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Key points Exercise is a potent physiological stimulus to clear blood glucose from the circulation into skeletal muscle. The mammalian target of rapamycin complex 2 (mTORC2) is an important regulator of muscle glucose uptake in response to insulin stimulation. Here we report for the first time that the activity of mTORC2 in mouse muscle increases during exercise. We further show that glucose uptake during exercise is decreased in mouse muscle that lacks mTORC2 activity. We also provide novel identifications of new mTORC2 substrates during exercise in mouse muscle. Abstract Exercise increases glucose uptake into insulin-resistant muscle. Thus, elucidating the exercise signalling network in muscle may uncover new therapeutic targets. The mammalian target of rapamycin complex 2 (mTORC2), a regulator of insulin-controlled glucose uptake, has been reported to interact with ras-related C3 botulinum toxin substrate 1 (Rac1), which plays a role in exercise-induced glucose uptake in muscle. Therefore, we tested the hypothesis that mTORC2 activity is necessary for muscle glucose uptake during treadmill exercise. We used mice that specifically lack mTORC2 signalling in muscle by deletion of the obligatory mTORC2 component Rictor (Ric mKO). Running capacity and running-induced changes in blood glucose, plasma lactate and muscle glycogen levels were similar in wild-type (Ric WT) and Ric mKO mice. At rest, muscle glucose uptake was normal, but during running muscle glucose uptake was reduced by 40% in Ric mKO mice compared to Ric WT mice. Running increased muscle phosphorylated 5′ AMP-activated protein kinase (AMPK) similarly in Ric WT and Ric mKO mice, and glucose transporter type 4 (GLUT4) and hexokinase II (HKII) protein expressions were also normal in Ric mKO muscle. The mTORC2 substrate, phosphorylated protein kinase C α (PKCα), and the mTORC2 activity readout, phosphorylated N-myc downstream regulated 1 (NDRG1) protein increased with running in Ric WT mice, but were not altered by running in Ric mKO muscle. Quantitative phosphoproteomics uncovered several additional potential exercise-dependent mTORC2 substrates, including contractile proteins, kinases, transcriptional regulators, actin cytoskeleton regulators and ion-transport proteins. Our study suggests that mTORC2 is a component of the exercise signalling network that regulates muscle glucose uptake and we provide a resource of new potential members of the mTORC2 signalling network.

Muscle-specific tyrosine kinase (MuSK) is involved in the formation and maintenance of the neuromuscular junction (NMJ), and is necessary for NMJ integrity. As muscle involvement is strikingly selective in pathological conditions in which MuSK is targeted, including congenital myasthenic syndrome with MuSK mutation and MuSK antibody-seropositive myasthenia gravis, we hypothesized that the postsynaptic response to MuSK-agrin signalling differs between adult muscles. Transcript levels of postsynaptic proteins were compared between different muscles in wild-type adult mice. MuSK expression was high in the soleus and sternomastoid muscles and low in the extensor digitorum longus (EDL) and omohyoid muscles. The acetylcholine receptor (AChR) alpha subunit followed a similar expression pattern, whereas expression of Dok-7, Lrp4 and rapsyn was comparable between the muscles. We subsequently examined muscles in mice that overexpressed a miniaturized form of neural agrin or MuSK. In these transgenic mice, the soleus and sternomastoid muscles responded with formation of ectopic AChR clusters, whereas such clusters were almost absent in the EDL and omohyoid muscles. Electroporation of Dok-7 revealed its important role as an activator of MuSK in AChR cluster formation in adult muscles. Together, our findings indicate for the first time that adult skeletal muscles harbour different endogenous levels of MuSK and that these levels determine the ability to form ectopic AChR clusters upon overexpression of agrin or MuSK. We believe that these findings are important for our understanding of adult muscle plasticity and the selective muscle involvement in neuromuscular disorders in which MuSK is diminished.

Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes. The high-affinity interaction with the coiled-coil domain of laminin is mediated by the N-terminal domain of agrin. We have adopted a structurally guided site-directed mutagenesis approach to map the laminin-binding site of NtA. Mutations of L117 and V124 in the C-terminal helix 3 showed that they are crucial for binding. Both residues are located in helix 3 and face the groove between the beta-barrel and the C-terminal helical segment of NtA. Remarkably, the distance between both residues matches a heptad repeat distance of two aliphatic residues which are solvent exposed in the coiled-coil domain of laminin. A lower but significant contribution originates from R43 and a charged cluster (E23, E24 and R40) at the open face of the beta-barrel structure. We propose that surface-exposed, conserved residues of the laminin gamma1 chain interact with NtA via hydrophobic and ionic interactions.

Agrin is required for appropriate pre- and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a "stop signal" for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite-outgrowth responses to purified agrin in vitro. We find that both full-length agrin and the C-terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N-CAM binds to full-length agrin, and suggest that alpha-dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full-length agrin inhibited both laminin- and N-cadherin-induced neurite growth from CG neurons. The N-terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N-terminal portion of agrin are sufficient for this function. Adhesion assays using protein-coated beads and agrin-expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N-CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N-terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither alpha-dystroglycan nor N-CAM, two known binding proteins for agrin, mediate this effect.

The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse-inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C-terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N-terminal fragment of agrin. Here we show that an expression construct encoding a 'mini'-agrin, in which the laminin-binding fragment was fused to the AChR-clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non-synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full-length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR epsilon-subunit. Altogether, our data show that a 'mini'-agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse-like structures that are reminiscent of those induced by full-length neural agrin or innervation by motor neurons.

Using selective metabolic labelling in a compartmental cell culture system two proteins, denoted axonin-1 and axonin-2, were found to be secreted by axons of dorsal root ganglia neurons from chicken embryos. Based on its characteristic coordinates and spot morphology in two-dimensional gel electrophoresis, axonin-1 was detected in the cerebrospinal fluid and the vitreous fluid, axonin-1 was purified 476-fold to homogeneity by a four-step chromatographic procedure. The identity of the purified protein as axonin-1 was confirmed by immunological methods. Axonin-1 is a glycoprotein that subdivides into at least 16 immunologically similar isoelectric variants; their molecular weight range extends from 132 to 140 kd and their pI range from 5.3 to 6.2. In the vitreous fluid of the embryo, axonin-1 could first be detected on the embryonic day 5 and highest concentrations were measured during the second half of embryonic life; in the vitreous fluid of the adult chicken, concentrations were approximately 20 times lower. The early onset of secretion and the time course of expression suggest a role for axonin-1 in the development of the nervous system.