DIRECT MEASUREMENT OF THE BINDING OF RAS TO NEUROFIBROMIN USING A SCINTILLATION PROXIMITY ASSAY

Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct bin ding are required. One such example is the relatively weak interaction between oncogenic Pas and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-fo rm of oncogenic Pas protein dissociates rapidly; hence, equilibrium bi nding must be quantitated. Scintillation proximity assay (SPA) technol ogy, a radioisotopic technique that requires no separation step, was u sed to characterize this interaction. Leu-61 Ras complexed with [H-3]G TP was generated by nucleotide exchange in the presence of a GTP-regen erating system. A SPA signal was obtained when radiolabeled Pas was mi xed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which poten tly reduces the affinity of interaction between Pas and NF1. The neutr alizing anti-Pas monoclonal antibody Y13-259 and the detergent n-dodec yl maltoside, a specific inhibitor of NF1 catalytic activity, both abo lished the SPA signal from the NF1/Ras assay but neither affected a co ntrol SPA signal in which a [H-3]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extend ed to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents. (C) 1994 Academic Press, Inc.