The main aim of this study is to determine the role played by the AChR gamma- and epsilon-subunits in regulating AChR stability and whether phosphorylation of the epsilon-subunit is involved in this process. The degradation rate of adult epsilon-containing AChRs (called Rs) that normally has a t1/2 of about 10 days accelerates upon denervation to a t1/2 of about 3 days but is restabilized by reinnervation. Elevation of intracellular cAMP mimics this effect of the nerve via a pathway involving protein kinase A. In contrast, the degradation rate of the gamma-containing or embryonic AChRs (called Rr) with a t1/2 of about 1d is unaffected by either innervation or cAMP. The molecular mechanisms underlying the differences in degradation rate is unknown. The current proposal plans to address the question of whether the epsilon-subunit is directly responsible for the appearance of the Rs AChR degradation rate and its stabilization by PKA mediated phosphorylation. A series of experiments are proposed in which cells from the myogenic cell line C2C12 will be transfected with epitope tagged epsilon-containing AChRs whose PKA phosphorylation site will be modified by site directed mutagenesis. Stable clones will be assayed to determine the degradation rates of the epitope-tagged epsilon-containing AChRs and their ability to be stabilized in the presence of elevated levels of intracellular cAMP. Chimeric constructs of the main intracellular loop of the epsilon-subunit with that of the gamma-subunit will determine if this region is sufficient to confer the slow degradation properties of the Rs AChR. The stability of the resultant receptors should establish the role of the epsilon-subunit in AChR degradation.
| Cohen, Tatiana V; Randall, William R (2004) NFATc1 activates the acetylcholinesterase promoter in rat muscle. J Neurochem 90:1059-67 |
