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 function using a library of human single chain variable region antibodies (scFv) that can be displayed on M13 phage or expressed as soluble protein. A panel of 21 independent MuSK-specific scFv, identified in a screen for binding to MuSK-Fc immunoadhesin, were examined for ability to induce proliferation in a factor dependent cell line (Ba/F3) through a chimeric receptor, MuSK-Mpl. 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 myotube cell line induced AChR clustering and tyrosine phosphorylation. These results provide direct evidence that MuSK activation is capable of triggering a key event in neuromuscular junction formation and further demonstrate that large libraries of phage-displayed scFv provide a robust method for generating highly specific agonist agents.
Xie
M H
MH
Department of Molecular Biology, Genentech, Inc., San Francisco, CA 94080, USA.
Yuan
J
J
Adams
C
C
Gurney
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D017104:Bacteriophage M13; D015153:Blotting, Western; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D015202:Protein Engineering; D011940:Receptor Aggregation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011950:Receptors, Cholinergic; D014443:Tyrosine; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases