Phosphorylation Of Acetylcholine Receptor Research Articles

Developmental Regulation of Tyrosine Phosphorylation of the Nicotinic Acetylcholine Receptor inTorpedoElectrocyte

Tyrosine phosphorylation is thought to play a critical role in the clustering of acetylcholine receptors (AChR) at the developing neuromuscular junction. Yet,in vitroapproaches have led to conflicting conclusions regarding the function of tyrosine phosphorylation of AChR β subunit in AChR clustering. In this work, we followedin situthe time course of tyrosine phosphorylation of AChR in developingTorpedoelectrocyte. We observed that tyrosine phosphorylation of the AChR β and δ subunits occurs at a late stage of embryonic development after the accumulation of AChRs and rapsyn in the membrane and the onset of innervation. Interestingly, in the mature postsynaptic membrane, we observed two populations of AChR differing both in their phosphotyrosine content and distribution. Our data are consistent with the notion that tyrosine phosphorylation of the AChR is related to downstream events in the pathway regulating AChR accumulation rather than to initial clustering events.

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.

Muscle-Specific Agrin Isoforms Reduce Phosphorylation of AChR γ and δ Subunits in Cultured Muscle Cells

The accumulation of nicotinic acetylcholine receptors (AChRs) at neuromuscular synapses is triggered by agrin, a protein that is synthesized by both nerve and muscle. Nerve-derived agrin, which contains an amino acid insert at a conserved splice site in the carboxy-terminal part of the protein, induces AChR aggregation and causes tyrosine phosphorylation of the AChR β subunit. In contrast, agrin isoforms synthesized by muscle cells lack such an insert and have no effect on AChR distribution. In order to identify possible functional roles of muscle-derived agrin we have analyzed further the effect of various fragments of recombinant agrin on AChR phosphorylation. A carboxy-terminal fragment of muscle agrin, c95A0B0, reduced AChR γ and δ subunit phosphorylation when added to C2C12 myotubes in culture. Although c95A0B0had no effect on AChR β subunit phosphorylation when added alone, it inhibited AChR β subunit phosphorylation and AChR aggregation by the nerve-specific agrin isoform c95A4B8. We conclude that muscle-derived agrin can influence, both directly and indirectly, AChR phosphorylation. Such changes may play a role in the formation, maintenance, or function of the neuromuscular junction.

Dimerization of the Muscle-specific Kinase Induces Tyrosine Phosphorylation of Acetylcholine Receptors and Their Aggregation on the Surface of Myotubes

During development of the neuromuscular junction, neuronal splice variants of agrin initiate the aggregation of acetylcholine receptors on the myotube surface. The muscle-specific kinase is thought to be part of an agrin receptor complex, although the recombinant protein does not bind agrin with high affinity. To specify its function, we induced phosphorylation and activation of this kinase in the absence of agrin by incubating myotubes with antibodies directed against its N-terminal sequence. Antibody-induced dimerization of the muscle-specific kinase but not treatment with Fab fragments was sufficient to trigger two key events of early postsynaptic development: acetylcholine receptors accumulated into aggregates, and their beta-subunits became phosphorylated on tyrosine residues. Heparin partially inhibited receptor aggregation induced by both agrin and anti-muscle-specific kinase antibodies. In contrast, it did not affect kinase or acetylcholine receptor phosphorylation. These data indicate that agrin induces postsynaptic differentiation by dimerizing the muscle-specific kinase. They also suggest that activation of the kinase domain can account for only part of agrin's effects. Dimerization of this molecule appears to activate an additional signal, most likely by organizing a scaffold for other postsynaptic proteins.

Intracellular Calcium Regulates Agrin-Induced Acetylcholine Receptor Clustering

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of acetylcholine receptors (AChRs), but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPTA inhibited agrin-induced AChR aggregation. In addition, preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPTA-loaded cells was dose-dependent and reversible, and no change in the number or mobility of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation of the AChR beta-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrin-induced AChR clustering was unaffected in cells loaded with EGTA, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.

Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle.

During synaptogenesis at the neuromuscular junction, a neurally released factor, agrin, causes the clustering of acetylcholine receptors (AChRs) in the muscle membrane beneath the nerve terminal. Agrin acts through a specific receptor which is thought to have a receptor tyrosine kinase, MuSK, as one of its components. In agrin-treated muscle cells, both MuSK and the AChR become tyrosine phosphorylated. To determine how the activation of MuSK leads to AChR clustering, we have investigated their interaction in cultured C2 myotubes. Immunoprecipitation experiments showed that MuSK is associated with the AChR and that this association is increased by agrin treatment. Agrin also caused a transient activation of the AChR-associated MuSK, as demonstrated by MuSK phosphorylation. In agrin-treated myotubes, MuSK phosphorylation increased with the same time course as phosphorylation of the beta subunit of the AChR, but declined more quickly. Although both herbimycin and staurosporine blocked agrin-induced AChR phosphorylation, only herbimycin inhibited the phosphorylation of MuSK. These results suggest that although agrin increases the amount of activated MuSK that is associated with the AChR, MuSK is not directly responsible for AChR phosphorylation but acts through other kinases.

Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: required role for the MuSK ectodomain?

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation-phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSK-based scaffold.

Rapsyn Is Required for MuSK Signaling and Recruits Synaptic Components to a MuSK-Containing Scaffold

Agrin-induced clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane is a key step in synaptogenesis at the neuromuscular junction. The receptor tyrosine kinase MuSK is a component of the agrin receptor, while the cytoplasmic protein rapsyn is necessary for the clustering of AChRs and all other postsynaptic membrane components studied to date. We show here that MuSK remains concentrated at synaptic sites in rapsyn-deficient mutant mice, suggesting that MuSK forms a primary structural scaffold to which rapsyn attaches other synaptic components. Using nonmuscle cells, we show that rapsyn–MuSK interactions are mediated by the ectodomain of MuSK, suggesting the existence of a transmembrane intermediate. In addition to rapsyn's structural role, we demonstrate that it is required for an early step in MuSK signaling, AChR phosphorylation. This signaling requires the kinase domain of MuSK, but not its ectodomain. Thus, MuSK may interact with rapsyn in multiple ways to play both structural and signaling roles in agrin-induced differentiation.

AChR phosphorylation and aggregation induced by an agrin fragment that lacks the binding domain for alpha-dystroglycan.

Agrin induces both phosphorylation and aggregation of nicotinic acetylcholine receptors (AChRs) when added to myotubes in culture, apparently by binding to a specific receptor on the myotube surface. One such agrin receptor is alpha-dystroglycan, although binding to alpha-dystroglycan appears not to mediate AChR aggregation. To determine whether agrin-induced AChR phosphorylation is mediated by alpha-dystroglycan or by a different agrin receptor, fragments of recombinant agrin that differ in affinity for alpha-dystroglycan were examined for their ability to induce AChR phosphorylation and aggregation in mouse C2 myotubes. The carboxy-terminal 95 kDa agrin fragment agrin-c95(A0B0), which binds to alpha-dystroglycan with high affinity, failed to induce AChR phosphorylation and aggregation. In contrast, agrin-c95(A4B8) which binds less strongly to alpha-dystroglycan, induced both phosphorylation and aggregation, as did a small 21 kDa fragment of agrin, agrin-c21(B8), that completely lacks the binding domain for alpha-dystroglycan. We conclude that agrin-induced AChR phosphorylation and aggregation are triggered by an agrin receptor that is distinct from alpha-dystroglycan.

Modulation of acetylcholine receptor function in TE671 (rhabdomyosarcoma) cells by non-AChR ligands: possible relevance to seronegative myasthenia gravis

The acetylcholine receptor (AChR) is the main target antigen in myasthenia gravis (MG), but about 15% of patients with typical, immunologically mediated MG do not have detectable anti-AChR antibodies. Previous studies showed that plasma from these ‘seronegative’ patients (SNMG) reduced AChR function in the human AChR-expressing TE671 cell line, and it was proposed that SNMG plasmas may act indirectly via phosphorylation of AChR. We show here that substances such as the β 2-adrenergic agonist, salbutamol, calcitonin-gene-related-peptide (CGRP), and cholera toxin, that increase intracellular cAMP via binding to specific cell-surface receptors, reduced AChR function in TE671 cells. Moreover, non-specific activation of cell surface proteins by lectins achieved similar results. These observations lead us to hypothesise that SNMG immunoglobulins act in TE671 cells by cross-linking of specific cell surface antigen(s) resulting in generation of intracellular cAMP and/or other second messengers. The role of such antibodies at the neuromuscular junction in vivo could be reduction in AChR function by desensitization and/or damage to the postsynaptic membrane following complement activation.

Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase.

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin-induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.

Monoclonal Antibodies as Site-Specific Probes for the Acetylcholine-receptor delta-Subunit Tyrosine and Serine Phosphorylation Sites

Phosphorylation of the nicotinic acetylcholine receptor (AChR) has been implicated in the assembly, clustering, regulation of function and degradation rate of the molecule. Torpedo AChR is phosphorylated at eight cytoplasmic residues, four of which are on the delta subunit. We have precisely mapped the epitopes of eleven monoclonal antibodies (mAbs) directed against the Torpedo AChR delta-subunit regions delta 350-396 and delta 484-493, which are therefore exposed on the surface of the intact AChR, and now have four highly specific tools for analysing the role of delta-subunit phosphorylation. More than 160 synthetic peptides attached to polyethylene rods were used for epitope mapping. Four mAbs bound within the region delta 350-380, which contains all of the delta-subunit phosphorylation sites (Ser361, Ser362, Tyr372 and Ser377). Specifically, the epitope for mAb 134 (delta 365-375) contains Tyr372, the epitope(s) for mAbs 139 and 166 (delta 376-381) contains Ser377, while the epitope for mAb 146 (delta 350-359) is close to Ser361 and Ser362 and includes parts of the corresponding phosphorylation consensus sequences. Using peptide analogues with single residue substitutions, Tyr372 was found to be essential for the binding of mAb 134 and Ser377 was found contributing to the binding of mAbs 139 and 166. Finally, tyrosine phosphorylation of Torpedo AChR selectively inhibited binding of mAb 134. These data, and the availability of the defined mAb probes, should facilitate the study of the functional role of single AChR phosphorylation sites.

Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor

Studies of the regulation of tyrosine phosphorylation at the neuromuscular junction during development and following denervation suggest that tyrosine phosphorylation of the nicotinic acetylcholine receptor is regulated by neuronal innervation of muscle. The finding that agrin, a neuronally derived extracellular matrix protein also induces tyrosine phosphorylation of the nicotinic receptor, suggests that nerve-induced tyrosine phosphorylation may be mediated by agrin. To study this further, we have examined the regulation of tyrosine phosphorylation of the nicotinic receptor by innervation in vitro using muscle-neuron cocultures. Innervation of chick myotubes by chick ciliary ganglia neurons induced tyrosine phosphorylation of the nicotinic receptor with the same subunit specificity seen with bath applied purified agrin. Both innervation and agrin-induced phosphorylation of the nicotinic receptor resulted in an increase in tyrosine and serine phosphorylation. In addition, thermolysin phosphopeptide maps of the subunits after innervation or agrin-treatment were identical. The similarity in the agrin- and nerve-induced phosphorylation of the acetylcholine receptor suggests that agrin mediates the nerve-induced phosphorylation during development in vivo and that phosphorylation of the acetylcholine receptor may play an important role in the development of the neuromuscular junction.

Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation.

Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin-induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.

Intracellular ATP modulates desensitization of acetylcholine receptors controlling chloride current in Lymnaea neurons.

Chloride current activated by nicotinic acetylcholine receptors (AChR) was examined in dialysed voltage-clamp neurons of Lymnaea stagnalis. Fast superfusion of acetylcholine (ACh) evoked an inward current rapidly rising to a peak followed by a decline due to desensitization. When adenosine triphosphate with Mg2+ (MgATP, 2-10 mM) was added intracellularly the peak of the ACh-induced current was increased and its decay was slowed down. ATP without Mg2+ did not affect desensitization. Mg2+ alone accelerated desensitization. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, increased the desensitization rate and decreased the peak current. MgATP after arsenate wash-out restored the initial characteristics of the response; a mixture of glycolytic substrates had a similar effect. A non-hydrolysable analogue of ATP, adenosine [gamma-thio]triphosphate mimicked ATP action after arsenate removal but was weaker; another non-hydrolysable analogue, adenylyl imidodiphosphate, did not affect desensitization at all. Intracellular treatment of the neurons with alkaline phosphatase accelerated current decay. The data suggest that a change in intracellular ATP concentration modulates AChR desensitization via an enzymatic process that might be phosphorylation of AChR or some associated protein(s). Involvement of Ca2+ homeostasis cannot be excluded. The results are compared with the data obtained on vertebrate tissues under conditions promoting phosphorylation.

Mechanism of agrin-induced acetylcholine receptor aggregation.

Agrin induces the formation of specializations on chick myotubes in culture at which several components of the postsynaptic apparatus accumulate, including acetylcholine receptors (AChRs). Agrin also induces AChR phosphorylation. Several lines of evidence suggest that agrin-induced phosphorylation of tyrosine residues in the beta subunit of the AChR is an early step in receptor aggregation: agrin-induced phosphorylation and aggregation have the same dose dependence; treatments that prevent aggregation block phosphorylation; phosphorylation begins before any detectable change in receptor distribution, reaches a maximum hours before aggregation is complete, and declines slowly together with the disappearance of aggregates after agrin is withdrawn; agrin slows the rate at which receptors are solubilized from intact myotubes by detergent extraction; and the change in receptor extractability parallels the change in phosphorylation. A model for agrin-induced AChR aggregation is presented in which phosphorylation of AChRs by an agrin-activated protein tyrosine kinase causes receptors to become attached to the cytoskeleton, which reduces their mobility and detergent extractability, and leads to the accumulation of receptors in the vicinity of the activated kinase, forming an aggregate.

Agonist-independent activation of acetylcholine receptor channels by protein kinase A phosphorylation.

Protein phosphorylation is a ubiquitous and one of the most effective means of regulating protein activity. Receptor phosphorylation is a key event in signal transduction. The question, therefore, that arises is whether this modulatory mechanism might produce functional changes in a membrane receptor in the absence of its naturally occurring ligand. To examine this issue, single-channel properties of purified acetylcholine receptors (AChRs) from Torpedo californica reconstituted in lipid bilayers were studied in the absence of ACh in both unphosphorylated preparations and after in vitro phosphorylation by a purified catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A). Notably, the spontaneous open-channel probability of phosphorylated AChRs is significantly higher than that of unphosphorylated AChRs. Channel activation by protein kinase A is correlated with AChR phosphorylation and is abolished by alpha-bungarotoxin. Analysis of probability distributions of the open dwell times indicates that, similar to unphosphorylated AChR has two distinct open states, short- and long-lived. The frequency of occurrence of the long openings over the short and the magnitude of both time constants increase after phosphorylation, as they do with agonist concentration. Thus, phosphorylation of AChR gamma and delta subunits activates AChR channel opening in the absence of ligand binding. This result is compatible with the notion that protein phosphorylation may effectively act as an intracellular ligand with the phosphorylation sites envisioned as cytoplasmic ligand binding sites.

Agrin induces phosphorylation of the nicotinic acetylcholine receptor

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR β, γ, and δ subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the γ and δ subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the β subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the β subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the β subunit may play a role in regulating AChR distribution.

Calcium influx in molluscan neurons enhances the acetylcholine-induced current: The role of calmodulin

1. Intracellular recordings were made from identified LPL1, RBc4, D1 and E4 neurons in perioesophageal ganglionic ring with buccal ganglia of the mollusc Helix pomatia. 2. The modulations of acetylcholine (ACh) receptor activity by calcium influx and cyclic adenosine monophosphate (cyclic AMP) in these neurons were investigated using two-microelectrode intracellular recording and voltage clamp techniques. 3. The elevation of calcium and cyclic AMP levels to the same identified LP11 and RBc4 neurons was shown to produce antagonistic effects on the ACh-induced chloride current. 4. The calcium influx in LP11 and RBc4 neurons triggered secondary mechanisms, which led to enhancement of the ACh-induced chloride current in these neurons. 5. Calcium influx and elevation of intracellular cyclic AMP levels both induced a decrease of ACh-induced chloride current in the same identified D1 and E4 neurons. 6. The calmodulin inhibitor chloropromazine (6.10 −5M) inhibited the enhancing effect of calcium influx on ACh-induced currents in LP11 and RBc4 neurons, but it was ineffective for D1 and E4 neurons. 7. Moreover, the inhibitor of calcium-phospholipid-dependent protein kinase polymixin B (10 −4M) had no effect on the modulations of ACh receptor activity by calcium influx in LP11, RBc4, D1 and E4 neurons. 8. These results suggest that calcium influx may activate calcium/calmodulin-dependent phosphorylation of ACh receptors on LP11 and RBc4 neurons, which evoke the essential enhancement of ACh-induced chloride current.

Cyclic AMP-dependent phosphorylation of a neuronal acetylcholine receptor alpha-type subunit

Chick ciliary ganglion neurons have nicotinic acetylcholine receptors (AChRs) that mediate synaptic transmission through the ganglion. A cAMP-dependent process has previously been shown to enhance the ACh response of the neurons 2- to 3-fold without requiring the synthesis of new receptors. We show here that the receptors can be phosphorylated in situ by a cAMP-dependent process. The phosphorylation occurs predominantly on components of 50 and 58 kDa. Both derive from putative ligand-binding alpha 3 subunits, with the smaller phosphorylated species probably representing a degradation product of the larger. The increase in receptor phosphorylation caused by incubating the neurons with a cAMP analog parallels the increase observed in the ACh response, with respect to both time course and relative extent. The phosphorylation of ciliary ganglion AChRs differs from that reported for electric organ AChRs, which occurs primarily on the non-ligand-binding gamma and delta subunits and increases the rate of agonist-induced receptor desensitization.