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pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HC[O.sub.3.sup.-]). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons ([H.sup.+]) and HC[O.sub.3.sup.-] at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HC[O.sub.3.sup.-] and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors, dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HC[O.sub.3.sup.-] levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb [H.sup.+] into the blood, sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom. cAMP | pH | proton pump | dogfish | gill www.pnas.org/cgi/doi/10.1073/pnas.0911790107

BACKGROUND: Organophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation. OBJECTIVES: We evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons. RESULTS: In undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication. CONCLUSIONS: Our results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects. KEY WORDS: adenylyl cyclase, brain development, chlorpyrifos, cholinergic antagonists, neurotoxicity, nicotine, organophosphate insecticides, oxidative stress, theophylline, vitamin E. Environ Health Perspect 115:1306-1313 (2007). doi:10.1289/ehp.10194 available via http://dx.doi.org/ [Online 14 June 2007], The nearly ubiquitous exposure of the human population to organophosphate pesticides has raised increasing concern about their propensity to elicit developmental neurotoxicity at exposures that go undetected because of the [...]

We previously identified a sperm-specific [Na.sup.+]/[H.sup.+] exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation. cAMP | sperm motility

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-[R.sub.s]) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-[R.sub.s] with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed [beta]-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-[R.sub.s]. These results present a structural basis for hPAC1-[R.sub.s] selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-[R.sub.s] receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation. NMR | vasoactive intestinal peptide | G protein-coupled receptor

Human sperm development and the role of adenylyl cyclase in this process is examined.

Heterogeneity of heart failure (HF) phenotypes indicates contributions from underlying common polymorphisms. We considered polymorphisms in the [[beta].sub.1]-adrenergic receptor ([[beta].sub.1]AR), a [beta]-blocker target, as candidate pharmacogenomic loci. Transfected cells, genotyped human nonfailing and failing ventricles, and a clinical trial were used to ascertain phenotype and mechanism. In nonfailing and failing isolated ventricles, [[beta].sub.1]-Arg-389 had respective 2.8 [+ or -] 0.3- and 4.3 [+ or -] 2.1-fold greater agonist-promoted contractility vs. [[beta].sub.1]-Gly-389, defining enhanced physiologic coupling under relevant conditions of endogenous expression and HF. The [beta]-blocker bucindolol was an inverse agonist in failing Arg, but not Gly, ventricles, without partial agonist activity at either receptor; carvedilol was a genotype-independent neutral antagonist. In transfected cells, bucindolol antagonized agonist-stimulated cAMP, with a greater absolute decrease observed for Arg-389 (435 [+ or -] 80 vs. 115 [+ or -] 23 fmol per well). Potential pathophysiologic correlates were assessed in a placebo-controlled trial of bucindolol in 1,040 HF patients. No outcome was associated with genotype in the placebo group, indicating little impact on the natural course of HF. However, the Arg-389 homozygotes treated with bucindolol had an age-, sex-, and race-adjusted 38% reduction in mortality (P = 0.03) and 34% reduction in mortality or hospitalization (P = 0.004) vs. placebo. In contrast, Gly-389 carriers had no clinical response to bucindolol compared with placebo. Those with Arg-389 and high baseline norepinephrine levels trended toward improved survival, but no advantage with this allele and exaggerated sympatholysis was identified. We conclude that [[beta].sub.1]AR-389 variation alters signaling in multiple models and affects the [beta]-blocker therapeutic response in HF and, thus, might be used to individualize treatment of the syndrome. adenylyl cyclase | G protein-coupled receptor | genetics | myocardium | signaling

Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven 'upstream' proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git[1.sup.+] gene. Girl is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git[1.sup.+] demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G[alpha]-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Girl and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.

Opioid drugs produce their pharmacological effects by activating inhibitory guanine nucleotide-binding regulatory protein-linked [mu], [delta], and [kappa] opioid receptors. One major effector for these receptors is adenylyl cyclase, which is inhibited upon receptor activation. However, little is known about which of the ten known forms of adenylyl cyclase are involved in mediating opioid actions. Here we show that all of the major behavioral effects of morphine, including locomotor activation, analgesia, tolerance, reward, and physical dependence and withdrawal symptoms, are attenuated in mice lacking adenylyl cyclase type 5 (AC5), a form of adenylyl cyclase that is highly enriched in striatum. Furthermore, the behavioral effects of selective [mu] or [delta] opioid receptor agonists are lost in AC[5.sup.-/-] mice, whereas the behavioral effects of selective [kappa] opioid receptor agonists are unaffected. These behavioral data are consistent with the observation that the ability of a [mu] or [delta] opioid receptor agonist to suppress adenylyl cyclase activity was absent in striatum of AC[5.sup.-/-] mice. Together, these results establish AC5 as an important component of [mu] and [delta] opioid receptor signal transduction mechanisms in vivo and provide further support for the importance of the cAMP pathway as a critical mediator of opioid action. striatum | opioid receptors | analgesia | addiction | cAMP

Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs[2.sup.+] were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git[2.sup.+]/cyr[1.sup.+]), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git[2.sup.-] mutant alleles used in the suppressor selection and mutations in git[1.sup.+], git[3.sup.+], git[5.sup.+], git[7.sup.+], git[10.sup.+], and git[11.sup.+], which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa[2.sup.+], which encodes the G[alpha] subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2 allele does suppress loss of gpa[2.sup.+]. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa[2.sup.R176H]-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection.

Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperature-sensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaA[DELTA]11). The mechanism in which cyaA[DELTA]11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaA[DELTA]11 mutant strain that contains a Cpx- and sigma E-inducible degP-lacZ reporter fusion decreased [beta]-galactosidase expression to levels observed in a cya[A.sup.+] strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaA[DELTA]11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.

Bicarbonate stimulates the activities of several class III adenylyl cyclases studied to date. However, we show here that bicarbonate decreased [V.sub.max] and substrate affinity in Cya1, a major adenylyl cyclase in the cyanobacterium Synechocystis sp. strain PCC 6803. This indicates that manifestation of the bicarbonate responsiveness is specifically modulated in Cya1.

Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55° to form two catalytic sites at their interface. Two α helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH., Adenylyl cyclases (ACs) synthesize the universal second messenger 3',5'-cyclic adenosine monophosphate (cAMP) (1). Most ACs belong to class III, such as all mammalian and many bacterial enzymes (2), and are [...]

We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCI) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant.

I. Introduction II. Native GAB[A.sub.B] Receptors A. Coupling to G proteins B. Coupling to [Ca.sup.2+] channels C. Coupling to [K.sup.+] channels D. Coupling to adenylyl cyclase E. Pharmacology of native [...]

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility, sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive.

Bicarbonate-responsive 'soluble' adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

In mammals, [Ca.sup.2+] and HC[O.sup.-.sub.3] ions play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. Mammalian germ cells contain a soluble adenylyl cyclase (sAC) with properties distinct from the well characterized membrane-bound enzymes Here we investigated whether the cyclase expressed in mature spermatozoa has the properties of sAC and whether it is regulated by [Ca.sup.2+]. In addition to an HC[O.sup.-.sub.3]-dependent activation, the cyclase endogenous to human spermatozoa is stimulated 2- to 3-fold by [Ca.sup.2+]. In a concentration-dependent manner (E[C.sub.50] [approximately equal to] 400 nM). In a similar fashion, [Ca.sup.2+] activates the recombinant rat and human full-length sAC with similar E[C.sub.50] values. The [Ca.sup.2+] stimulation was also observed when sAC was activated with HC[O.sup.-.sub.3], was independent of calmodulin, and was associated with an increase in [V.sub.max] without changes in [K.sub.m] for ATP-[Mg.sup.2+]. An increase in intracellular [Ca.sup.2+] by ionophore or by a muscarinic cholinergic receptor agonist increases cAMP in cells transfected with FL-hsAC, but not in mock-transfected cells. Similarly, both [Ca.sup.2+] and HC[O.sup.-.sub.3] stimulate cAMP accumulation in human spermatozoa. These findings provide evidence that human spermatozoa express a cyclase with the properties of sAC and that [Ca.sup.2+] can substitute for HC[O.sup.-.sub.3] in the stimulation of this enzyme, underscoring an important role for sAC in the control of sperm functions.

The sympathetic nervous system is designed to respond to stress. Adenylyl cyclase (AC) is the keystone of sympathetic transmission, yet its role in response to acute overload in the heart or in the pathogenesis of heart failure is controversial. We examined the effects of pressure overload, induced by thoracic aortic banding, in mice in which type 5 AC, a major cardiac AC isoform, was disrupted (AC[5.sup.-/-]). Left ventricular weight/tibial length ratio (LVW/TL) was not different between the WT and AC[5.sup.-/-] at baseline and increased progressively and similarly in both groups at 1 and 3 wk after aortic banding. However, LV ejection fraction (LVEF) fell in WT at 3 wk after banding (from 70 [+ or -] 2.8 to 57 [+ or -] 3.9%, P < 0.05), and this decrease was associated with LV dilatation, indicating incipient cardiac failure. In contrast, AC[5.sup.-/-] mice did not exhibit a fall in LVEF from 74 [+ or -] 2.2%. The number of apoptotic myocytes was similar at baseline, but it increased roughly 4-fold in WT at both 1 and 3 wk after banding, and significantly less, P < 0.05, in AC[5.sup.-/-]. Importantly, the increase in apoptosis occurred before the decline in LVEF in WT. The protective mechanism seems to involve Bcl-2, which was up-regulated significantly more in AC[5.sup.-/-] mice with pressure overload. Our findings suggest that limiting type 5 AC plays a protective role in response to pressure overload and the development of heart failure, potentially through limiting the incidence of myocardial apoptosis.

The intracellular levels of cAMP play a critical role in the meiotic arrest of mammalian oocytes. However, it is debated whether this second messenger is produced endogenously by the oocytes or is maintained at levels inhibitory to meiotic resumption via diffusion from somatic cells. Here, we demonstrate that adenylyl cyclase genes and corresponding proteins are expressed in rodent oocytes. The mRNA coding for the AC3 isoform of adenylyl cyclase was detected in rat and mouse oocytes by RT-PCR and by in situ hybridization. The expression of AC3 protein was confirmed by immunocytochemistry and immunofluorescence analysis in oocytes in situ. Cyclic AMP accumulation in denuded oocytes was increased by incubation with forskolin, and this stimulation was abolished by increasing intraoocyte [Ca.sub.2+] with the ionophore A23187. The [Ca.sub.2+] effects were reversed by an inhibitor of [Ca.sub.2+], calmodulin-dependent kinase II. These regulations of cAMP levels indicate that the major cyclase that produces cAMP in the rat oocyte has properties identical to those of recombinant or endogenous AC3 expressed in somatic cells. Furthermore, mouse oocytes deficient in AC3 show signs of a defect in meiotic arrest in vivo and accelerated spontaneous maturation in vitro. Collectively, these data provide evidence that an adenylyl cyclase is functional in rodent oocytes and that its activity is involved in the control of oocyte meiotic arrest. Keywords: cAMP; Adenylyl cyclase; Oocyte; Meiosis; Cell cycle

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with [Beta][Gamma] subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe [Beta][Gamma]-mediated inhibition of enzyme activity, which is stimulated by either constitutively active [G.sub.s[Alpha]] ([[G.sub.s[Alpha].sup.*]]) or [Ca.sup.2+]/calmodulin (CAM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by [Beta][Gamma]subunits, the presence of both of these regions formed another [Beta][Gamma] interaction site that was sufficient to observe [[G.sub.s[Alpha].sup.*]]- or [Ca.sup.2+]/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of [Beta][Gamma] subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of [Beta][Gamma] on various effectors. Moreover, the C1 region of ACI was sufficient to observe [G.sub.i[Alpha]1]-elicited inhibition of [Ca.sup.2+]/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by [G.sub.i[Alpha]1], the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of [G.sub.i[Alpha]1] are mediated on the C1 regions of both ACI and ACV. The effects of [Beta][Gamma] on ACI can be mediated by interactions with the C1a region and a [Beta][Gamma] interacting site formed by the C1b and C2 domains of this enzyme.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation. Key Words: adenylyl cyclase B; protein kinase A; rapidly developing mutants; terminal differentiation; Dictyostelium discoideum.

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein [Alpha] subunit (G[Alpha]), thereby generating two potential signaling molecules, G[Alpha]-GTP and free G[Beta][Gamma]. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative G[Alpha] mutation. Here we describe a mutant [Alpha] subunit designed to inhibit receptor-mediated hormonal activation of [G.sub.s], the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the [Alpha] subunit ([[Alpha].sub.s]) of [G.sub.s] three separate mutations chosen because they impair [[Alpha].sub.s] function in complementary ways: the [A.sup.366]S mutant reduces affinity of [[Alpha].sub.s] for binding GDP, whereas the [G.sup.226]A and [E.sup.268]A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) [G.sub.s]-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active [[Alpha].sub.s] mutant ([[Alpha].sub.s]-[R.sup.201]C) or to agonists for receptors that activate [G.sub.q] or [G.sub.i]. This [[Alpha].sub.s] triple mutant and cognate [G.sub.[Alpha]] mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.

Mammals have nine differentially regulated isoforms of G protein-responsive transmembrane-spanning adenylyl cyclases. We now describe the existence of a distinct class of mammalian adenylyl cyclase that is soluble and insensitive to G protein or Forskolin regulation. Northern analysis indicates the gene encoding soluble adenylyl cyclase (sAC) is preferentially expressed in testis. As purified from rat testis cytosol, the active form of sAC appears to be a fragment derived from the full-length protein, suggesting a proteolytic mechanism for sAC activation. The two presumptive catalytic domains of sAC are closely related to cyanobacterial adenylyl cyclases, providing an evolutionary link between bacterial and mammalian signaling molecules.

Cell cycle progression is regulated by cAMP in several cell types. Cellular cAMP levels depend on the activity of different adenylyl cyclases (ACs), which have varied signal-receiving capabilities. The role of individual ACs in regulating proliferative responses was investigated. Native NIH 3T3 cells contain AC6, an isoform that is inhibited by a variety of signals. Proliferation of exogenous AC6-expressing cells was the same as in control cells. In contrast, expression of AC2, an isoform stimulated by protein kinase C (PKC), resulted in inhibition of cell cycle progression and increased doubling time. In AC2-expressing cells, platelet-derived growth factor (PDGF) elevated cAMP levels in a PKC-dependent manner. PDGF stimulation of mitogen-activated protein kinases 1 and 2 (MAPK 1,2), DNA synthesis, and cyclin D1 expression was reduced in AC2-expressing cells as compared with control cells. Dominant negative protein kinase A relieved the AC2 inhibition of PDGF-induced DNA synthesis. Expression of AC2 also blocked H-ras-induced transformation of NIH 3T3 cells. These observations indicate that, because AC2 is stimulated by PKC, it can be activated by PDGF concurrently with the stimulation of MAPK 1,2. The elevation in cAMP results in inhibition of signal flow from the PDGF receptor to MAPK 1,2 and a significant reduction in the proliferative response to PDGF. Thus, the molecular identity and signal receiving capability of the AC isoforms in a cell could be important for proliferative homeostasis.

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

The [Alpha] subunit of the stimulatory heterotrimeric G protein ([G.sub.s[Alpha]]) is critical for the [Beta]-adrenergic receptor activation of the cAMP messenger system. The role of [G.sub.s[Alpha]] in regulating cardiac [Ca.sup.2+] channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac [G.sub.s[Alpha]] were used to assess the role of [G.sub.s[Alpha]] on the whole-cell [Ca.sup.2+] currents ([I.sub.Ca]). Cardiac myocytes from transgenic mice had a 490% higher peak [I.sub.Ca] compared with those of either wild-type controls or [G.sub.s[Alpha]]-nonexpressing littermates. The effect of [G.sub.s[Alpha]] overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTP[Gamma]S-activated [G.sub.s[Alpha]]. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated [G.sub.s[Alpha]]. The data indicate that [G.sub.s[Alpha]] overexpression is associated with a constitutive enhancement of [I.sub.Ca] which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.

The process of development in Dictyostelium is prompted by starvation-induced DdMyb2 activation. This leads to adenylyl activity and produces cAMP, which triggers differentiation-inducing signals. The DdMyb2 gene is a DNA-binding motif of Myb-related transcription factors. Mutants without DdMyb2 can be rescued with ectopic expression of ACA.

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) have fundamental roles in a wide range of cellular processes. Whereas GCs use GTP as a substrate to form cGMP, ACs catalyze the analogous conversion of ATP to cAMP. Previously, a model based on the structure of adenylate cyclase was used to predict the structure of the nucleotide-binding pocket of a membrane guanylyl cyclase, RetGC-1. based on this model, we replaced specific amino acids in the guanine-binding pocket of GC with their counterparts from AC. A change of two amino acids, E925K together with C995D, is sufficient to completely alter the nucleotide specificity from GTP to ATP. These experiments strongly validate the AC-derived RetGC-1 structural model and functionally confirm the role of these residues in nucleotide discrimination.

Cellular content of cAMP generated by activation of adenylyicyclase (AC; EC 4.6.1.1) is a key determinant of functional responsiveness in the heart and other tissues. We have tested two hypotheses regarding the relationship between AC content and [Beta]-adrenergic receptor ([Beta]AR)-mediated signal transduction in cardiac myocytes. First, that AC content limits adrenergic signal transduction, and, second, that increased AC, independent of ([Beta]AR) number and G-protein content, yields a proportional increase in [Beta]AR-mediated transmembrane signaling. We used recombinant adenovirus to increase AC isoform VI ([AC.sub.VI]) expression in neonatal cardiac myocytes. Cells that overexpressed [AC.sub.VI] responded to agonist stimulation with marked increases in cAMP production in proportion to protein expressed. In parallel experiments performed on cells transfected with lacZ (control) or [AC.sub.VI], [3H]forskolin binding, used to assess AC protein expression, was amplified 6-fold, while [Beta]AR-stimulated cAMP production from these cells was increased 7-fold. No changes in [Beta]AR number, or in the heterotrimeric GTP-binding proteins, G[Alpha]s or G[Alpha][i.sub.2], were observed. Previous studies indicate that increased cardiac expression of [Beta]AR or G[Alpha]s does not yield proportional increases in transmembrane adrenergic signaling. In contrast, the current data demonstrate that increased [AC.sub.VI] expression provides a proportional increase in [Beta]-adrenergic signal transduction. Our results show that the amount of AC sets a limit on transmembrane [Beta]-adrenergic signaling. We speculate that similar functional responses are possible in other cell types in which AC plays an important physiological role.

Escherichia coli samples are studied in an experiment to determine if the protein IIA(super Glc) of the phosphoenolpyruvate:sugar phosphotransferase system can affect the basal activity of adenylyl cyclase. The samples were mutated via site-directed mutagenesis to glutamate and glutamine. Results show that the tested protein did not affect basal activity. However, partial and full activators of adenylyl cyclase were identified.

Cyclic AMP's significance in higher plants has been doubted as they have low adenylyl cyclase activity and hardly detectable amounts of cAMP. Activation T-DNA tagging was used to create tobacco cell lines than proliferate without the phytohormone auxin in the culture media. Axi 141 isolated a complementary NA encoding adenylyl cylase and sequence analysis showed that the tobacco adenylyl cyclase may be soluble and contains leucine-rich repeats, with similarities to adenylyl cyclase from the Schizosaccharomyces pombe yeast.

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein [Alpha] subunit ([G.sub.s Alpha]) and forskolin was determined to resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substance recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by [G.sub.s Alpha]., Adenylyl cyclase is the effector enzyme responsible for converting adenosine triphosphate (ATP) to adenosine 3,5-monophosphate (cAMP), a ubiquitous second messenger that mediates diverse cellular responses primarily by activating cAMP-dependent protein [...]

Receptors activate adenylyl cyclases through the G[[Alpha].sub.s] subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by G[[Alpha].sub.s]. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of G[[Alpha].sub.s]. Similar treatment of AC1 or AC2 did not affect Gas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of G[[Alpha].sub.s] stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of G[[Alpha].sub.s]. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from G[[Alpha].sub.s]. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.

The adenylyl and guanylyl cyclases catalyze the formation of 3[prime],5[prime]-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5[prime]-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase [C.sub.2] catalytic domain was used to model by homology a mammalian adenylyl cyclase [C.sub.1]-[C.sub.2] domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. [Mg.sup.2+]ATP or [Mg.sup.2+]GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a [Mg.sup.2+] ion. The D310S and D310A mutants have 10-fold reduced [V.sub.max] and altered [[Mg.sup.2+]] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.

Many G protein [Alpha] subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The [G.sub.[Alpha]] protein that activates adenylyl cyclase, [[Alpha].sub.s], is not myristoylated but can be reversibly palmitoylated. It appears that [[Alpha].sub.s] contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to

cAMP assay and measurement of expression of cAMP-regulated genes were conducted to determine the relationship between external glucose concentration and cAMP levels inside Escherichi coli. Results reveal that E. coli controls its cAMP levels through two or more transitions influenced by external glucose concentration and does not respond to decreasing saturation of glucose transport. Furthermore, results present implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The mammalian adenylyl cyclase has two conserved regions, C(sub 1) and C(sub 2), which are responsible for forskolin- and G-protein-stimulated catalysis. The C(sub 2) catalytic region of type II rat adenylyl cyclase contains an alpha/beta class fold that has a central cleft. At the end of the cleft are two forskolin molecules that bind in hydrophobic pockets while at the center are charged residues implicated in adenosine triphosphate binding. Forskolin seems to activate adenylyl cyclase by promoting the assembly of the active dimer and by direct interaction within the catalytic cleft. Other adenylyl cyclase regulators work at the dimer interface or on a flexible C-terminal region.

Insertion mutations in two Vibrio cholerae genes, cya and crp, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP), respectively, derepressed the expression of a chromosomal cholera toxin (CT) promoter-lacZ fusion at the nonpermissive temperature of 37 [degrees] C. In the classical biotype strain O395, the crp mutation increased the production of both CT and toxin-coregulated pilus (TCP) in vitro under a variety of growth conditions not normally permissive for their expression. The most dramatic increase in CT and TCP was observed with the crp mutant in Luria-Bertani (LB) medium pH 8.5, at 30 [degrees] C. El Tor biotype strains differ from classical strains in that they do not produce CT or TCP when grown in LB media. Incorporation of the crp mutation into El Tor strain C6706 permitted production of these proteins in LB medium pH 6.5, at 30 [degrees] C. In the infant mouse cholera model, the crp mutation decreased colonization in both biotypes at least 100-fold relative to the wild-type strains. The data presented here suggest a model whereby cAMP-CRP negatively regulates the expression of CT and TCP in both classical and El Tor biotypes under certain environmental conditions and also influences pathogenesis by regulating other processes necessary for optimal growth in vivo.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:003:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the [C.sub.1a] domain of type I adenylyl cyclase and the [C.sub.2] domain of the type II enzyme to assess their interactions with each other and with the activators [G.sub.s[Alpha]] and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated [G.sub.s[Alpha]]. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by [G.sub.s[Alpha]] and forskolin is synergistic. These data suggest a model wherein either [G.sub.s[Alpha]] or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the [C.sub.1] and [C.sub.2] domains of the enzyme.

