Separation of regulatory and catalytic subunits of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase(s) of rabbit skeletal muscle

The cyclic 3′, 5′-adenosine monophosphate-dependent (cAMP-dependent) protein kinase(s) from rabbit skeletal muscle has been separated into catalytic and regulatory subunits by affinity chromatography utilizing a casein-Sepharose column in the presence of cAMP. The isolated catalytic subunit manifests full activity in the absence of cAMP but its requirement for this nucleotide is regained when the enzyme is reconstituted by addition of the regulatory subunit. Evidence is presented for the existence of more than a single type of regulatory or cAMP-binding subunit in muscle.     

Mechanism(s) of activation of secretory phospholipase A(2)s in mouse keratinocytes.

The differential activation of different members of the phospholipase A(2) (PLA(2)) superfamily and their regulation are important as one or more of them regulates the production of eicosanoids and others may contribute to the formation of other lipid mediators. We previously reported the existence of two forms of secretory or sPLA(2) in mouse keratinocytes, namely type I and type II sPLA(2). We show here that mouse keratinocyte sPLA(2)s were potently activated by protease treatment and inhibited by protease inhibitors. We also observed that G protein effectors induced substantial release of oleic acid (OA) from prelabeled mouse keratinocytes. A G(i)/G(0) protein activator significantly enhanced the hydrolysis of OA and this increase was not responsive to either pertussis toxin or cholera toxin treatment. Although there was a significant negative correlation between intracellular cAMP levels and OA hydrolysis, experimentally increasing cAMP with forskolin treatment had no effect on sPLA(2) activity. Arachidonic acid but not its metabolites was also shown to marginally activate keratinocyte sPLA(2) by 1.5-fold. These results lead to the conclusion that mouse keratinocyte sPLA(2)s can be regulated primarily by proteolytic activation and a G protein pathway.     

Fish gonadotropin(s). I. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads.

1. The effect of salmon gonadotropin(s) (SG) on cyclic AMP (cAMP) levels in immature trout gonadal tissue of both sexes was measured by radioimmunoassay. 2. A dose-response line was obtained to SG in gonads of both male and female trout. 3. As little as 0.45 SG units (1 SG unit = 1 mug NIH-LH-S18 in the chick bioassay) significantly increased cAMP formation in the presence of 8 mM theophylline; mammalian LH, FSH, LTH, ACTH, TSH and HCG were inactive. 4. The assay for SG was investigated with respect to time of incubation and two phosphodiesterase inhibitors; some conditions for the cAMP radioimmunoassay (cAMP-RIA) were compared.     

The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network

Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.     

Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3)

cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed inEscherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (<1 μM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [³²P]-cGMP. The IC₅₀ values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP. These authors contributed equally to this work.     

Ethanol inhibits palmitoylation of G protein G alpha(s)

Neurobiological actions of ethanol have been linked to perturbations in cyclic AMP (cAMP)-dependent signaling processes. Chronic ethanol exposure leads to desensitization of cAMP production in response to physiological ligands (heterologous desensitization). Ethanol-induced alterations in neuronal expression of G proteins G(s) and G(i) have been invoked as a cause of heterologous desensitization. However, effects of ethanol on G protein expression vary considerably among different experimental protocols, various brain regions and diverse neuronal cell types. Dynamic palmitoylation of G protein alpha subunits is critical for membrane localization and protein-protein interactions, and represents a regulatory feature of G protein function. We studied the effect of ethanol on G alpha(s) palmitoylation. In NG108-15 rat neuroblastoma x glioma hybrid cells, acute exposure to pharmacologically relevant concentrations of ethanol (25-100 mm) inhibited basal and prostaglandin E1-stimulated incorporation of palmitate into G alpha(s). Exposure of NG108-15 cells to ethanol for 72 h induced a shift in G alpha(s) to its non-palmitoylated state, coincident with an inhibition of prostaglandin E1-induced cAMP production. Both parameters were restored following 24 h of ethanol withdrawal. Chronic ethanol exposure also induced the depalmitoylation of G alpha(s) in human embryonic kidney (HEK)293 cells that overexpress wild-type G alpha(s) and caused heterologous desensitization of adenylyl cyclase. By contrast, HEK293 cells that express a non-palmitoylated mutant of G alpha(s) were insensitive to heterologous desensitization after chronic ethanol exposure. In summary, the findings identify a novel effect of ethanol on post-translational lipid modification of G alpha(s), and represent a mechanism by which ethanol might affect adenylyl cyclase activity.     

Biochemical mechanism(s) of stunning in conscious dogs

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% (P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.     

A G(s)-linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor-G(s) signaling

The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.     

The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides -7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one-to-one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst-inducing substance(s) and cAMP at the aggregation stage.     

Synthesis and characterization of amphiphilic monodisperse compounds and poly(ethylene imine)s: influence of their microstructures on the antimicrobial properties

Amphiphilic monodisperse compounds (series B-I and B-II) and poly(ethylene imine)s (PEI-I, PEI-II, and PEI-III) with different microstructures were prepared from primary amines or poly(ethylene imine) with functional carbonates bearing cationic, hydrophobic, or amphiphilic groups. Their inhibition potential against proliferation of E. coli , S. aureus , and B. subtilis was investigated and their hemolytic activities were determined. The influence of the microstructures, the alkyl chain length and the distribution of cationic and hydrophobic groups, on their antimicrobial efficacy was studied. Amphiphilic compounds with long alkyl chains (C14-C18) directly linked to the cationic groups (series B-I) are more effective against both Gram-positive and Gram-negative bacteria than amphiphilic compounds in which the hydrophobic and cationic groups (series B-II) are connected by a spacer. Poly(ethylene imine)s with amphiphilic grafts (B-I) called PEI-II are more effective than amphiphilic PEIs with the same alkyl chain but with randomly linked cationic and hydrophobic graft called PEI-I or with the amphiphilic grafts (B-II) called PEI-III. The influence of the inoculum size on the MIC value was investigated exemplarily with compounds of series B-I against S. aureus .     

