Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state

Epac proteins (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors (GEFs) for the small GTP-binding proteins Rap1 and Rap2 that are directly regulated by the second messenger cyclic AMP and function in the control of diverse cellular processes, including cell adhesion and insulin secretion. Here we report the three-dimensional structure of full-length Epac2, a 110-kDa protein that contains an amino-terminal regulatory region with two cyclic-nucleotide-binding domains and a carboxy-terminal catalytic region. The structure was solved in the absence of cAMP and shows the auto-inhibited state of Epac. The regulatory region is positioned with respect to the catalytic region by a rigid, tripartite beta-sheet-like structure we refer to as the 'switchboard' and an ionic interaction we call the 'ionic latch'. As a consequence of this arrangement, the access of Rap to the catalytic site is sterically blocked. Mutational analysis suggests a model for cAMP-induced Epac activation with rigid body movement of the regulatory region, the features of which are universally conserved in cAMP-regulated proteins.     

Cyclic AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium

Chloride (Cl-) secretion by the airway epithelium regulates, in part, the quantity and composition of the respiratory tract fluid, thereby facilitating mucociliary clearance. The rate of Cl- secretion is controlled by apical membrane Cl- channels. Apical Cl- channels are opened and Cl- secretion is stimulated by a variety of hormones and neurotransmitters that increase intracellular levels of cyclic AMP (cAMP). In cystic fibrosis (CF), a common lethal genetic disease of Caucasians, airway, sweat-gland duct, secretory-coil and possibly other epithelia are anion impermeable. This abnormality may explain several of the clinical manifestations of the disease. The Cl- impermeability in CF-airway epithelia has been localized to the apical cell membrane, where regulation of Cl- channels is abnormal: hormonal secretagogues stimulate cAMP accumulation appropriately but Cl- channels fail to open. Here we report that the purified catalytic subunit of cAMP-dependent protein kinase plus ATP opens Cl- channels in excised, cell-free patches of membrane from normal cells, but fails to open Cl- channels in CF cells. These results indicate that in normal cells, the cAMP-dependent protein kinase phosphorylates the Cl- channel or an associated regulatory protein, causing the channel to open. The failure of CF Cl- channels to open suggests a defect either in the channel or in such an associated regulatory protein.     

Mutant alpha subunits of Gi2 inhibit cyclic AMP accumulation

One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.     

A molecular mechanism for long-term sensitization in Aplysia

Sensitization of the gill- and siphon-withdrawal reflex in Aplysia is thought to result from a set of molecular processes with different time courses: short-term sensitization is explained by cyclic AMP-dependent modulation of ion-channel function in sensory neurons lasting minutes; memory that endures for hours or longer, by the expression and distribution within the neurons of new gene products. Because gene induction and axonal transport are relatively slow, there may also be a need for a distinct form of intermediate memory to bridge the short- and long-term processes. We now report that a protocol producing long-term sensitization results in a decrease in the amount of regulatory subunits of the cAMP-dependent protein kinase in animals 24 h after training, with no effect on the catalytic subunit. The loss appears to be post-translational. Because a decrease in the ratio of regulatory to catalytic subunits would result in elevated kinase activity after cAMP has returned to its unstimulated concentration in sensory cells, it could be the molecular mechanism of intermediate memory.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

Insulin stimulates sugar transport in giant muscle fibres of the barnacle

Insulin stimulates sugar transport in vertebrate skeletal muscle but the mechanism of insulin action is unknown. It has been reported that Na transport in giant muscle fibers of the barnacle (Balanus nubilis) is sensitive to insulin but no one has examined the sensitivity of sugar tansport to insulin in this preparation. We show here that insulin does, indeed, stimulate sugar transport in barnacle muscle. The great advantage of barnacle muscle over all other muscles used so far for investigating the mechanism of insulin action is its large size, which facilitates measurements on single cells and permits the experimenter to control the intracellular environment of the muscle fibre by the technique of internal dialysis. Using single muscle fibres it is possible to show that acceleration of sugar transport by insulin is associated with a fall in ionized Ca, a fall in cyclic AMP and a rise in cyclic GMP. Working with internally dialysed muscle fibres we find that insulin only increases sugar transport when the dialysis solution contains ATP. In the absence of insulin, sugar transport is dialysed muscle is increased by a rise in ionized Ca, a fall in cyclic AMP and, when the internal Ca is elevated, by a rise in cyclic GMP.     

Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.     

