A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle.

Abstract

Abstract. In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.

Potassium channel Kir4.1 macromolecular complex in retinal glial cells

“…2B, top). Syntrophins have been shown to migrate at 58 kDa on SDS-PAGE (Froehner et al, 1987;Adams et al, 1995;Suzuki et al, 1995), which is consistent with the band seen on our blot.…”

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

“…Antibodies and Antisera-Primary antibodies used include affinitypurified rabbit polyclonal anti-Kir2.2 (6,24), mouse monoclonal anti-SAP97, anti-Mint1 antibody, and anti-dystrobrevin (BD Transduction Laboratories), mouse monoclonal anti-CASK antibody, anti-PSD-95 family antibody (pan-MAGUK; clone K28/86.2), and rabbit polyclonal anti-GluR1 (Upstate Biotechnology), rabbit polyclonal anti-SAP97 (PA-741, Affinity Bioreagents), rabbit polyclonal anti-Veli/MALS-1, -2, and -3 (Zymed Laboratories Inc.), and mouse monoclonal anti-syntrophin 1351 (25), mouse monoclonal anti-dystrophin (MANDRA1, Sigma), mouse monoclonal anti-␤-dystroglycan (Novocastra), horseradish peroxidase-linked anti-GST antibody (Santa Cruz Biotechnology). The rabbit anti-abLIM antiserum generated against a GST-abLIM fusion protein (26) (generous gift from Dr. Tiansen Li) and rabbit anti-Pals2 antiserum generated against a GST-Pals2 fusion protein (27) (generous gift from Dr. Ben Margolis) were pre-absorbed against GST-Kir2.2 fusion protein linked to glutathione-agarose prior to use in immunoblotting.…”

Tyrosine Phosphorylation of Nicotinic Acetylcholine Receptor Mediates Grb2 Binding

“…Postsynaptic membranes of this tissue have provided a rich source for the identification and characterization of many synaptic proteins, including rapsyn, syntrophin, and agrin (Sobel et al, 1977;Godfrey et al, 1984;Froehner et al, 1987). To identify potential binding partners for Grb2 in Torpedo postsynaptic membranes, we used a protein overlay assay in which GST fusion proteins were used to probe membrane proteins separated by SDS-PAGE and transferred to nitrocellulose.…”