Adenylyl Cyclase Type Research Articles

Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

A Guanylyl Cyclase from Paramecium with 22 Transmembrane Spans: EXPRESSION OF THE CATALYTIC DOMAINS AND FORMATION OF CHIMERAS WITH THE CATALYTIC DOMAINS OF MAMMALIAN ADENYLYL CYCLASES

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.

Differential superactivation of adenylyl cyclase isozymes after chronic activation of the CB(1) cannabinoid receptor.

Many types of cells exhibit increased adenylyl cyclase (AC) activity after chronic agonist treatment of G(i/o)-coupled receptors. This phenomenon, defined as AC superactivation or sensitization, has mostly been studied for the opioid receptors and is implicated in opiate addiction. Here we show that this phenomenon is also observed on chronic activation of the CB(1) cannabinoid receptor. Moreover, using COS-7 cells cotransfected with CB(1) receptor and individual AC isozymes, we could show selective superactivation of AC types I, III, V, VI, and VIII. The level of superactivation was dependent on the concentration of agonist and time of agonist exposure and was not dependent on the AC stimulator used. No superactivation of AC types II, IV, or VII was observed in COS-7 cells cotransfected with CB(1). The superactivation of AC type V was abolished by pretreatment with pertussis toxin and by cotransfection with the carboxy terminus of beta-adrenergic receptor kinase, which serves as a scavenger of G(betagamma) dimers, implying a role for the G(i/o) proteins and especially G(betagamma) dimers in the cannabinoid-induced superactivation of AC.

Specific expression of soluble adenylyl cyclase in male germ cells.

The cAMP signaling pathway is an important mediator of extracellular signals in organisms from prokaryotes to higher eukaryotes. In mammals two types of adenylyl cyclase synthesize cAMP; a ubiquitous family of transmembrane isoforms regulated by G proteins in response to extracellular signals, and a recently isolated soluble enzyme insensitive to heterotrimeric G protein modulation. Using the very sensitive reverse transcription-polymerase chain reaction (RT-PCR), soluble adenylyl cyclase (sAC) expression is detectable in almost all tissues examined; however, Northern analysis and in situ hybridization indicate that high levels of sAC message are unique to male germ cells. Elevated levels of sAC mRNA are first observed in pachytene spermatocytes and expression increases through spermiogenesis. The accumulation of high levels of message in round spermatids suggests sAC protein plays an important role in the generation of cAMP in spermatozoa, implying possible roles in sperm maturation through the epididymis, capacitation, hypermotility, and/or the acrosome reaction.

Ca2+, Sr2+, and Ba2+ Identify Distinct Regulatory Sites on Adenylyl Cyclase (AC) Types VI and VIII and Consolidate the Apposition of Capacitative Cation Entry Channels and Ca2+-sensitive ACs

Ca(2+)-sensitive adenylyl cyclases may act as early integrators of the two major second messenger-signaling pathways mediated by Ca(2+) and cAMP. Ca(2+) stimulation of adenylyl cyclase type I (ACI) and adenylyl cyclase type VIII (ACVIII) is mediated by calmodulin and the site on these adenylyl cyclases that interacts with calmodulin has been defined. By contrast, the mechanism whereby Ca(2+) inhibits adenylyl cyclase type V (ACV) and adenylyl cyclase type VI (ACVI) is unknown. In this study, Ca(2+), Sr(2+), and Ba(2+) were compared to probe the involvement of E-F hand-like domains in both Ca(2+) stimulation and inhibition of ACVIII and ACVI, respectively. HEK 293 cells transfected with ACVIII cDNA and C6-2B glioma cells (where the endogenous adenylyl cyclases is predominantly ACVI) were used to compare the effects of these three cations in in vitro and in vivo measurements. The in vitro data identified two Ca(2+) regulatory sites for both ACVIII and ACVI. Strikingly different potency series for these cations at mediating high affinity stimulation and inhibition of ACVIII and ACVI, respectively, effectively rule out the possibility that calmodulin or proteins utilizing similar Ca(2+)-binding motifs mediate inhibition of ACVI. On the other hand, the low affinity inhibition that is common to both ACVIII and ACVI showed virtually identical potency profiles for the IIa cation series, indicating a common site of action. Remarkably, whereas Sr(2+) was rather ineffective at regulating these cyclases (particularly ACVI) in vitro, adequate concentrations accumulated in the vicinity of these enzymes as a consequence of capacitative cation entry to partially regulate both of these activities in vivo. This latter finding consolidates earlier observations that Ca(2+)-sensitive adenylyl cyclases detect and respond to capacitative cation entry rather than global cytosolic cation concentrations.

