CLUSTERING AND IMMOBILIZATION OF ACETYLCHOLINE-RECEPTORS BY THE 43-KDPROTEIN - A POSSIBLE ROLE FOR DYSTROPHIN-RELATED PROTEIN

Recombinant acetylcholine receptors (AChRs) expressed on the surface o f cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D . Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC) . 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich memb rane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskel eton. Myotubes of the mouse cell line C2 spontaneously display occasio nal AChR/43k-rich membrane domains that ranged in diameter up to 15 mu m, but expressed many more when 43k was overexpressed following transf ection of 43k cDNA. However, the membrane domains induced by recombina nt 43k were predominantly small (less-than-or-equal-to 2 mum). We were then interested in whether the cytoskeletal component, dystrophin rel ated protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fair-brot her, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthe rs, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofl uorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myot ubes. Surprisingly, anti-DRP staining was concentrated mainly at the l arge, but not the small AChR clusters on C2 myotubes suggesting that D RP may be principally involved in permitting the growth of AChR cluste rs.