INHIBITION OF FGF-STIMULATED PHOSPHATIDYLINOSITOL HYDROLYSIS AND NEURITE OUTGROWTH BY A CELL-MEMBRANE PERMEABLE PHOSPHOPEPTIDE

Background: Activated receptor tyrosine kinases bind downstream effect or molecules with high affinity. Provided that they can be introduced into cells, peptides corresponding to these high-affinity sites should be able to compete for the interaction and thereby inhibit specific s ignal transduction cascades. The high-affinity binding site for phosph olipase C gamma (PLC gamma) on the activated fibroblast growth factor receptor (FGFR) is centred around the tyrosine at position 766 ((766)T yr), and peptides corresponding to this site inhibit PLC gamma binding to the receptor in vitro. A 16 amino-acid peptide from the third heli x of the Antennapedia homeodomain protein has recently been shown to b e able to act as an internalization vector that can deliver other pept ides into cells. Here, we have designed a peptide that contains both t he internalization sequence and the FGFR high-affinity binding site fo r PLC gamma, and tested it in cultures of cerebellar neurons for its a bility to inhibit the activation of PLC gamma by basic FGF. Results: T he peptide containing the FGFR high-affinity binding site for PLC gamm a inhibited phospholipid hydrolysis stimulated by basic FGF with a max imal effect at 1 mu g ml(-1). Phosphorylation of (766)Tyr was required for this effect. The phosphorylated peptide had no effect on phosphol ipid hydrolysis stimulated by platelet-derived growth factor, neurotro phin-3 and bradykinin. The phosphorylated peptide also inhibited neuri te outgrowth stimulated by FGF, but had no effect on neurite outgrowth stimulated by agents that activate the FGFR signal transduction casca de downstream from the activation of PLC gamma. Conclusions: Cell-perm eable peptides can be designed that inhibit the function of receptor t yrosine kinases. In this context we have developed a peptide that prev ents the FGFR from activating PLC gamma, and have used this peptide to obtain the first direct evidence that activation of PLC gamma is requ ired for the neurite outgrowth response stimulated by basic FGF.