Increased adenylyl cyclase type 1 mRNA, but not adenylyl cyclase type 2 in the rat hippocampus following antidepressant treatment

The adenylyl cyclase (AC) system is affected by several types of antidepressant treatments, and increased activity in this system is linked to the therapeutic action of antidepressants. The present study was undertaken to compare the effects of single-dose and long-term treatment with the selective serotonin reuptake inhibitor, citalopram (10 mg/kg, i.p.), on the AC system in the male rat brain of Wistar rats. Furthermore, we compared the effects of long-term citalopram and lithium treatments on the AC system. Long-term citalopram, but not single-dose administration, increased the AC type 1 mRNA in the hippocampus, whereas type 2 mRNA was unaffected. Long-term lithium treatment also increased AC1 in the hippocampus. However, long-term citalopram treatment did not increase AC type 1 protein, basal or forskolin-stimulated AC activity, but GTP increased AC activity in the hippocampus. This may indicate enhanced AC/G protein coupling. Thus, citalopram may increase cAMP signalling by acting on components of the AC system without affecting the protein level of the AC type 1.

New dimensions in G protein signalling: Gβ5 and the RGS proteins

The βγ complex of G-proteins regulates effectors independently of the Gα subunits, such that upon activation G proteins give may signal downstream along one or both pathways. The Gβ5 isoform exhibits much less homology with other Gβ isoforms (∼50%) and is preferentially expressed in brain. The Gβ5 isoform exhibits novel properties in its activation of effector pathways such as MAPK, phospholipase C-β, and adenylyl cyclase type II when compared to Gβ1. Recently specific native complexes between Gβ5 and the regulator of G protein signaling (RGS) protein-7 (RGS7) and between Gβ5L (a splice variant with a 42 amino acid N-terminal extension) and RGS9 have been isolated from different retinal fractions. Such findings are not accounted for by current models as only the Gα subunits and not Gβ had been previously implicated in RGS protein function. These recent novel observations further reinforce the view of Gβ5 as a unique and highly specialized G protein subunit.

Immunohistochemical localization of adenylyl cyclase isoforms in the lateral wall of the rat cochlea

The enzymatic activity of adenylyl cyclase (AC) is attributable to nine isoforms with individual pharmacology and tissue distribution. Polyclonal antibodies for AC isoforms I–IV, VII and VIII were applied to sections of cochlear lateral wall, a tissue involved in ion transport contributing to the unique ion content of endolymph and electrical potential of scala media. Within the stria vascularis, immunoreactivity primarily to Ca 2+/calmodulin-independent isoforms II, IV and VII was localized to sites consistent in position to the basolateral extensions of marginal cells. Little immunoreactivity was observed in the stria vascularis for Ca 2+/calmodulin-dependent isoforms I, III and VIII. Within the spiral ligament, type II and type IV fibrocytes exhibited moderate staining for ACII, IV and VII, less staining for VIII and little for I and III. Immunoreactivity to ACII, IV, VII and VIII was observed in type I fibrocytes. The outer sulcus cells and root processes were highly immunoreactive for isoforms I and VIII, but not for III or the Ca 2+/calmodulin-independent isoforms. The differential pattern of immunoreactivity in the lateral wall overall appears to reflect subfamily-specific expression with Ca 2+/calmodulin-independent isoforms expressed in the stria vascularis and Ca 2+/calmodulin-dependent isoforms expressed in the outer sulcus cells and root processes. cAMP-mediated modulation of ion transport by marginal cells is predicted to exhibit, in the microenvironment of basolateral membrane infoldings, pharmacological characteristics of the AC type II subfamily (II, IV and VII), including activation by protein kinase C (II and VII).