Activation-induced subcellular redistribution of G alpha(s) is dependent upon its unique N-terminus

The heterotrimeric G protein subunit, alpha(s), can move reversibly from plasma membranes to cytoplasm in response to activation by GPCRs or activating mutations. We examined the importance of the unique N-terminus of alpha(s) in this translocation in cultured cells. alpha(s) contains a single site for palmitoylation in its N-terminus, and this was replaced by different plasma membrane targeting motifs. These N-terminal alpha(s) mutants were targeted properly to plasma membranes, capable of coupling activated GPCRs to effectors, and able to constitutively stimulate cAMP production when they also contained an activating mutation. However, when activated by a constitutively activating mutation or by agonist-activated beta-AR, these N-terminal alpha(s) mutants failed, for the most part, to undergo redistribution from plasma membranes to cytoplasm, as assayed by immunofluorescence microscopy, or from a particulate to soluble fraction, as assayed by subcellular fractionation. These results highlight the importance of the extreme N-terminus of alpha(s) and its single site of palmitoylation for facilitating activation-induced translocation and provide insight into the mechanism of this G protein trafficking event.     

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.     

Effects of seminiferous tubule secreted factor(s) on Leydig cell cyclic AMP production in mature rat

The effects of spent media from seminiferous tubules (STM) on Percoll-purified rat Leydig cells were investigated. Intracellular and extracellular cyclic AMP (cAMP) accumulation and testosterone production were measured. After a 5 h incubation period, STM reduces both the basal and LH-dependent cAMP levels (38 and 20%, respectively for intra- and extracellular cAMP) while, simultaneously, a stimulation of testosterone production is observed (47 to 50%, respectively in the absence or presence of LH). The reduction of cAMP levels observed after 5 h is likely to be due to the potentiating effect of the STM factor on the LH-dependent initial rise of the cAMP level which, in turn, induces a desensitization of the Leydig cell adenylate cyclase. This substance is a thermolabile protein (Mr greater than 50 000) produced by the Sertoli cell, independent of FSH and testosterone controls, and different from the LHRH-like substance.     

Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity

Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.     

Role of dynamics in the autoinhibition and activation of the exchange protein directly activated by cyclic AMP (EPAC)

The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.     

Involvement of both G(q/11) and G(s) proteins in gonadotropin-releasing hormone receptor-mediated signaling in L beta T2 cells

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in L beta T2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LH beta genes in these cells. Signaling via the G(i) subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LH beta induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters G beta gamma-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of G alpha(q) (Q209L) and was blocked by a cell-permeable peptide that uncouples G alpha(q) from GPCRs. Furthermore, chronic activation of G alpha(q) signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples G alpha(s) from receptors was also able to inhibit ERK, c-Fos, and LH beta, indicating that both G(q/11) and G(s) proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on G(s) and G(q/11) and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of G alpha(q) (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both G(q) and G(s) signaling to regulate gene expression in L beta T2 cells.     

Dexamethasone potentiates high-affinity beta-agonist binding and g(s)alpha protein expression in airway smooth muscle

Corticosteroids enhance beta-adrenergic responses by actions at both beta-adrenoceptor (beta-AR) and post-beta-AR sites. The present study investigated the effects of dexamethasone on beta-AR density, high-affinity beta-agonist binding, G(s)alpha and G(i)alpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta-AR density 1.6- to 1.9-fold as assessed by the saturation binding of [(3)H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta-AR at two sites, a high-affinity site with a density of 5.9 +/- 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 +/- 1. 0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 +/- 2.2 and 25.9 +/- 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased G(s)alpha protein levels from 0.99 +/- 0.14 to 1.46 +/- 0.17 microg/mg protein without affecting G(i)alpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta-adrenergic signaling pathways in airway smooth muscle.     

Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Protein kinase A (PKA), a central locus for cAMP signaling in the cell, is composed of regulatory (R) and catalytic (C) subunits. The C-subunits are maintained in an inactive state by binding to the R-subunit dimer in a tetrameric holoenzyme complex (R(2)C(2)). PKA is activated by cAMP binding to the R-subunits which induces a conformational change leading to release of the active C-subunit. Enzymatic activity of the C-subunit is thus regulated by cAMP via the R-subunit, which toggles between cAMP and C-subunit bound states. The R-subunit is composed of a dimerization/docking (D/D) domain connected to two cAMP-binding domains (cAMP:A and cAMP:B). While crystal structures of the free C-subunit and cAMP-bound states of a deletion mutant of the R-subunit are known, there is no structure of the holoenzyme complex or of the cAMP-free state of the R-subunit. An important step in understanding the cAMP-dependent activation of PKA is to map the R-C interface and characterize the mutually exclusive interactions of the R-subunit with cAMP and C-subunit. Amide hydrogen/deuterium exchange mass spectrometry is a suitable method that has provided insights into the different states of the R-subunit in solution, thereby allowing mapping of the effects of cAMP and C-subunit on different regions of the R-subunit. Our study has localized interactions with the C-subunit to a small contiguous surface on the cAMP:A domain and the linker region. In addition, C-subunit binding causes increased amide hydrogen exchange within both cAMP-domains, suggesting that these regions become more flexible in the holoenzyme and are primed to bind cAMP. Furthermore, the difference in the protection patterns between RIalpha and the previously studied RIIbeta upon cAMP-binding suggests isoform-specific differences in cAMP-dependent regulation of PKA activity.