A possible role for dephosphorylation in glucocorticoid receptor transformation

Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation.     

Soluble form of acetylcholinesterase from rabbit enterocytes: comparison of its molecular properties with those of the plasma membrane species

A soluble form of acetylcholinesterase was shown to be present in rabbit enterocytes. The enzyme was obtained from a high-speed supernatant (105,000 X g centrifugation) after homogenization of intestinal mucosa without detergent. It was shown to possess no obvious hydrophobic character and could be classified as a low-salt-soluble (LSS) acetylcholinesterase. Sucrose gradient centrifugation revealed a single enzyme species with a sedimentation coefficient of 3.9 +/- 0.2S. By gel filtration performed in HPLC the enzyme was eluted as a protein corresponding to an Mr of 72,000 +/- 3,000. It could be precipitated with concanavalin A by affinoelectrophoresis, but the catalytic activity was not affected by the lectin. Our results are consistent with a G1 globular form for this soluble acetylcholinesterase which differs very clearly from detergent-soluble forms also found recently in the plasma membranes of rabbit enterocytes.     

Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor.

The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentamer composed of two ligand-binding alpha-subunits and one beta-, gamma-, and delta-subunit. To examine the functional roles of the non-alpha-subunits, we have expressed, in stable cell lines, AChRs lacking either a gamma- or a delta-subunit. Most previous work has examined how these changes in subunit composition affect single channel properties. Here, we take advantage of the stable expression system to produce large amounts of AChR and, for the first time, examine ligand binding to altered AChRs on intact cells. The changes in subunit composition affect both ligand affinity and cooperativity of the receptor, suggesting important roles for the gamma- and delta-subunits, both in shaping the ligand binding site and maintaining cooperative interactions between alpha-subunits.     

Epibatidine activates muscle acetylcholine receptors with unique site selectivity.

We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.     

Arterial acid-base status during digestion and following vascular infusion of NaHCO(3) and HCl in the South American rattlesnake, Crotalus durissus

Digestion is associated with gastric secretion that leads to an alkalinisation of the blood, termed the "alkaline tide". Numerous studies on different reptiles and amphibians show that while plasma bicarbonate concentration ([HCO(3)(-)](pl)) increases substantially during digestion, arterial pH (pHa) remains virtually unchanged, due to a concurrent rise in arterial PCO(2) (PaCO(2)) caused by a relative hypoventilation. This has led to the suggestion that postprandial amphibians and reptiles regulate pHa rather than PaCO(2). Here we characterize blood gases in the South American rattlesnake (Crotalus durissus) during digestion and following systemic infusions of NaHCO(3) and HCl in fasting animals to induce a metabolic alkalosis or acidosis in fasting animals. The magnitude of these acid-base disturbances were similar in magnitude to that mediated by digestion and exercise. Plasma [HCO(3)(-)] increased from 18.4+/-1.5 to 23.7+/-1.0 mmol L(-1) during digestion and was accompanied by a respiratory compensation where PaCO(2) increased from 13.0+/-0.7 to 19.1+/-1.4 mm Hg at 24 h. As a result, pHa decreased slightly, but were significantly below fasting levels 36 h into digestion. Infusion of NaHCO(3) (7 mmol kg(-1)) resulted in a 10 mmol L(-1) increase in plasma [HCO(3)(-)] within 1 h and was accompanied by a rapid elevation of pHa (from 7.58+/-0.01 to 7.78+/-0.02). PaCO(2), however, did not change following HCO(3)(-) infusion, which indicates a lack of respiratory compensation. Following infusion of HCl (4 mmol kg(-1)), plasma pHa decreased by 0.07 units and [HCO(3)(-)](pl) was reduced by 4.6 mmol L(-1) within the first 3 h. PaCO(2), however, was not affected and there was no evidence for respiratory compensation. Our data show that digesting rattlesnakes exhibit respiratory compensations to the alkaline tide, whereas artificially induced metabolic acid-base disturbances of same magnitude remain uncompensated. It seems difficult to envision that the central and peripheral chemoreceptors would experience different stimuli during these conditions. One explanation for the different ventilatory responses could be that digestion induces a more relaxed state with low responsiveness to ventilatory stimuli.     

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.     

Novel class III phosphoribosyl diphosphate synthase: structure and properties of the tetrameric, phosphate-activated, non-allosterically inhibited enzyme from Methanocaldococcus jannaschii

The prs gene encoding phosphoribosyl diphosphate (PRPP) synthase of the hyperthermophilic autotrophic methanogenic archaeon Methanocaldococcus jannaschii has been cloned and expressed in Escherichia coli. Subsequently, M.jannaschii PRPP synthase has been purified, characterised, crystallised, and the crystal structure determined. The enzyme is activated by phosphate ions and only ATP or dATP serve as diphosphoryl donors. The K(m) values are determined as 2.6 mM and 2.8 mM for ATP and ribose 5-phosphate, respectively, and the V(max) value as 2.20 mmol (minxmg of protein)(-1). ADP is a potent inhibitor of activity while GDP has no effect. A single ADP binding site, the active site, is present per subunit. The crystal structure of the enzyme reveals a more compact subunit than that of the enzyme from the mesophile Bacillus subtilis, caused by truncations at the N and C terminus as well as shorter loops in the M.jannaschii enzyme. The M.jannaschii enzyme displays a tetrameric quaternary structure in contrast to the hexameric quaternary structure of B.subtilis PRPP synthase. Soaking of the crystals with 5'-AMP and PRPP revealed the position of the former compound as well as that of ribose 5-phosphate. The properties of M.jannaschii PRPP synthase differ widely from previously characterised PRPP synthases by its tetrameric quaternary structure and the simultaneous phosphate ion-activation and lack of allosteric inhibition, and, thus, constitute a novel class of PRPP synthases.     

Pharmacoproteomics Profile in Response to Acamprosate Treatment of an Alcoholism Animal Model

Acamprosate is an FDA‐approved medication for the treatment of alcoholism that is unfortunately only effective in certain patients. Although acamprosate is known to stabilize the hyper‐glutamatergic state in alcoholism, pharmacological mechanisms of action in brain tissue remains unknown. To investigate the mechanism of acamprosate efficacy, the authors employ a pharmacoproteomics approach using an animal model of alcoholism, type 1 equilibrative nucleoside transporter (ENT1) null mice. The results demonstrate that acamprosate treatment significantly decreased both ethanol drinking and preference in ENT1 null mice compared to that of wild‐type mice. Then, to elucidate acamprosate efficacy mechanism in ENT1 null mice, the authors utilize label‐free quantification proteomics comparing both genotype and acamprosate treatment effects in the nucleus accumbens (NAc). A total of 1040 protein expression changes are identified in the NAc among 3634 total proteins detected. The proteomics and Western blot result demonstrate that acamprosate treatment decreased EAAT expression implicating stabilization of the hyper‐glutamatergic condition in ENT1 null mice. Pathway analysis suggests that acamprosate treatment in ENT1 null mice seems to rescue glutamate toxicity through restoring of RTN4 and NF‐κB medicated neuroimmune signaling compared to wild‐type mice. Overall, pharmacoproteomics approaches suggest that neuroimmune restoration is a potential efficacy mechanism in the acamprosate treatment of certain sub‐populations of alcohol dependent subjects.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.