cAMP-dependent protein kinase and heterologous desensitization of the adenylyl cyclase

Previous experiments had shown no differences in desensitization in cells with mutations of the adenylyl cyclase or the cAMP-dependent protein kinase and had ruled out this kinase as a mediator of desensitization; however, the assays of adenylyl cyclase had been made at high concentrations of free magnesium. The work presented in this dissertation documents a role for cAMP-dependent protein kinase which became apparent with assays at low concentrations of free magnesium. (1) The adenylyl cyclase in membranes from wild type S49 lymphoma cells showed substantial desensitization after incubation of the intact cells with low concentrations of epinephrine (5-20 nM). This desensitization was heterologous, that is it reduced the subsequent responses of the adenylyl cyclase to both epinephrine and prostaglandin-E$\sb1$. (2) The adenylyl cyclase in membranes of S49 cyc$\sp-$ cells, which do not make cAMP in response to hormones, and S49 kin$\sp-$ cells, which lack cAMP-dependent protein kinase activity, showed no heterologous desensitization following incubation of the intact cells with low concentrations of hormones. (3) Heterologous desensitization of the adenylyl cyclase was induced by incubations of wild type cells with forskolin, which activates the adenylyl cyclase downstream of the hormone receptors, or dibutyryl-cAMP, which activates the cAMP-dependent protein kinase directly. (4) Site-directed mutagenesis was used to delete the cAMP-dependent protein kinase consensus phosphorylation sequences on the $\beta$-adrenergic receptor. Heterologous desensitization occurred in intact L-cells expressing the wild type receptor or the receptor lacking the C-terminal phosphorylation site; however, only homologous desensitization occurred when the phosphorylation site on the third intracellular loop of the receptor was deleted. (5) To test directly the effects of cAMP-dependent protein kinase on the adenylyl cyclase the catalytic subunit of the kinase was purified from bovine heart and incubated with adenylyl cyclase in plasma membrane preparations. In this cell-free system the kinase caused rapid heterlogous reductions of the responsiveness of the S49 wild type adenylyl cyclase. Additionally, the adenylyl cyclase in kin$\sp-$ membranes, which showed only homologous desensitization in the intact cell, was desensitization by cell-free incubation with the kinase.^ The epinephrine responsiveness was not affected in L-cell membranes expressing the $\beta$-adrenergic receptor lacking the cAMP-dependent protein kinase consensus sequence on the third intracellular loop. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Effects of variable Gs expression on $\beta\sb2$-adrenergic receptor stimulated adenylyl cyclase response

An exact knowledge of the kinetic nature of the interaction between the stimulatory G protein (G$\sb{\rm s}$) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. In particular, insight as to the association of these proteins could lead to progress in tumor biology where single spontaneous mutations in G proteins have been associated with the formation of tumors (118). The question this work attempts to answer is whether the adenylyl cyclase activation by epinephrine stimulated $\beta\sb2$-adrenergic receptors occurs via G$\sb{\rm s}$ proteins by a G$\sb{\rm s}$ to C shuttle or G$\sb{\rm s}$-C precoupled mechanism. The two forms of activation are distinguishable by the effect of G$\sb{\rm s}$ levels on epinephrine stimulated EC50 values for cyclase activation.^ We have made stable transfectants of S49 cyc$\sp-$ cells with the gene for the $\alpha$ protein of G$\sb{\rm s}$ $(\alpha\sb{\rm s})$ which is under the control of the mouse mammary tumor virus LTR promoter (110). Expression of G$\sb{\rm s}\alpha$ was then controlled by incubation of the cells for various times with 5 $\mu$M dexamethasone. Expression of G$\sb{\rm s}\alpha$ led to the appearance of GTP shifts in the competitive binding of epinephrine with $\sp{125}$ICYP to the $\beta$-adrenergic receptors and to agonist dependent adenylyl cyclase activity. High expression of G$\sb{\rm s}\alpha$ resulted in lower EC50's for the adenylyl cyclase activity in response to epinephrine than did low expression. By kinetic modelling, this result is consistent with the existence of a shuttle mechanism for adenylyl cyclase activation by hormones.^ One item of concern that remains to be addressed is the extent to which activation of adenylyl cyclase occurs by a "pure" shuttle mechanism. Kinetic and biochemical experiments by other investigators have revealed that adenylyl cyclase activation, by hormones, may occur via a Gs-C precoupled mechanism (80, 94, 97). Activation of adenylyl cyclase, therefore, probably does not occur by either a pure "'Shuttle" or "Gs-C Precoupled" mechanism, but rather by a "Hybrid" mechanism. The extent to which either the shuttle or precoupled mechanism contributes to hormone stimulated adenylyl cyclase activity is the subject of on-going research. ^