ETn insertion in the mouse Adcy1 gene: transcriptional and phylogenetic analyses.

Early retrotransposons (ETn) are murine transposable elements, bearing some structural similarity to integrated proviruses, and can be insertional mutagens. We have recently identified the causative mutation of the barrelless (Adcy1brl) phenotype as an integration of a 5.7-kb ETn in an intron of the adenylyl cyclase type I (Adcy1) gene. In the present study, Northern blot analysis shows that the ETn insertion results in loss of the normal Adcy1 transcript, a finding consistent with the loss-of-function Adcy1brl mutation, and generation of shorter transcripts. These aberrant transcripts are the products of abnormal RNA splicing and termination owing to the inserted sequence, and transcription initiation within the 3' long terminal repeat (LTR) of the ETn. The DNA sequences of the LTRs were compared in phylogenetic analyses with LTRs from 22 other ETn-related sequences. Three distinct families of ETn sequences can be identified on the basis of their LTRs. The ETn found in Adcy1brl is a member of a family that includes all classified ETn elements known to have recently transposed. Further, of the four known solitary (solo) LTRs, we have identified two that show evidence of recombination between LTRs from different ETn families.

Acute and chronic activation of the μ-opioid receptor with the endogenous ligand endomorphin differentially regulates adenylyl cyclase isozymes

While acute activation of G i/o-coupled receptors leads to inhibition of adenylyl cyclase, chronic activation of such receptors produces an increase in cyclic AMP accumulation, particularly evident upon withdrawal of the inhibitory agonist. This phenomenon has been referred to as adenylyl cyclase superactivation and is believed to play an important role in opiate addiction. Nine adenylyl cyclase isozymes have been recently identified and shown by us to be differentially regulated by acute and chronic inhibitory receptor activation. Using COS-7 cells cotransfected with various adenylyl cyclase isozymes, we examined here whether the endomorphins (the most recently discovered of the four classes of endogenous opioid peptides, and which interact selectively with the μ receptor) are able to induce inhibition/superactivation of representatives from the various adenylyl cyclase isozyme classes. Here, we show that adenylyl cyclase types I and V were inhibited by acute endomorphin application and superactivated upon chronic exposure, while adenylyl cyclase type II was stimulated by acute and “superinhibited” by chronic endomorphin exposure. These results show that the endomorphins are capable of regulating adenylyl cyclase activity and that different adenylyl cyclase isozymes respond differently to these endogenous ligands.

Reduced adenylyl cyclase immunolabeling and activity in postmortem temporal cortex of depressed suicide victims

Background: Previous studies have found altered receptor/G protein-modulated adenylyl cyclase (AC) activity in subjects with mood disorders. Methods: To investigate whether these effects are associated with altered levels of specific isoforms of AC, we measured AC isoform I, IV and V/VI immunoreactivities in postmortem temporal cortex from nine depressed suicide victims, nine subjects with bipolar disorder (BD) and 18 age-matched non-psychiatric controls. Basal, GTPγS- and forskolin-stimulated AC activities were measured in the temporal cortex from the nine depressed suicide victims and their controls. Results: Western blotting revealed significant reductions in immunolabeling in AC type IV (−49%; p<0.05) in depressed suicide subjects compared to age-matched controls, but no differences were found in AC type I or type V/VI. There were no statistically significant differences in AC type I, IV or V/VI immunoreactivities between BD and matched control subjects. Functionally, there was a significant reduction in forskolin-stimulated AC activity in depressed suicide subjects compared to controls, which may be, in part, related to higher basal AC activity in the former group. Limitations: Our sample size was small with diverse subject characteristics. Conclusions: These preliminary findings suggest altered levels and/or function in AC type IV may contribute to disturbances in the postreceptor cAMP signaling cascade in depression.

