DIRECT DEMONSTRATION OF MUSK INVOLVEMENT IN ACETYLCHOLINE-RECEPTOR CLUSTERING THROUGH IDENTIFICATION OF AGONIST SCFV

MuSK is a tyrosine kinase localized to the postsynaptic surface of the neuromuscular junction. We have searched for modulators of MuSK funct ion using a library of human single chain variable region antibodies ( scFv) that can be displayed on M13 phage or expressed as soluble prote in, A panel of 21 independent MuSK-specific scFv, identified in a scre en for binding to MuSK-Fe immunoadhesin, were examined for ability to induce proliferation in a factor dependent cell line (Ba/F3) through a chimeric receptor, MuSK-MpI, Four of the scFv induced a proliferative response, suggesting an ability to induce dimerization of MuSK, These scFv were also able to induce tyrosine phosphorylation of full-length MuSK and retained this ability when re-engineered to be expressed as authentic (and dimeric) human IgG molecules., Addition of agonist scFv to a cultured myo tube cell line induced AChR clustering and tyrosine phosphorylation. These results provide direct evidence that MuSK acti vation is capable of triggering a key event in neuromuscular junction formation and further demonstrate that large libraries of phage-displa yed scFv provide a robust method for generating highly specific agonis t agents.