Cloning, characterization and regulation of the soluble guanylyl cyclase alpha1 and beta1 genes

Cell signaling by nitric oxide (NO) through soluble guanylyl cyclase (sGC) and cGMP production regulates physiological responses such as smooth muscle relaxation, neurotransmission, and cell growth and differentiation. Although the NO receptor, sGC, has been studied extensively at the protein level, information on regulation of the sGC genes remains elusive. In order to understand the molecular mechanisms involved at the level of gene expression, cDNA and genomic fragments of the murine sGCα1 subunit gene were obtained through library screenings. Using the acquired clones, the sGCα 1 gene structure was determined following primer extension, 3 ′RACE and intron/exon boundary analyses. The basal activity of several 5′-flanking regions (putative promoter regions) for both the α1 and β1 sGC subunits were determined following their transfection into mouse N1E-115 neuroblastoma and rat RENE1Δ14 uterine epithelial cells using a luciferase reporter plasmid. Using the sGC sequences, real-time RT-PCR assays were designed to measure mRNA levels of the sGC α1 and β1 genes in rat, mouse and human. Subsequent studies found that uterine sGC mRNA and protein levels decreased rapidly in response to 17β-estradiol (estrogen) in an in vivo rat model. As early as 1 hour following treatment, mRNA levels of both sGC mRNAs decreased, and reached their lowest level of expression after 3 hours. This in vivo response was completely blocked by the pure estrogen receptor antagonist, ICI 182,780, was not seen in several other tissues examined, did not occur in response to other steroid hormones, and was due to a post-transcriptional mechanism. Additional studies ex vivo and in various cell culture models suggested that the estrogen-mediated decreased sGC mRNA expression did not require signals from other tissues, but may require cell communication or paracrine factors between different cell types within the uterus. Using chemical inhibitors and molecular targeting in other related studies, it was revealed that c-Jun-N-terminal kinase (JNK) signaling was responsible for decreased sGC mRNA expression in rat PC12 and RFL-6 cells, two models previously determined to exhibit rapid decreased sGC mRNA expression in response to different stimuli. To further investigate the post-transcriptional gene regulation, the full length sGCα1 3′-untranslated region (3′UTR) was cloned from rat uterine tissue and ligated downstream of the rabbit β-globin gene and expressed as a chimeric mRNA in the rat PC12 and RFL-6 cell models. Expression studies with the chimeric mRNA showed that the sGCα 1 3′UTR was not sufficient to mediate the post-transcriptional regulation of its mRNA by JNK or cAMP signaling in PC12 and RFL-6 cells. This study has provided numerous valuable tools for future studies involving the molecular regulation of the sGC genes. Importantly, the present results identified a novel paradigm and a previously unknown signaling pathway for sGC mRNA regulation that could potentially be exploited to treat diseases such as uterine cancers, neuronal disorders, hypertension or various inflammatory conditions. ^

CCTeta: A novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of 3′,5 ′-cyclic guanosine monophosphate (cGMP) from 5′ -guanosine triphosphate (GTP). In this thesis, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit η and the α1β1 isoform of sGC. Using the yeast-two-hybrid system, CCTη was found to interact with the N-terminal portion of β1 subunit of sGC. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast-two-hybrid system, CCTη was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTη and Sf9 lysate expressing sGC resulted in a 33% inhibition of sodium nitroprusside (SNP)-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTη had no effect on this activity. Furthermore, CCTη had no effect on the activity of αβCys105 sGC a constitutively active mutant that lacks a heme group. Of note is the fact that the full-length CCTη-expressing bacterial lysate inhibited the activity of sGC-expressing Sf9 lysate by 48% compared with GST alone. This indicates that the amino terminal 94 amino acids of CCTη are important to the inhibition of sGC activity. Lastly, a 45% inhibition of sGC activity by CCTη was seen in vivo in BE2 cells stably transfected with CCTη and treated with SNP. The fact that the inhibition of sGC was more pronounced with bacterial lysate expressing CCTη versus the purified CCTη implies that some factor in the bacterial lysate enhances the inhibitory effect of CCTη. Because the level of inhibition seen in bacterial lysate and in vivo experiments is similar, might imply that the factor that aids in CCTη effect on sGC is conserved. Together, these data suggest that CCTη is a novel type of sGC inhibitor that inhibits sGC by modifying the binding of NO to the heme group or the subsequent conformational changes induced by NO binding. ^