Constitutive Gi2-dependent Activation of Adenylyl Cyclase Type II by the 5-HT1A Receptor: INHIBITION BY ANXIOLYTIC PARTIAL AGONISTS

The 5-HT1A receptor is implicated in depression and anxiety. This receptor couples to G(i) proteins to inhibit adenylyl cyclase (AC) activity but can stimulate AC in tissues (e.g. hippocampus) that express ACII. The role of ACII in receptor-mediated stimulation of cAMP formation was examined in HEK-293 cells transfected with the 5-HT1A receptor, which mediated inhibition of basal and G(s)-induced cAMP formation in the absence of ACII. In cells cotransfected with 5-HT1A receptor and ACII plasmids, 5-HT1A agonists induced a 1. 5-fold increase in cAMP level. Cotransfection of 5-HT1A receptor, ACII, and Galpha(i2), but not Galpha(i1), Galpha(i3), or Galpha(o), resulted in an agonist-independent 6-fold increase in the basal cAMP level, suggesting that G(i2) preferentially coupled the receptor to ACII. The 5-HT1B receptor also constitutively activated ACII. Constitutive activity of the 5-HT1A receptor was blocked by pertussis toxin and the Gbetagamma antagonist, betaCT, suggesting an important role for Gbetagamma-mediated activation of ACII. The Thr-149 --> Ala mutation in the second intracellular domain of the 5-HT1A receptor disrupted Gbetagamma-selective activation of ACII. Spontaneous 5-HT1A receptor activity was partially attenuated by 5-HT1A receptor partial agonists with anxiolytic activity (e.g. buspirone and flesinoxan) but was not altered by full agonists or antagonists. Thus, anxiolytic activity may involve inhibition of spontaneous 5-HT1A receptor activity.

Molecular identification of adenylyl cyclase 3 in bovine corpus luteum and its regulation by prostaglandin F2alpha-induced signaling pathways.

The involvement of cAMP in various aspects of ovarian steroidogenic cells functions has been extensively studied. However, the adenylyl cyclase (AC) types expressed in ovarian cells, of any species, are not yet determined. The present study was undertaken to identify AC types present in bovine luteal cells and their regulation by various stimuli. AC isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain by Northern blotting analysis, whereas the bovine corpus luteum (CL) only expressed AC3 and 6 mRNAs, with AC3 being more abundant than AC6. The use of AC3-specific primers in RT-PCR reaction verified the presence of AC3 mRNA in both bovine and rat CL tissue as well as in bovine steroidogenic luteal cells. Because these two AC isoforms, AC3 and 6, exhibit distinct regulatory patterns we have next examined the effects of various signaling pathways on AC activity in luteal cells. These studies have shown that: 1) prostaglandin (PG) F2alpha and phorbol 12-myristate 13-acetate markedly elevated agonist-stimulated cAMP synthesis (these effects were inhibited by addition of highly specific PKC inhibitor, bisindolylmaleimide); 2) depletion of Ca2+ from the incubation medium inhibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 mM) significantly stimulated cAMP production in luteal cells; and 4) the effects of Ca2+ on cAMP synthesis were evident only in the presence of forskolin. These regulatory characteristics of AC activity are consistent with the molecular identification of ACs indicating the presence of AC3 in luteal cells. The reported data may delineate the cross-talk between physiological activators of AC in the CL (such as LH, PGE2, and PGI2) and other ligands (such as PGF2alpha and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward understanding the mode of action of a wide array of hormones regulating ovarian cells.

