Postsynaptic membranes from the electric organ of Torpedo californica, rich in the nicotinic acetylcholine receptor, were shown to contain an endogenous tyrosine protein kinase. This endogenous kinase phosphorylated three major proteins with molecular masses corresponding to 50 kDa, 60 kDa, and 65 kDa. The phosphorylation of these three proteins occurred exclusively on tyrosine residues under the experimental conditions used and was abolished by 0.1% Nonidet P-40 and stimulated by Mnf+. The 50-kDa, 60-kDa, and 65-kDa phosphoproteins were demonstrated to be the 13, v, and 6 subunits, respectively, of the nicotinic acetylcholine receptor by purification of the phosphorylated receptor using affinity chromatography. The endogenous tyrosine kinase specifically phosphorylated the 13, y, and 6 subunits rapidly to a final stoichiometry of -0.5 mol of phosphate per mol of subunit. Two-dimensional phosphopeptide mapping of the phosphorylated 13, y, and 6 subunits, after limit proteolysis with trypsin or thermolysin, indicated that each subunit was phosphorylated on a single site. Locations are proposed for the amino acid residues phosphorylated on the receptor by the tyrosine-specific protein kinase and by two other protein kinases (cAMP-dependent protein kinase and protein kinase C) which phosphorylate the receptor.Protein phosphorylation has been widely accepted as one of the principal regulatory mechanisms in the control of biological processes (1). It has recently been suggested that phosphorylation of membrane receptors plays an important role in the regulation of signal transduction systems. The nicotinic acetylcholine receptor (2, 3), the 3-adrenergic receptor (4), the epidermal growth factor (EGF) receptor (5), the insulin receptor (6), and the platelet-derived growth factor (PDGE) receptor (7) have been demonstrated to be phosphorylated by endogenous protein kinases. The nicotinic acetylcholine receptor is phosphorylated on serine residues (8, 9), whereas the EGF receptor, the insulin receptor, and the PDGF receptor are phosphorylated on tyrosine (10-12), serine, and threonine residues (13-15). In fact, it appears that the EGF (5), insulin (16), and PDGF (7) receptors themselves are tyrosine-specific protein kinases that are stimulated by their respective physiological ligands.In View of the potential physiological significance of phosphorylation of membrane receptors, we have carried out a detailed investigation of the phosphorylation of the nicotinic acetylcholine receptor (9, 17). This receptor, a neurotransmitter-regulated ion channel, is one of the most well-characterized membrane receptors and has served as a model system for the investigation of the structure, function, and regulation of membrane receptors (18). The purified receptor is a 255-kDa polypeptide complex, which consists of four subunits, a (40 kDa), /3 (50 kDa), y (60 kDa), and 8 (65 kDa), in a stoichiometry of a2PY8 (19). This pentameric polypeptide complex is functionally complete and displays all of the known biological properties of...

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The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32Plorthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the 1 and 6 subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the 6 subunit 20-fold over basal phosphorylation and induced phosphorylation of the a subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ,LM in the presence of 35 ,uM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of 6 subunit phosphorylation was almost maximal within 5 min, whereas stimulation of a subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the 6 subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the 6 subunit increases the rate of AcChoR desensitization in rat myotubes.

Protein kinase C (PKC) is a family of enzymes involved in synapse formation and signal transduction at the neuromuscular junction. Two PKC isoforms, classical PKC ␣ and novel PKC , have been shown to be enriched in skeletal muscle or localized to the endplate. We examined the role of nerve in regulating the expression of these PKC isoforms in rat skeletal muscle by denervating diaphragm muscle and measuring PKC protein expression at various postoperative times. nPKC protein levels decreased 65% after denervation, whereas cPKC ␣ levels increased 80% compared with control hemidiaphragms. These results suggest that innervation regulates PKC and ␣ isoform expression in skeletal muscle. To explore further how nerve regulates PKC expression, we characterized PKC isoform expression in rat myotubes deprived of neural input. Myoblast expression of nPKC was low, and the increase in nPKC expression that occurred during differentiation into myotubes resulted in levels of nPKC significantly below adult skeletal muscle. cPKC ␣ expression in myoblasts increased during differentiation to levels that exceeded expression in adult skeletal muscle. Coculturing myotubes with a neuroblastoma X glioma hybrid clonal cell line (NG108-15) increased nPKC expression, but not cPKC ␣, suggesting that nPKC in skeletal muscle and myotubes is regulated by nerve contact or by a factor(s) provided by nerve. Treating myotubes with tetrodotoxin did not affect either basal-or NG108-15 cell-stimulated nPKC expression. Together these results suggest that expression of nPKC in skeletal muscle is regulated by a transynaptic interaction with nerve that specifically influences nPKC expression.

Despite the wide use of offloading orthotic devices in orthopedic and neurologic populations, their influence on postural control has received little attention. We, therefore, tested the hypotheses that a nonpneumatic walking boot (WB) increases body motion during balance tests and that adding a heel lift to the noninvolved limb reduces body motion when wearing a WB by correcting the leg length discrepancy. Twelve healthy subjects performed three different types of balance tests, including quiet stance (eyes open or closed, firm or foam surface), functional reach (anterior or lateral directions), and treadmill walking (unperturbed or perturbed). Perturbed walking was used to specifically challenge balance and was created with a treadmill mounted to a continuously rotating platform at 0.5 and 1.5 Hz. In each condition, subjects wore either athletic shoes (control) or a WB covering the lower leg or a WB with heel lift in the opposite shoe. Frontal and sagittal plane upper-and lower-body motion was measured with tilt sensors. The WB significantly increased root-mean-square body motion in quiet stance and walking conditions and significantly decreased anterior functional reach. Body motion with the heel lift was significantly reduced compared with the WB in quiet stance conditions but was similar to the WB in functional reach and walking. Results suggest that the WB influenced balance across all tests by increasing body motion, but the contributing factors (leg length discrepancy, reduced ankle range of motion, or reduced base of support because of the rocker bottom) differed across test conditions. These conclusions add to our understanding of how an offloading orthotic device impacts balance. (J Prosthet Orthot. 2014;26:54Y60.) Figure 4. A, Sample platform and medial-lateral (ML) upper-body motion from a subject in one unperturbed and one perturbed walking control condition. B, Across-subject means T 1 SE of the root-mean-square (RMS) of upper-body motion in ML and anterior-posterior (AP) directions. RMS was significantly increased when subjects wore the walking boot (WB) compared with control in both perturbed and unperturbed walking. Statistically significant effects of the orthotic conditions are denoted by asterisks.Goodworth et al.