THE ROLE OF THE CYTOPLASMIC DOMAINS OF INDIVIDUAL SUBUNITS OF THE ACETYLCHOLINE-RECEPTOR IN 43 KDA PROTEIN-INDUCED CLUSTERING IN COS CELLS

The 43 kDa protein, a cytoplasmic peripheral membrane protein, is clos ely associated with the acetylcholine receptor (AChR) at the neuromusc ular junction, where it is thought to anchor the receptor in the posts ynaptic membrane. We have used the 43 kDa protein-induced clustering o f AChRs that occurs when both proteins are transiently expressed in CO S cells to investigate which parts of the AChR might interact with the 43 kDa protein. By constructing chimeric subunits, we showed that the cytoplasmic domains of neither the epsilon nor delta subunits are req uired for 43 kDa protein-induced clustering. Systematic mutational ana lysis of the long cytoplasmic loops of the alpha and beta subunits sho wed that most of the loops can be altered without affecting the abilit y of the AChR to be clustered; in each case, however, one or more sequ ences could not be tested, because mutation in these regions prevented AChR assembly. Our results suggest either that these regions are invo lved in clustering or that the 43 kDa protein can interact with multip le, alternative sites on the cytoplasmic surface of the AChR. Our expe riments also show that the postulated sites of tyrosine phosphorylatio n in the beta subunit and of serine phosphorylation in the a subunit c an be mutated without affecting 43 kDa protein-induced AChR clustering .