Regulators of G Protein Signaling 6 and 7: PURIFICATION OF COMPLEXES WITH Gβ5 AND ASSESSMENT OF THEIR EFFECTS ON G PROTEIN-MEDIATED SIGNALING PATHWAYS

Regulators of G protein signaling (RGS) proteins that contain DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains form a subfamily that includes the mammalian RGS proteins RGS6, RGS7, RGS9, and RGS11. We describe the cloning of RGS6 cDNA, the specificity of interaction of RGS6 and RGS7 with G protein beta subunits, and certain biochemical properties of RGS6/beta5 and RGS7/beta5 complexes. After expression in Sf9 cells, complexes of both RGS6 and RGS7 with the Gbeta5 subunit (but not Gbetas 1-4) are found in the cytosol. When purified, these complexes are similar to RGS11/beta5 in that they act as GTPase-activating proteins specifically toward Galpha(o). Unlike conventional G(betagamma) complexes, RGS6/beta5 and RGS7/beta5 do not form heterotrimeric complexes with either Galpha(o)-GDP or Galpha(q)-GDP. Neither RGS6/beta5 nor RGS7/beta5 altered the activity of adenylyl cyclases types I, II, or V, nor were they able to activate either phospholipase C-beta1 or -beta2. However, the RGS/beta5 complexes inhibited beta(1)gamma(2)-mediated activation of phospholipase C-beta2. RGS/beta5 complexes may contribute to the selectivity of signal transduction initiated by receptors coupled to G(i) and G(o) by binding to phospholipase C and stimulating the GTPase activity of Galpha(o).

Localization of the olfactory cyclic nucleotide-gated channel subunit 1 in normal, embryonic and regenerating olfactory epithelium

The spatial and temporal expression of subunit 1 of the olfactory cyclic nucleotide-gated channel was investigated using affinity-purified anti-fusion protein antibodies. Immunoreactivity was most prominent in the ciliary layer of the olfactory epithelium, but high protein expression was also seen along the entire length of olfactory receptor neuronal axons to the level of the glomeruli. Electron microscopy showed that the long, thin distal compartments of olfactory cilia labeled more prominently than their thicker proximal segments. This was true as soon as these distal parts began to develop. Using light microscopy, developmental expression of olfactory cyclic nucleotide-gated channel subunit 1 could be detected in discrete populations of olfactory receptor neurons by embryonic day 14. Other signaling molecules are expressed either later (G olf) or only at the level of the epithelial surface and not in axons (adenylyl cyclase type III). Following unilateral lesions of the olfactory bulb, olfactory cyclic nucleotide-gated channel subunit 1 immunoreactivity was present early and throughout developing olfactory receptor neurons; adenylyl cyclase type III immunoreactivity, in contrast, was detectable only later, and again present only in the cilial layer. These results support the hypothesis that this subunit of the olfactory cyclic nucleotide-gated channel may be involved in olfactory axon guidance, in addition to its well-described role in olfactory signal transduction.

Identification of NFI-binding sites and cloning of NFI-cDNAs suggest a regulatory role for NFI transcription factors in olfactory neuron gene expression

Olfactory receptor neurons are responsible for the detection and signal transduction of odor ligands. Several genes associated with this activity are preferentially or exclusively expressed in these neurons. Among these genes are those coding for olfactory receptors, adenylyl cyclase type III, the cyclic nucleotide gated olfactory channel 1 (OcNC-1), Gα olf and the olfactory marker protein (OMP). Promoter analyses of these genes identified a binding site for the new transcription factor family O/E whose initial member, Olf-1, is abundantly expressed in olfactory neurons. We report here that the proximal promoters of three of these genes, that are selectively expressed in olfactory neurons, each contains a functional NFI binding site and that the sites have different affinities for NFI proteins indicating a regulatory role for NFI proteins in olfactory gene expression. We further demonstrate, by cloning, that all four NFI genes are expressed in the olfactory nasal mucosa. Analysis by in situ hybridization illustrates that at least three of these gene products are expressed in the neuroepithelium in which the olfactory neurons reside. NFI proteins are capable of functioning as positive or negative regulators of transcription depending on the tissue, cell-type, age, and gene in question. These multivalent functions of NFI could be achieved by temporally and spatially regulated expression of distinct subsets of NFI isoforms. It now remains to characterize the tissue and cell specific patterns of expression of distinct NFI transcription factors during ontogeny and their roles in regulating gene expression.

