Direct demonstration of MuSK involvement in acetylcholine receptor clustering through identification of agonist ScFv

Nat Biotechnol. 1997 Aug;15(8):768-71. doi: 10.1038/nbt0897-768.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / metabolism
  • Bacteriophage M13
  • Blotting, Western
  • Cell Line
  • Dimerization
  • Enzyme Activation
  • Flow Cytometry
  • Gene Library
  • Humans
  • Immunoglobulin Fc Fragments / metabolism
  • Immunoglobulin Fragments / metabolism*
  • Immunoglobulin Variable Region / metabolism*
  • Mice
  • Molecular Sequence Data
  • Muscle Fibers, Skeletal / metabolism
  • Neuromuscular Junction / metabolism
  • Neuromuscular Junction / ultrastructure
  • Phosphorylation
  • Protein Engineering
  • Receptor Aggregation
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Receptors, Cholinergic / metabolism*
  • Tyrosine / metabolism

Substances

  • Antibodies
  • Immunoglobulin Fc Fragments
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Receptors, Cholinergic
  • Tyrosine
  • MUSK protein, human
  • Receptor Protein-Tyrosine Kinases