Scaffolding of adenylyl cyclase by A Kinase Anchoring Proteins

Adenylyl cyclase (AC) converts ATP into cAMP, which activates protein kinase A (PKA). Activation of PKA leads to the phosphorylation of specific substrates. The mechanism of specificity of PKA phosphorylation baffled researchers for many years. The discovery of A Kinase Anchoring Proteins (AKAPs) has helped to unravel this mystery. AKAPs function to target PKA to specific regions within the cell. They also anchor other enzymes, receptors, or channels leading to tightly regulated signaling modules. Several studies have suggested an important role for activated PKA in these complexes, including the AKAPs yotiao and muscle AKAP (mAKAP). Yotiao, a plasma membrane AKAP, anchors PP1, NMDA receptors, IP3 receptors, and heart potassium channel subunit KCNQI. PKA phosphorylation of NMDA receptors as well as KCNQI leads to increased channel activity. Patients with mutations in KCNQI or yotiao that cause loss of targeting of KCNQI develop long QT syndrome, which can be fatal. mAKAP anchors several CAMP/PKA-regulated pathways to the nuclear envelope in cardiac myocytes. The necessity of activated PKA in these complexes led to the hypothesis that AC is also anchored. The results indicate that AC does associate with yotiao in brain and heart, specifically with AC types I-III, and IX. Co-expression of AC II or III with yotiao leads to inhibition of each isoform's activity. Binding assays revealed that yotiao binds to the N-terminus of AC II and that this region can reverse the inhibition of AC II, but not AC III, indicating unique binding sites on yotiao. AC II binds directly to as 808-957 of yotiao. Y808-957 acts as a dominant negative as the addition of it to rat brain membranes results in a ∼40% increase in AC activity. Additionally, AC was also found to associate with mAKAP in heart, specifically with AC types II and V. The binding site of AC was mapped to 275-340 of mAKAP, while mAKAP binds to the soluble domains of AC V as a complex. These results indicate that interactions between AC and AKAPs are specific and that AC plays an important role in AKAP-targeted signaling. ^

CHARACTERIZATION OF SELECTIVE INHIBITION OF ADENYLYL CYCLASE ACTIVITY BY SMALL MOLECULE INHIBITOR NKY80

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. Decreasing AC5 activity has been shown to have potential therapeutic benefit, including reduced stress on the heart, pain relief, and attenuation of morphine dependence and withdrawal behaviors. However, AC structure is well conserved, and there are currently no isoform selective AC inhibitors in clinical use. P-site inhibitors inhibit AC directly at the catalytic site, but with an uncompetitive or noncompetitive mechanism. Due to this mechanism and nanomolar potency in cell-free systems, attempts at ligand-based drug design of novel AC inhibitors frequently use P-site inhibitors as a starting template. One small molecule inhibitor designed through this process, NKY80, is described as an AC5 selective inhibitor with low micromolar potency in vitro. P-site inhibitors reveal important ligand binding “pockets” in the AC catalytic site, but specific interactions that give NKY80 selectivity are unclear. Identifying and characterizing unique interactions between NKY80 and AC isoforms would significantly aid the development of isoform selective AC inhibitors. I hypothesized that NKY80’s selective inhibition is conferred by AC isoform specific interactions with the compound within the catalytic site. A structure-based virtual screen of the AC catalytic site was used to identify novel small molecule AC inhibitors. Identified novel inhibitors are isoform selective, supporting the catalytic site as a region capable of more potent isoform selective inhibition. Although NKY80 is touted commercially as an AC5 selective inhibitor, its characterization suggests strong inhibition of both AC5 and the closely related AC6. NKY80 was also virtually docked to AC to determine how NKY80 binds to the catalytic site. My results show a difference between NKY80 binding and the conformation of classic P-site inhibitors. The selectivity and notable differences in NKY80 binding to the AC catalytic site suggest a catalytic subregion more flexible in AC5 and AC6 that can be targeted by selective small molecule inhibitors.

ATP- and carbachol -stimulated 1,2-diacylglycerol production during sensitization of adenylyl cyclase

Stimulation of LM5 cells with the phorbol ester 4$\beta$-phorbol 12-myristate 13-acetate (PMA), causes a 2-4 fold sensitization of hormonally-stimulated adenylyl cyclase (AC) activity. This effect is thought to be due to protein kinase C (PKC)-mediated phosphorylation of either G$\sb{\rm i}$ or the catalytic subunit of AC. PKC are components of the phosphatidylinositol-4,5-bisphosphate phospholipase C (PIP$\sb2$-PLC) pathway. The currently accepted model of this pathway is that its activation by an agonist results in the production of inositol 1,4,5-triphosphate (IP$\sb3$) which causes Ca$\sp{++}$ mobilization, and 1,2-diacylglycerols (DAG) which activate PKC. Based on this model, we predicted that stimulation of purinergic and muscarinic receptors with the agonists ATP and carbachol (CCh), respectively in the LM5 cells, should sensitize AC. Surprisingly we found that only stimulation of the purinergic receptors in these cells caused a sensitization of PGE$\sb1$-stimulated AC measured in cell-free assays.^ We hypothesized that ATP-and CCh-stimulated differential DAG production contributes to the effectiveness of these two agonists to sensitize PGE$\sb1$-stimulated AC activity. To test this hypothesis directly, we performed a combined high-performance liquid chromatography and gas-liquid chromatography analysis of the DAG produced in the LM5 cells in response to stimulation with ATP and CCh.^ We found that both ATP and CCh increased levels of 23 species of DAG. Relative to the control levels (0.261 nmol DAG/100 nmol phospholipid) the CCh-induced increase in DAG levels was 280% (0.738 $\pm$ 0.051 nmol DAG/100 nmol phospholipid) whereas the ATP-induced levels increased 180% (0.441 t 0.006 nmol DAG/100 nmol phospholipid). Neither agonist created new species or eliminated the existing ones. The major species which comprised $\approx$50% of the total cellular DAG in all of the groups were 16:0-18:1, 18:0-18:1, 18:1-18:1, and 18:0-20:4. CCh was more effective than ATP at stimulating these major DAG species.^ It is concluded that factor(s) other than DAG contribute(s) to the differences between ATP-and CCh-sensitization of PGE$\sb1$-stimulated AC activity in the LM5 cells. ^

Nicotinamide nucleotide and adenylate concentrations in mante, siphon and gill tissue of old and young Laternula elliptica individuals under control and experimental conditions

Future oceans are predicted to contain less oxygen than at present. This is because oxygen is less soluble in warmer water and predicted stratification will reduce mixing. Hypoxia in marine environments is thus likely to become more widespread in marine environments and understanding species-responses is important to predicting future impacts on biodiversity. This study used a tractable model, the Antarctic clam, Laternula elliptica, which can live for 36 years, and has a well-characterized ecology and physiology to understand responses to hypoxia and how the effect varied with age. Younger animals had a higher condition index, higher adenylate energy charge and transcriptional profiling indicated that they were physically active in their response to hypoxia, whereas older animals were more sedentary, with higher levels of oxidative damage and apoptosis in the gills. These effects could be attributed, in part, to age-related tissue scaling; older animals had proportionally less contractile muscle mass and smaller gills and foot compared with younger animals, with consequential effects on the whole-animal physiological response. The data here emphasize the importance of including age effects, as large mature individuals appear to be less able to resist hypoxic conditions and this is the size range that is the major contributor to future generations. Thus, the increased prevalence of hypoxia in future oceans may have marked effects on benthic organisms' abilities to persist and this is especially so for long-lived species when predicting responses to environmental perturbation.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsα

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (Gsα) to stimulate enzyme activity. Concentration–response curves with Gsα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572–F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042–T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042–T1051) in the C2 domain of the enzyme to modulate Gsα-elicited stimulation of activity.