Modulation of nicotinic acetylcholine receptor turnover by tyrosine phosphorylation in rat myotubes Anna Sava a , Ilaria Barisone a , Debora Di Mauro b , Guido Fumagalli a, * , Carlo Sala c a Department of Medicine & Public Health, Section of Pharmacology, School of Medicine, University of Verona, Policlinico Borgo Roma, 37134 Verona, Italy b Department of Biomorphology, University of Messina, V.le Gazzi, 90125 Messina, Italy c CNR Center of Cellular and Molecular Pharmacology, via Vanvitelli 32, 20129 Milan, Italy Received 4 July 2001; received in revised form 20 August 2001; accepted 24 August 2001 Abstract The muscle nicotinic acetylcholine receptor (AChR) turns over at different rates depending on stage of synaptogenesis and innervation. Tyrosine phosphorylation modulates desensitization, interaction with cytoskeleton and lateral mobility in the membrane of AChR. To determine whether tyrosine phosphorylation also modulates the turnover of AChR, myotubes in vitro were exposed to the tyrosine phosphatase inhibitor pervanadate. Our data indicate that a transient increase of phosphotyrosine levels stabilized a fraction of AChRs. The effects were limited to the non-1 subunit-contain- ing AChRs already present in the membrane. Tyrosine phosphorylation of the receptor occurred on the b subunit, was transient and stable molecules were not selectively tyrosine phosphorylated. The data indicate that modulation of phosphotyrosine levels in muscle cells provides signals to control AChR metabolic stability. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nicotinic acetylcholine receptor; Neuromuscular junction; Turnover; Myotubes; Tyrosine phosphatases; Pervanadate; Bioti- nylation In skeletal muscles trophic factors such as agrin and ARIA/Neuregulin control the formation and maintenance of the neuromuscular junction (NMJ) by triggering the accu- mulation of the acetylcholine receptor (AChR), ion channels and specific cytoskeletal proteins in the postsynaptic membrane (for review see Ref. [12]). Their effects are mediated by tyrosine kinase receptors and accumulation of phosphotyrosine residues has been detected at the NMJ as well as at aggregates of AChRs that spontaneously form in vitro in aneurally cultured myotubes [3,9]. Aggregated AChRs have limited lateral mobility [15], a condition that can also be induced by the tyrosine phosphatase inhibitor pervanadate [7]. The rapid postnatal growth of the NMJ is also sustained by metabolic stabilization of AChRs in the postsynaptic membrane; indeed, the rapidly degrading receptors present in the embryonic muscle are stabilized and/or replaced by metabolically stable AChRs at birth [13]. A number of studies has shown that two metabolically distinct forms of AChR exist in cultured myotubes and, in vivo, at the adult NMJ (for review see Ref. [11]). The half-lives and the rela- tive amounts of both unstable receptors (Rr, half-life ~1 day) and stable receptors (Rs, half-life of 3–10 days) in embryonic and adult muscles are selectively controlled by innervation, muscle activity, intracellular calcium concen- tration, cAMP and activation of purinergic P2X receptor [1,4,5,8,11]. Whether modulation of phosphotyrosine levels plays a role in the metabolic stabilization of AChR indepen- dently of muscle activity is the open question addressed by this study. The study was done on skeletal muscle cells obtained by collagenase (0.05%; Sigma, St Louis, MO) digestion of hindlimb muscles of Sprague–Dawley rat embryos (E18), plated on dishes coated with 0.7 mg/cm 2 Matrigel (Becton Dickinson, Bedford, MA) and cultured in DMEM supple- mented with 10% horse serum [10]. To determine AChR metabolic properties, myotubes were labelled 6 days after plating with 10 nM 125 I-a-bungarotoxin ( 125 I-BTX; Amer- sham, Buckinghamshire, UK; specific activity .200 Ci/ mmol). Non-specific binding was determined by preincubat- ing with a 100-fold excess of non-radioactive BTX. Decay Neuroscience Letters 313 (2001) 37–40 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)02244-3 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 139-045-8027-605; fax: 139-045- 581-111. E-mail address: gfumagalli@sfm.univr.it (G. Fumagalli).