Cardiac-directed adenylyl cyclase expression improves heart function in murine cardiomyopathy.

We tested the hypothesis that increased cardiac myocyte adenylyl cyclase (AC) content increases cardiac function and response to catecholamines in cardiomyopathy. Transgenic mice with cardiac-directed expression of AC type VI (ACVI) were crossbred with mice with cardiomyopathy induced by cardiac-directed Gq expression. Gq mice had dilated left ventricles, reduced heart function, decreased cardiac responsiveness to catecholamine stimulation, and impaired beta-adrenergic receptor (betaAR)-dependent and AC-dependent cAMP production. Gq/AC mice showed improved basal cardiac function in vivo (P=0.01) and ex vivo (P<0.0005). When stimulated through the betaAR, cardiac responsiveness was increased (P=0.02), and cardiac myocytes showed increased cAMP production in response to isoproterenol (P=0.03) and forskolin (P<0.0001). Increasing myocardial ACVI content in cardiomyopathy restores cAMP-generating capacity and improves cardiac function and responsiveness to betaAR stimulation.

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca 2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca 2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.

Quantitative reduction of type I adenylyl cyclase in human alcoholics

The amounts of adenylyl cyclase type I (AC I) were examined in various parts of the postmortem brains from alcoholics who prior to death had been abstinent from alcohol for at least 6 months and compared with controls using immunoblot analysis with anti-AC I specific antibody. It was revealed that a significant reduction of AC I was observed in both frontal and temporal cortices. On the other hand, in other areas (occipital cortex, caudate nucleus, putamen, and hippocampus) the amounts were comparable between alcoholics and controls. In the next step, we examined two subtypes of human AC mRNA levels (AC I and AC VIII) in blood cells by quantitative RT-PCR using [α- 32P]dCTP with two sets of the synthetic oligonucleotide primers based on the DNA sequences reported elsewhere (Villacres, E.C. et al., Genomics 16 (1993) 473–478; J. Parma et al., Biochem. Biophys. Res. Commun. 179 (1991) 455–462). The amounts of amplified DNAs of both AC I and AC VIII were significantly smaller in alcoholics than in controls. On the other hand, the amounts of amplified DNA of β-actin DNA were almost equal between alcoholics and controls. It appears from these results that a reduction in the amount of AC subtypes may be a biological marker for alcoholics.

Calmodulin-binding Sites on Adenylyl Cyclase Type VIII

Ca2+ stimulation of adenylyl cyclase type VIII (ACVIII) occurs through loosely bound calmodulin. However, where calmodulin binds in ACVIII and how the binding activates this cyclase have not yet been investigated. We have located two putative calmodulin-binding sites in ACVIII. One site is located at the N terminus as revealed by overlay assays; the other is located at the C terminus, as indicated by mutagenesis studies. Both of these calmodulin-binding sites were confirmed by synthetic peptide studies. The N-terminal site has the typical motif of a Ca2+-dependent calmodulin-binding domain, which is defined by a characteristic pattern of hydrophobic amino acids, basic and aromatic amino acids, and a tendency to form amphipathic alpha-helix structures. Functional, mutagenesis studies suggest that this binding makes a minor contribution to the Ca2+ stimulation of ACVIII activity, although it might be involved in calmodulin trapping by ACVIII. The primary structure of the C-terminal site resembles another calmodulin-binding motif, the so-called IQ motif, which is commonly Ca2+-independent. Mutagenesis and functional assays indicate that this latter site is a calcium-dependent calmodulin-binding site, which is largely responsible for the Ca2+ stimulation of ACVIII. Removal of this latter calmodulin-binding region from ACVIII results in a hyperactivated enzyme state and a loss of Ca2+ sensitivity. Thus, Ca2+/calmodulin regulation of ACVIII may be through a disinhibitory mechanism, as is the case for a number of other targets of Ca2+/calmodulin.