Characterization of protein phosphorylation in acetylcholine receptor-enriched membrane preparations from torpedo fuscomaculata

Summary Erythrocyte soluble phosphodiesterase activities show at least two interconvertible states: aggregated and non-aggregated. In the former state the existence of a high affinity component for cyclic AMP is favoured and the system is more active with cyclic AMP than with cyclic GMP as substrate. When the system is in the non-aggregated state, no activity with cyclic GMP is detected. Conversion to the non-aggregated state is enhanced by dilution or by addition of magnesium ions.Upon isoelectric focusing of erythrocyte soluble extracts, phosphodiesterase activities can be resolved in two peaks: one specific for cyclic AMP located at pH 4.66 and the other specific for cyclic GMP focused at pH 4.95. Evidence would indicate that aggregation affects the enzyme specific for cyclic AMP.     

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.     

Protein phosphatase activity in acetylcholine receptor-enriched membranes.

Phosphorylation of the acetylcholine receptor (AChR)¹ has been demonstrated in AChR-enriched membranes prepared from the electric organ of $\text{Torpedo californica}$. In this report we show that the same AChR-enriched membrane fraction also contains phosphoprotein phosphatase activity which dephosphorylates both the endogenous AChR and exogenous phosphorylated casein. Release of [³²P] PO₄ from phosphorylated casein was shown to be inhibited by F^? as well as GTP. cAMP and cGMP were without effect. Dephosphorylation of the membrane-bound AChR was also inhibited by F^?. This phosphorylation-dephosphorylation mechanism may play a role in mediating the function of the AChR at the synapse.     

Extraction of peripheral proteins from nicotinic acetylcholine receptor-enriched membranes

The solubilisation of membrane proteins from nicotinic acetylcholine receptor-enriched membranes from the electric organ of Torpedo marmorata was studied. Chaotropic ions were shown to be ineffective in extracting peripheral proteins from these membranes. Two different anhydrides, 2,3-dimethylmaleic and 3,4,5,6-tetrahydrophthalic anhydride, released certain peripheral membrane proteins but not the integral receptor protein. Treatment of membranes containing $\text{> 3}\text{nmol}$ α-bungarotoxin binding sites per mg protein with anhydride resulted in a 43 kDa polypeptide as the major constituent of the solubilised material. The nature of the 43 kDa polypeptide is discussed. Gentle anhydride treatment did not change the α-bungarotoxin and carbamoylcholine binding properties of the receptor.     

Location of terbium binding sites on acetylcholine receptor-enriched membranes

Acetylcholine receptor-enriched membranes bind 45 terbium cations per receptor. The Tb(III) X-ray scattering factor changes by as much as 30% over a 50 eV range about the L3 absorption edge. We exploit these changes to modulate the contribution of these ions to the X-ray diffraction pattern of oriented receptor-enriched membranes by varying the incident X-ray energy. Difference Fourier analysis of the meridional diffraction amplitudes at two X-ray energies revealed six localized regions of Tb(III) density across the membrane. Most significant is the finding of 18 Tb(III) ions near the entrance and 11 ions near the exit of the ion channel as well as 4 or 5 Tb(III) ions localized in the channel itself. This evidence strongly suggests the presence of anionic carboxylate side-chains on the channel lining.     

The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue

The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.     

Apparent cooperative effects in acetylcholine receptor-mediated ion flux electroplax membrane preparations.

The kinetics of acetylcholine receptor-mediated flux of ²²sodium ions from microsacs has been measured in the presence of activators (carbamylcholine and decamethonium) and an inhibitor (d-tubocurarine) of neural transmission. The dependence of the first-order rate constant, k_obs, for ²²sodium ion efflux on either decamethonium or carbamylcholine concentration does not exhibit cooperativity. The apparent cooperativity observed by Kasai and Changeux in dose-response curves for ²²sodium flux from the same preparation is adequately accounted for by the contribution which efflux from non-excitable microsacs, the main component of the preparation, makes to the measurements. d-Tubocurarine was found to be a non-competitive inhibitor of decamethonium-activated ²²sodium efflux. The results of the kinetic measurements are in agreement with equilibrium measurements of the interaction of decamethonium with the same microsac preparation, i.e. adherence to a classic Langmuir binding isotherm and separate binding sites for activators and inhibitors of neural activity. The results indicate a direct relationship between ligand binding and receptor-mediated ion flux. How these two processes contribute to electrophysiological measurements is not apparent.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.     

Effect of cholinergic ligands on the lipids of acetylcholine receptor-rich membrane preparations from torpedo californica

Ion permeation, triggered by ligand-receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long-lived fluorescent probe pyrene (fluorescence lifetime ～400 nsec) has been inserted into the lipid phase of acetylcholine receptor-rich membrane (AcChR-M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10-nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR-M produces varying effects on the properties of the pyrene probe in the lipid phase. It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity or permeability of the lipids in the bilayer in contact with pyrene. On the other hand, local anesthetics do affect these properties.     

ACTH and brain membrane phosphorylation: a model for modulation by neuropeptides

It has been hypothesized that changes in the phosphorylation of synaptic membrane constituents (proteins and lipids) may affect transmission in certain types of synapses. In this paper some of the recent evidence that neuropeptides like ACTH may bring about their behavioral activity by influencing brain protein and lipid phosphorylation is reviewed. An ACTH-sensitive, cAMP-independent protein kinase was isolated from rat brain synaptosomal plasma membranes. This enzyme was partially characterized and it was observed that its activity greatly depended on the presence of calcium ions. One of its substrate proteins B-50 (MW 48,000; IEP 4.5) may play a key role in the turnover of a special class of membrane phospholipids i.e. the (poly)phosphoinositides. Evidence was obtained to suggest that the degree of phosphorylation of the B-50 protein determines the conversion of diphosphoinositol to triphosphoinositol. A model which links the protein phosphorylation to lipid phosphorylation and which points to a functional role for peptides in the regulation of the permeability of brain membranes for calcium ions will be discussed. As the structure-activity relationship for the peptide effects on grooming behavior closely resembles that on phosphorylation, it is assumed that this neurochemical event may indeed be of relevance to the biological activity of the peptide. As the ion permeability may be altered by the peptide it can be suggested that this may lead to modulation of transynaptic information processing in the brain.     

Protein phosphorylation of nicotinic acetylcholine receptors

The nicotinic acetylcholine receptor (nAcChR) is a ligand-gated ion channel found in the postsynaptic membranes of electric organs, at the neuromuscular junction, and at nicotinic cholinergic synapses of the mammalian central and peripheral nervous system. The nAcChR from Torpedo electric organ and mammalian muscle is the most well-characterized neurotransmitter receptor in biology. It has been shown to be comprised of five homologous (two identicle) protein subunits (alpha 2 beta gamma delta) that form both the ion channel and the neurotransmitter receptor. The nAcChR has been purified and reconstituted into lipid vesicles with retention of ion channel function and the primary structure of all four protein subunits has been determined. Protein phosphorylation is a major posttranslational modification known to regulate protein function. The Torpedo nAcChR was first shown to be regulated by phosphorylation by the discovery that postsynaptic membranes contain protein kinases that phosphorylate the nAcChR. Phosphorylation of the nAcChR has since been shown to be regulated by the cAMP-dependent protein kinase, protein kinase C, and a tyrosine-specific protein kinase. Phosphorylation of the nAcChR by cAMP-dependent protein kinase has been shown to increase the rate of nAcChR desensitization, the process by which the nAcChR becomes inactivated in the continued presence of agonist. In cultured muscle cells, phosphorylation of the nAcChR has been shown to be regulated by cAMP-dependent protein kinase, a Ca2+-sensitive protein kinase, and a tyrosine-specific protein kinase. Stimulation of the cAMP-dependent protein kinase in muscle also increases the rate of nAcChR desensitization and correlates well with the increase in nAcChR phosphorylation. The AcChR represents a model system for how receptors and ion channels are regulated by second messengers and protein phosphorylation.     

