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.

Cross-talk between tyrosine kinase and G-protein-linked receptors. Phosphorylation of beta 2-adrenergic receptors in response to insulin.

Protein kinases play a pivotal role in the propagation and modulation of transmembrane signaling pathways. Two major classes of receptors, G-protein-linked and tyrosine kinase receptors not only propagate signals but also are substrates for phosphorylation in response to stimulation by agonist ligands. Insulin (operating via tyrosine kinase receptors) and catecholamines (operating by G-protein-linked receptors) are counterregulatory with respect to lipid and carbohydrate metabolism. How, on a cellular level, these two distinct classes of receptors may cross-regulate each other remains controversial. In the present work we identify a novel cross-talk between members of two distinct classes of receptors, tyrosine kinase (insulin) and G-protein-linked (beta-adrenergic) receptors. Treatment of DDT1 MF-2 hamster vas deferens smooth muscle cells with insulin promoted a marked attenuation (desensitization) of beta-adrenergic receptor-mediated activation of adenylylcyclase. Measured by immune precipitation of beta 2-adrenergic receptors from cells metabolically labeled with [32P]orthophosphate, the basal state of receptor phosphorylation was increased 2-fold by insulin. Phosphoamino acid analysis revealed that for insulin-stimulated cells, the beta 2-adrenergic receptors showed increased phosphorylation on tyrosyl and decreased phosphorylation on threonyl residues. Phosphorylation of the beta-adrenergic receptor was rapid and peaked at 30 min following stimulation of cells by insulin. beta-Adrenergic receptor phosphorylation and attenuation of catecholamine-sensitive adenylylcyclase provide a biochemical basis for the counterregulatory effects of insulin upon catecholamine action.