1 because the SH2 domain of SHP-1 selectively binds to the phosphoylated ITIM.…”