Functional reconstitution of purified muscarinic receptors and inhibitory guanine nucleotide regulatory protein

Muscarinic receptors trigger several different responses including an increase in concentration of cyclic GMP, a decrease in cyclic AMP concentration, breakdown of polyphosphoinositides and changes in ion permeability. It is not yet clear whether these reactions occur sequentially or independently and which directly coupled to the muscarinic receptor. Several lines of evidence indicate that muscarinic receptors in many, if not all, cell types are coupled to the inhibitory guanine nucleotide regulatory protein (Ni or Gi) of adenylate cyclase. To provide direct evidence for this coupling, we have reconstituted muscarinic receptors purified from porcine brain with Ni purified from rat brain in a phospholipid vesicle. Here, we report that the GTPase activity of Ni is stimulated by carbachol. This action is blocked by the simultaneous addition of atropine and is not observed when the Ni protein is ADP-ribosylated. We conclude that one function of the muscarinic receptor is the activation of Ni.     

Mutation of protein kinase A causes heterochronic development of Dictyostelium.

In heterochronic mutants the relative timing of developmental events is altered compared with the wild type. This generally results in a disordered embryo, though heterochronic mutations may also be an important source of evolutionary variation. In the rapidly developing (rde) mutants of Dictyostelium, stalk and spore cells differentiate before morphogenesis is complete. We have traced the lesion in one class of these mutants to the regulatory subunit of cyclic AMP-dependent protein kinase (pk-A). Inactivation of this protein results in the unrestrained activity of the catalytic subunit, so prematurely triggering terminal cell differentiation.     

Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.     

Structural basis of protein phosphatase 1 regulation

The coordinated and reciprocal action of serine/threonine (Ser/Thr) protein kinases and phosphatases produces transient phosphorylation, a fundamental regulatory mechanism for many biological processes. The human genome encodes a far greater number of Ser/Thr protein kinases than of phosphatases. Protein phosphatase 1 (PP1), in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell-cycle progression and muscle relaxation. PP1 has evolved effective catalytic machinery but lacks substrate specificity. Substrate specificity is conferred upon PP1 through interactions with a large number of regulatory subunits. The regulatory subunits are generally unrelated, but most possess the RVxF motif, a canonical PP1-binding sequence. Here we reveal the crystal structure at 2.7 A resolution of the complex between PP1 and a 34-kDa N-terminal domain of the myosin phosphatase targeting subunit MYPT1. MYPT1 is the protein that regulates PP1 function in smooth muscle relaxation. Structural elements amino- and carboxy-terminal to the RVxF motif of MYPT1 are positioned in a way that leads to a pronounced reshaping of the catalytic cleft of PP1, contributing to the increased myosin specificity of this complex. The structure has general implications for the control of PP1 activity by other regulatory subunits.     

Chronic ethanol causes heterologous desensitization of receptors by reducing alpha s messenger RNA

One of the biochemical results of ethanol exposure is a change in the amount of the intracellular second messenger cyclic AMP (cAMP) produced in response to receptor stimulation. In general, acute ethanol exposure increases the amount of cAMP produced on stimulation of receptors coupled to the enzyme adenylyl cyclase via the GTP-binding protein Gs, whereas chronic ethanol exposure has the opposite effect (results for receptors coupled via Gi have been more variable). We previously reported that adaptation to continuous ethanol exposure reduces receptor-stimulated cAMP production by 25-35% in a neuroblastoma cell line (NG108-15), and an even greater reduction of 75% was observed in lymphocytes taken from actively-drinking alcoholics. This reduction in receptor-stimulated cAMP levels was recently confirmed in platelets from alcoholics. None of these studies, however, determined whether more than one receptor coupled to adenylyl cyclase activity was affected in the same cell. Here we report that chronic ethanol exposure causes desensitization of heterologous receptors coupled to Gs as cAMP production mediated by prostaglandin E1 as well as by adenosine is reduced by approximately 30% in NG108-15 cells. We show that, after chronic ethanol exposure, the activity of the alpha subunit of Gs is decreased by 29%, the amount of alpha s protein is decreased by 38.5%, and alpha s messenger RNA is decreased by 30%. Thus, cellular adaptation to ethanol involves a reduction in alpha s mRNA and, as a consequence, reduced cAMP production by heterologous receptors coupled to Gs. Such changes in cAMP production may account for the tolerance and physical dependence on ethanol in alcoholism.     

Fluorescence ratio imaging of cyclic AMP in single cells.

Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.     

