Cytoplasmic components of acetylcholine receptor clusters of cultured rat myotubes: the 58-kD protein.
A 58-kD protein, identified in extracts of postsynaptic membrane from Torpedo electric organ, is enriched at sites where acetylcholine receptors (AChR) are concentrated in vertebrate muscle (Froehner, S. C., A. A. Murnane, M. Tobler, H. B. Peng, and R. Sealock. 1987. J. Cell Biol. 104:1633-1646). We have studied the 58-kD protein in AChR clusters isolated from cultured rat myotubes. Using immunofluorescence microscopy we show that the 58-kD protein is highly enriched at AChR clusters, but is also present in regions of the myotube membrane lacking AChR. Within clusters, the 58-kD protein codistributes with AChR, and is absent from adjacent membrane domains involved in myotube-substrate contact. Semiquantitative fluorescence measurements suggest that molecules of the 58-kD protein and AChR are present in approximately equal numbers. Differential extraction of peripheral membrane proteins from isolated AChR clusters suggests that the 58-kD protein is more tightly bound to cluster membrane than is actin or spectrin, but less tightly bound than the receptor-associated 43-kD protein. When AChR clusters are disrupted either in intact cells or after isolation, the 58-kD protein still codistributes with AChR. Clusters visualized by electron microscopy after immunogold labeling and quick-freeze, deep-etch replication show that, within AChR clusters, the 58-kD protein is sharply confined to AChR-rich domains, where it is present in a network of filaments lying on the cytoplasmic surface of the membrane. Additional actin filaments overlie, and are attached to, this network. Our results suggest that within AChR domains of clusters, the 58-kD protein lies between AChR and the receptor-associated 43-kD protein, and the membrane-skeletal proteins, beta-spectrin, and actin.
Bloch
R J
RJ
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
Resneck
W G
WG
O'Neill
A
A
Strong
J
J
Pumplin
D W
DW
D000818:Animals; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D004557:Electric Organ / Q000166:cytology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D016253:Microscopy, Immunoelectron; D009132:Muscles / Q000737:chemistry* / Q000648:ultrastructure; D051381:Rats; D011950:Receptors, Cholinergic / Q000032:analysis*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D011950:Receptors, Cholinergic