Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth.

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.

Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells.

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.

Calnexin-dependent enhancement of nicotinic acetylcholine receptor assembly and surface expression.

The muscle-type nicotinic acetylcholine receptor (AChR)2 is a pentameric membrane ion channel assembled in the endoplasmic reticulum from four homologous subunits by mechanisms that are insufficiently understood. Nascent AChR subunits were recently found to form complexes with the endoplasmic reticulum-resident molecular chaperone calnexin. To determine the contribution of this interaction to AChR assembly and surface expression, we have now used transient transfection of mouse AChR subunits and calnexin into non-muscle cells. Co-transfection of calnexin along with AChR subunits into COS and HEK 293 cells was found to enhance AChR subunit folding and assembly, and to decrease degradation rates of newly synthesized AChR alpha-subunits, resulting in elevated surface expression of assembled AChR. Moreover, inhibition of the interaction between endogenous calnexin and AChR by castanospermine resulted in decreased AChR subunit folding, assembly, and surface expression in muscle and HEK 293 cells. Together, these findings provide evidence that calnexin directly contributes to AChR biogenesis by promoting subunit folding and assembly.

Arrest of subunit folding and assembly of nicotinic acetylcholine receptors in cultured muscle cells by dithiothreitol.

In this study we have used cultured muscle cells to investigate the role of disulfide bond formation in the sequence of molecular events leading to nicotinic acetylcholine receptor (AChR) assembly and surface expression. We have observed that disulfide bond formation in newly synthesized AChR alpha-subunits occurs 5-20 min after translation and that this modification can be blocked by dithiothreitol (DTT), a membrane-permeant thiol-reducing agent. DTT treatment was found to arrest AChR alpha-subunit conformational maturation, assembly, and appearance on the cell surface, showing that these events are dependent on prior formation of disulfide bonds. Subunits prevented from maturation by the reducing agent do not irreversibly misfold or aggregate, since upon removal of DTT, AChR alpha-subunits undergo formation of disulfide bonds and resume folding, oligomerization, and surface expression. We have previously found that nascent alpha-subunits form transient complexes with the molecular chaperone calnexin immediately after subunit synthesis (Gelman, M.S., Chang, W., Thomas, D. Y., Bergeron, J. J. M., and Prives, J. M. (1995) J. Biol. Chem. 270, 15085-15092) and have now observed that both the formation and the subsequent dissociation of these complexes are unaffected by DTT treatment. Thus, alpha-subunits appear to dissociate from calnexin independently of their undergoing disulfide bond formation and achieving conformational maturation. This finding together with the absence of irreversible misfolding of DTT-arrested alpha-subunits suggests that calnexin may act to prevent misfolding by aiding in the initial folding events and is not an essential participant in the late stages of alpha-subunit maturation.

Role of the endoplasmic reticulum chaperone calnexin in subunit folding and assembly of nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (AChR) is a pentameric complex assembled from four different gene products by mechanisms that are inadequately understood. In this study we investigated the role of the endoplasmic reticulum (ER)-resident molecular chaperone calnexin in AChR subunit folding and assembly. We have shown that calnexin interacts with nascent AChR alpha-subunits (AChR-alpha) in muscle cell cultures and in COS cells transfected with mouse AChR-alpha. In chick muscle cells maximal association of labeled alpha-subunits with calnexin was observed immediately after a 15-min pulse with [35S]methionine/cysteine and subsequently declined with a t1/2 of approximately 20 min. The decrease in association with calnexin was concomitant with the folding of the alpha-subunit to achieve conformational maturation shortly before assembly. Brefeldin A did not inhibit AChR subunit assembly or the dissociation of calnexin from the assembling subunits, confirming that the ER is the site of AChR assembly and that calnexin dissociation is not affected under conditions in which the exit of assembled AChR from the ER is blocked. These results indicate that calnexin participates directly in the molecular events that lead to AChR assembly.

Phosphorylation and assembly of nicotinic acetylcholine receptor subunits in cultured chick muscle cells.

