We propose to use a dominant negative genetic assay to derive an assembly pathway of the neuromuscular junction, characterized on the postsynaptic side by high density clusters of the acetylcholine receptor. Fragments of two types of genes will be used, the acetylcholine receptor genes and the 43k gene. Briefly, foreign DNA will be introduced into immature muscle cells in cultur and expressed at a high level. Then the cells will be allowed to become myotubes. Two types of receptor genes will block surface expression of the acetylcholine receptor. Second, both receptor and 43k gene fragments could interfere with clustering of the receptor on the myotube surface. Specifically, we will establish a high level expression system for foreign DNA in muscle cells. Using this, we will define the portions of the 43k protein involved in clustering, including identifying the region which interacts with the receptor beta- subunit. Similarly, the portion of the beta-subunit which crosslinks to 43k will be examined. Additionally, the role of the extracellular portions of the receptor subunits in assembly of the receptor will be analyzed. We will use alpha-bungarotoxin binding and immunological staining to assess the changes produced by the introduced gene fragments. By these analyses, we will define domains of proteins which govern the assembly of a highly complex cellular structure. Apart from scientific interest in macromolecular assembly, interest in the neuromuscular junction stems from the fact that the junction is the point at which the dystrophin protein is concentrated in the myotube, and the site of the lesion for the disease myasthenia gravis. Technology and results from this project should have direct application to gene therapy on muscular dystrophy.
