Identification of natural ligands for SH2 domains from a phage display cDNA library

The cytoplasmic domain of the Fc gamma receptor IIB (Fc gamma RIIB) can be successfully displayed on the surface of filamentous phage, and after phosp horylation in vitro, can interact specifically with the SH2 domains of SHP- 2, a cytoplasmic tyrosine phosphatase. When full-length Fc gamma RIIB is ex pressed on phage, however, this interaction is greatly compromised, illustr ating that characteristics of the full-length sequence are not well tolerat ed by the phage display system. Many associations in cell physiology are dr iven by similar interactions involving small modular binding domains or lig ands, and so a fragmented cDNA library will facilitate display of such doma ins free of sequences which compromise their expression. A fragmented leuko cyte cDNA display library of 10(8) clones was constructed. This library was phosphorylated in vitro with fyn kinase and was selected against the tande m SH2 domains of SHP-2 in the search for additional ligands. A depletion st rategy to remove non-specific clones was employed, using SHP-2 Sepharose, p rior to ill vitro phosphorylation and selection. This permitted the emergen ce of clones encoding the cytoplasmic domain of PECAM-1, another natural li gand for SHP-2. The importance of dual phosphorylation of tyrosine residues at positions 663 and 686 was confirmed in competition ELISA experiments us ing phosphorylated phage and synthetic peptides. Thus, phage display of fra gmented cDNA libraries permits the identification and characterisation of p hosphorylated ligands of modular binding domains based on their functional interaction. (C) 2000 Academic Press.