Modulation of epidermal growth factor-dependent protein phosphorylation in cell membrane preparations by receptor down regulation

We have reported previously [6] that epidermal growth factor (EGF)-induced down regulation of EGF receptors in normal rat kidney (NRK) cells results in a selective decrease in the in vitro EGF-dependent ³²P-phosphorylation of two membrane phosphoproteins of Mr I70K and Mr I50K. In this report, we further characterized the modulation of ³²P-phosphorylation of the 170K- and 150K-dalton proteins by down regulation with EGF in NRK cells. While EGF binding to its receptors was a necessary condition to induce loss of EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins, it was not sufficient. Thus, reduction in the temperature of the incubation of cells with EGF from 37°C to 4°C abolished the loss of EGF-dependent phosphorylation of the 170K- and 150K-dalton membrane proteins. When EGF was removed from the medium the EGF-dependent phosphorylation of the 170K- and l50K-dalton proteins was quickly replenished; by 3 hr one-half of the “down regulated” phosphorylation was restored. All EGF-dependent phosphorylating capacity of the 170K- and l50K-dalton protein bands returned by 6 hr after removal of the growth factor. The loss of EGF-dependent phosphorylation of the 170K- and I50K-dalton proteins occurred at physiological EGF concentrations (0.25–25 ng/ml) that span the concentration range which is mitogenic for NRK cells. Exposure of confluent nondividing NRK cells to 1 ng/ml EGF, followed by incubation for 5 hr at 37°C. led to a 50% reduction in the EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins. Maximal reduction (～95%) in the EGF-dependent phosphorylation of the 170K- and 150K-dalton proteins was noted with 10 ng/ml EGF for 5 hr. The EGF-induced loss of EGF-dependent phosphorylation was specific: several other growth factors did not produce phosphorylation loss of the 170K-     

Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor.

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.     

Intracellular modulation of membrane channels by cyclic AMP-mediated protein phosphorylation in peptidergic neurons of Aplysia

The peptidergic bag cell neurons of the opisthobranch mollusc Aplysia control egg laying and its correlated behavior by release of the neuroactive peptide, egg-laying hormone, during the extended electrical discharge termed afterdischarge. This paper examines the evidence for the involvement of cyclic AMP (cAMP) and protein phosphorylation in the mediation of this electrical afterdischarge. It is concluded that an important component in the mechanism of afterdischarge is the suppression of a potassium channel, mediated by cAMP-dependent protein kinase-induced protein phosphorylation. The exact identity of the potassium channel remains to be worked out.     

Rat liver membrane preparations

The quality of rat liver plasma membranes was examined using three different methods of preparation. Hypotonic Ca⁺⁺-free preparation media were compared with hypotonic Ca⁺⁺ media and with isotonic Ca⁺⁺ media. The plasma membranes prepared in hypotonic Ca⁺⁺ free media were as pure or better than the other two preparation types, while the (Na⁺K⁺)-MgATPase, MgATPase, and the ADPase specific activities were higher. The AMPase specific activities were of similar value for all three types of preparation.     

Fast cation flux from Torpedocalifornica membrane preparations: Implications for a functional role for acetylcholine receptor dimers

Closed membrane vesicles derived from the innervated face of $\text{Torpedo}$$\text{californica}$ electroplax respond to the cholinergic agonist carbamylcholine by a rapid efflux of cations. This response is detected by release of ²²Na enclosed within the vesicles and is considerably faster than previously reported for this $\text{in}\text{vitro}$ system. It is considered likely that the rapid response is analogous to physiological phenomena since it has the appropriate pharmacological characteristics; it desensitizes upon prolonged contact with the agonist and it has a dose-response curve in a physiological range. It is further shown that a dimeric form of the acetylcholine receptor, stabilized by chemical modification methods, is fully active in terms of the carbamylcholine elicited response.