Regulation of muscle acetylcholine receptor turnover by β subunit tyrosine phosphorylation

Abstract

At the neuromuscular junction (NMJ), the postsynaptic localization of muscle acetylcholine receptor (AChR) is regulated by neural signals and occurs via several processes including metabolic stabilization of the receptor. However, the molecular mechanisms that influence receptor stability remain poorly defined. Here, we show that neural agrin and the tyrosine phosphatase inhibitor, pervanadate slow the degradation of surface receptor in cultured muscle cells. Their action is mediated by tyrosine phosphorylation of the AChR β subunit, as agrin and pervandate had no effect on receptor half-life in AChR-β(3F/3F) muscle cells, which have targeted mutations of the β subunit cytoplasmic tyrosines. Moreover, in wild type AChR-β(3Y) muscle cells, we found a linear relationship between average receptor half-life and the percentage of AChR with phosphorylated β subunit, with half-lives of 12.7 and 23 h for nonphosphorylated and phosphorylated receptor, respectively. Surprisingly, pervanadate increased receptor half-life in AChR-β(3Y) myotubes in the absence of clustering, and agrin failed to increase receptor half-life in AChR-β(3F/3F) myotubes even in the presence of clustering. The metabolic stabilization of the AChR was mediated specifically by phosphorylation of βY390 as mutation of this residue abolished β subunit phosphorylation but did not affect δ subunit phosphorylation. Receptor stabilization also led to higher receptor levels, as agrin increased surface AChR by 30% in AChR-β(3Y) but not AChR-β(3F/3F) myotubes. Together, these findings identify an unexpected role for agrin-induced phosphorylation of β(Y390) in downregulating AChR turnover. This likely stabilizes AChR at developing synapses, and contributes to the extended half-life of AChR at adult NMJs.

Control of Neuronal Excitability by Cell Surface Receptor Density and Phosphoinositide Metabolism

“…Previous biochemical analysis has shown that superior cervical ganglion neurons have a resting surface density of approximately 16 receptors/μm 2 ( Kruse et al, 2016 ). Receptor turnover in neuronal cells is highly dynamic though ( Devreotes and Fambrough, 1975 ; Rudell and Ferns, 2013 ) and variations in receptor density are therefore likely to cause different amounts of phospholipase C activation and subsequent hydrolysis of PI(4,5)P 2 as well as generation of second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate. We used our model to develop predictions how alterations in M 1 R surface density would influence hydrolysis of PI(4,5)P 2 and action potential generation.…”

An Inside Job: Molecular Determinants for Postsynaptic Localization of Nicotinic Acetylcholine Receptors

“…Rapsyn binding to the βY390 motif may be especially important, as mutation of this site increases the detergent extractability of the receptor, implying that it is anchored less effectively to the postsynaptic cytoskeleton [ 88 , 89 ]. Third, rapsyn increases the metabolic stability of nAChR in cultured heterologous and muscle cells [ 92 , 93 ] and at the NMJ [ 71 , 73 ]. In heterologous cells this appears to be mediated by rapsyn binding to the MA-helix, as it does not require beta subunit tyrosine phosphorylation [ 92 ].…”

“…In heterologous cells this appears to be mediated by rapsyn binding to the MA-helix, as it does not require beta subunit tyrosine phosphorylation [ 92 ]. In muscle cells, agrin-induced stabilization of the nAChR requires βY390 phosphorylation but not clustering, suggesting that it involves rapsyn binding specifically to the Y390 motif [ 93 ]. Presumably both rapsyn interactions would combine to stabilize the nAChR and decrease its turnover at the NMJ ( Figure 2 C).…”

Cell-surface translational dynamics of nicotinic acetylcholine receptors

“…Phosphorylation occurs specifically at tyrosine residue 390 of the β subunit, and is induced by agrin. This unexpected role of agrin in downregulating AChR turnover most likely stabilizes nAChRs at developing synapses and contributes to the extended half-life of the receptors at adult NMJs (Rudell and Ferns, 2013). Phosphorylation-induced global conformational changes have been recently proposed to be a universal phenomenon among LGICs, and also to play a role in pathophysiological phenomena such as nicotine addiction in the specific case of the nAChR (Talwar and Lynch, 2014).…”