To elucidate the mechanisms involved in the localization of neurotransmitter receptors under nerve terminals, we will analyze the interactions between acetylcholine receptors (AChRs) and cytoskeletal components in the nicotinic post-synaptic membranes isolated from Torpedo electric organ. In those isolated membranes, as at the vertebrate neuromuscular junction, AChRs are constrained and unable to diffuse freely, but upon removal of peripheral proteins from that membrane, constraints on AChR mobility are effectively removed. It is our hypothesis that interactions between AChRs and peripheral membrane proteins serve to restrict receptor mobility, and we will characterize the structures of those peripheral proteins and identify the protein interactions that account for AChR immobilization. By immunological techniques four peripheral proteins (43K, 58K, 87K, and 270K) have been identified on the cytoplasmic face of the Torpedo post-synaptic membrane and also at the vertebrate neuromuscular junction. Experiments will focus on the 43K protein, which is present in stoichiometric amount with AChRs, and the 270K protein that has been shown by immunological criteria to be related to dystrophin, the protein product of the Duchenne/Becker muscular dystrophy gene locus. Immunoelectron microscopy will be used to identify the filamentous cytoskeletal elements associated with the innervated surface of the Torpedo electrocyte and to determine the disposition of 27OK/dystrophin as well as 58K and 87K proteins relative to AChR/43K, the lipid bilayer and the cytoskeleton. Biochemical and immunochemical techniques will be used to define the mechanism of association between 43K protein and AChRs and also with lipid. The 270K protein will be isolated and characterized by protein microsequencing to determine whether that protein contains all the structural domains of muscle dystrophin. Biochemical and immunochemical techniques will be used to determine whether 27OK/dystrophin interacts directly with AChRs or other integral membrane proteins of the post-synaptic membrane and to determine whether it binds to the actin cytoskeleton. To determine whether 58K and 87K proteins are related to previously described muscle proteins, they will be isolated and characterized by protein microsequencing.
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