The assembly of the nicotinic acetylcholine receptor (AChR), an oligomeric cell surface protein, was studied in cultured muscle cells. To measure this process, the incorporation of metabolically labeled alpha-subunit into oligomeric AChR was monitored in pulse-chase experiments, either by the shift of this subunit from the unassembled (5 S) to the assembled (9 S) position in sucrose density gradients, or by its coprecipitation with antisera specific for the delta-subunit. We have found that AChR assembly is initiated 15-30 min after subunit biosynthesis and is completed within the next 60 min. The alpha-subunit is not overproduced, as all detectable pulse-labeled alpha-subunit can be chased into the oligomeric complex, suggesting that AChR assembly in this system is an efficient process. The rate of AChR assembly is decreased by metabolic inhibitors and by monensin, an ionophore that impairs the Golgi apparatus. We have observed that the gamma- and delta-subunits of AChR are phosphorylated in vivo. The delta-subunit is more highly phosphorylated in the unassembled than in the assembled state, indicating that its phosphorylation precedes assembly and that its dephosphorylation is concomitant with AChR assembly. These findings suggest that subunit assembly occurs in the Golgi apparatus and that phosphorylation/dephosphorylation mechanisms play a role in the control of AChR subunit assembly.

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture.

We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.

Development of electrophysiological and biochemical membrane properties during differentiation of embryonic skeletal muscle in culture.

Newly fused chick myotubes undergo simultaneous and rapid changes in cell membrane properties during synchronous differentiation in culture. These changes are coordinately regulated and include increases in acetylcholine receptor, acetylcholinesterase, and resting potential, as well as the appearance of action potentials in discrete membrane areas upon stimulation. Subsequently, the acetylcholine receptor reaches maximal levels, whereas the development of electrical properties is marked by a further increase in resting potential, changes in the characteristics of the elicited action potential, and the recruitment of additional membrane areas for action potential generation. Maturation of electrical excitability, marked by the acquisition of the ability to fire repetitively and to conduct action potentials along the membrane, occurs well after resting potential has reached a maximum. During post-maturational development, myotubes exhibit spontaneous electrical and contractile activity, and levels of acetylcholine receptor accessible to externally applied 125I-labeled alpha-bungarotoxin decrease markedly. It is suggested that electrophysiological membrane maturation is autonomously regulated with no requirement for neuronal intervention and involves the coordinated biosynthesis of discrete membrane components and their subsequent organization in the myotube membrane.

Differentiation of cell membranes in cultures of embryonic chick breast muscle.

Three components of differentiated muscle membrane, the acetylcholine receptor, acetylcholinesterase (EC 3.1.1.7; acetylcholine hydrolase), and adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)], appear simultaneously during myogenesis in cultures of embryonic chick muscle, after the main period of rapid cell fusion. However, unlike the cytoplasmic proteins of differentiated muscle, the elaboration of these membrane components is unaltered when fusion is blocked by lowering the calcium concentration in the medium. These results suggest that membrane differentiation and cytoplasmic differentiation are regulated independently during muscle development.

Affinity labeling of the acetylcholine receptor in the electroplax: electrophoretic separtion in sodium dodecyl sulfate.

Electroplax, single cells dissected from electric tissue of Electrophorus, are labeled in a two-step procedure: reduction by dithiothreitol followed by alkylation by the affinity label 4-(N-maleimido)-alpha-benzyltri-[methyl-(3)H]methylammonium iodide, either alone or in combination with [2,3-(14)C]N-ethylmaleimide. Electrophoresis in sodium dodecyl sulfate on polyacrylamide gel of an extract, prepared with this detergent, of single-labeled or of double-labeled cells results in a major peak of (3)H activity, with a mobility corresponding to a polypeptide of molecular weight 42,000. In addition, in the double-labeled samples, there is a unique peak in the ratio of (3)H to (14)C that is coincident with the (3)H peak. The electrophoretic patterns of extracts of cells in which affinity alkylation of the reduced receptor has been suppressed by dithiobischoline, an affinity oxidizing agent, by cobratoxin, an irreversible ligand, or by hexamethonium, a reversible ligand, show a considerably diminished peak of (3)H activity in the region of molecular weight 42,000. This is the predominant difference between the electrophoretic patterns of extracts of unprotected and of protected cells. Furthermore, extracts of cells protected with dithiobischoline before labeling with both tritiated affinity label and [(14)C]N-ethylmaleimide do not show the peak in the (3)H to (14)C ratio seen in the absence of protection. Thus, by several diverse criteria, the peak of (3)H activity corresponding to a molecular weight of 42,000 contains affinity-labeled acetylcholine receptor or receptor subunit.