Structural basis for AMP binding to mammalian AMP-activated protein kinase

AMP-activated protein kinase (AMPK) regulates cellular metabolism in response to the availability of energy and is therefore a target for type II diabetes treatment. It senses changes in the ratio of AMP/ATP by binding both species in a competitive manner. Thus, increases in the concentration of AMP activate AMPK resulting in the phosphorylation and differential regulation of a series of downstream targets that control anabolic and catabolic pathways. We report here the crystal structure of the regulatory fragment of mammalian AMPK in complexes with AMP and ATP. The phosphate groups of AMP/ATP lie in a groove on the surface of the gamma domain, which is lined with basic residues, many of which are associated with disease-causing mutations. Structural and solution studies reveal that two sites on the gamma domain bind either AMP or Mg.ATP, whereas a third site contains a tightly bound AMP that does not exchange. Our binding studies indicate that under physiological conditions AMPK mainly exists in its inactive form in complex with Mg.ATP, which is much more abundant than AMP. Our modelling studies suggest how changes in the concentration of AMP ([AMP]) enhance AMPK activity levels. The structure also suggests a mechanism for propagating AMP/ATP signalling whereby a phosphorylated residue from the alpha and/or beta subunits binds to the gamma subunit in the presence of AMP but not when ATP is bound.     

Endogenous ADP-ribosylation of Gs subunit and autonomous regulation of adenylate cyclase

Although cholera toxin induces a marked stimulation of adenylate cyclase activity in rat adipocyte plasma membranes, the holotoxin induces only a slight increase of cyclic AMP accumulation in intact cells. A similar apparent anomaly is seen with pertussis toxin, which has been shown to inhibit the Gi subunit of adenylate cyclase, and has a greater effect on cAMP accumulation and lipolysis than the activation by cholera toxin of the Gs subunit. To understand better the way in which these bacterial toxins are modifying the adipocyte cells, we prepared adipocyte plasma membranes and submitted them to ADP-ribosylation by cholera and pertussis toxins. During the incubation of control cells, we found endogenous ADP-ribosylation of Gs as a result of sustained stimulation of Gi by adenosine. Our results point to a possible homoeostatic system in which the autonomous adjustment of the basal activity of Gs as a function of that of Gi, under the control of feedback inhibitory ligands, ensures a steady production of cAMP within the cell.     

Modulation of single Ca2+-dependent K+-channel activity by protein phosphorylation

There is considerable evidence that cyclic AMP can modulate the electrical activity of excitable cells and that protein phosphorylation by the catalytic subunit (CS) of cAMP-dependent protein kinase is a necessary step in these modulatory effects. In analogy to alterations in enzyme activities following phosphorylation, it seems possible that direct phosphorylation of ion-channel proteins may alter their gating properties, giving rise to the observe changes in electrical activity. However, the results obtained so far do not indicate whether it is ion channels themselves that are phosphorylated, or whether phosphorylation is simply an early step in some cascade of events which leads ultimately to modulation of channel activity. The development of single-channel recording techniques has provided a way to investigate this question. Here we describe effects of CS on the activity of individual CA2+-dependent K+ channels from the nervous system of the land snail Helix measured in isolated membrane patches and in artificial phospholipid bilayers. The results demonstrate that cAMP-dependent protein phosphorylation produces long-lasting changes in the activity of individual channels, and indicate that the relevant phosphorylation site is closely associated with the channel.     

Cyclic nucleotides may mediate taste transduction

Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. But due to technical difficulties of inserting glass electrodes into the mammalian taste cell, little is known about the mechanisms of taste transduction. Reliable intracellular recordings are necessary to determine the characteristics of taste cells. This has been accomplished previously in the mouse and is reported here. Recent experiments indicated that cyclic nucleotides can act on the inner surface of the membranes of a variety of cells to alter their ion-channel activity, and these substances might act as intracellular transmitters in taste cells. But tight junctions found at the apical membrane of mammalian taste cells do not allow stimuli to enter the taste bud, making it difficult to alter the environment of the taste cell by perfusing with chemical solutions. Here we report that cyclic AMP, cyclic GMP, EGTA or tetraethyl-ammonium electrophoretically injected into the mouse taste cell induce membrane depolarization and increased membrane resistance. These results suggest that a cyclic nucleotide enzymatic cascade, modulated by calcium ions, may mediate the potassium permeability that controls taste, in a way analogous to visual and olfactory transduction.     

Transmission of hormonal stimulation by cell-to-cell communication.

Rat ovarian granulosa cells and mouse myocardial cells respond to cell-specific hormones by cyclic AMP-dependent mechanisms. In coculture, these heterologous cells communicate by means of gap junctions. Exposure of the cocultures to a hormone specific for one cell type causes the heterologous cells to respond through a cell contact-dependent mechanism. These studies suggest that this cross-stimulation results from the intercellular communication of a mediator that is common to both cell types. The communicated mediator may be cyclic AMP.