Previous research has shown low cAMP levels in Escherichia coli grown on glucose 6-phosphate (Glc6P). To explain this phenomenon, the mechanism leading to the low cAMP level when Glc6P is the carbon source is investigated. Results indicate that Flc6P is not directly responsible for the low cAMP level in E. coli but rather that the level is determined by a flux in the Glc6P through the UhpT.

Wild-type Yersinia enterocolitica was engineered into murine PU-1.8 macrophages to derive mutant antihost proteins, designated as YopE and YopH. These proteins were fused to a calmodulin-dependent adenylate cyclase and were examined by enzyme immunoassay. Y. enterocolitica secreted YopE-cyclase and YopH-cyclase hybrids with an N-terminal domain of 15 residues and 17 residues, respectively. Internalization into the cytosol of macrophages via the cyoA gene fusion approach required an N-terminal domain of 50 residues for YopE and 71 residues for YopH. These results suggest that the hybrids consist of a secretion, a translocation and an effector domain.

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a [cdc25.sup.ts] ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras1[delta] proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2[delta], in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1[delta], in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1[delta], Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.

Study reports the expression of adenylyl cyclase protein at the location and abundance of adenylyl cyclase protein at the neuronal and synaptic sites. Microscopic analysis reveals distinctive labeling of axons and dendritic fields. However, analysis through electron microscope reveals a large but selective concentration of immunoreactivity in specialized sections, especially in postsynaptic densities of dendritic spines in areas such as the CA1 region of the hippocampus.

A soluble adenylyl cyclase was constructed by linkage of portions of the cytosolic domains of the mammalian type I and type II enzymes. The soluble enzyme was stimulated by both forskolin and the α subunit of the heterotrimeric guanine nucleotide-binding protein (G protein) [G.sub.s] ([G.sub.s]α). Expression of the construct complemented the catabolic defect in a strain of Escherichia coli that is deficient in adenylyl cyclase activity. The active, approximately 60-kilodalton enzyme accumulated in the cytoplasmic fraction of E. coli to yield activities in excess of 1 nanomole per minute per milligram of protein. The two sets of transmembrane helices of mammalian adenylyl cyclases are thus not necessary for the catalytic or the most characteristic regulatory activities of the enzyme. This system may be useful for both genetic and biochemical analysis of G protein-regulated adenylyl cyclases., The structures of G protein-regulated adenylyl cyclases are complex, consisting of two intensely hydrophobic domains ([M.sub.1] and [M.sub.2]), each hypothesized to contain six transmembrane helices, and two ~40-kD cytosolic domains [...]

Adenylyl cyclase, a second messenger generator in cellular signal transduction, is regulated by cytosolic calcium ions. An inhibition of adenylyl cyclase by Ca2+ results in an increase of cellular cyclic AMP levels. In contrast to the earlier view of a prominent role of G-protein alpha-subunits, findings suggest a predominant role of protein kinase C, Ca2+ and beta gamma subunits of G proteins in the regulation of adenylyl cyclases. Adenylyl cyclases are huge polypeptides similar to transporter molecules.

A study of the ability of sequences within the epidermal growth factor (EGF) receptor to activate the stimulatory GTP-binding protein Gs of adenylyl cyclase shows that the juxtamembrane region comprising a 13-aa peptide in the cytosolic domain of the EGF receptor promotes GTP binding and GTPase activity of Gs. GTP binding to Gi is not activated by this peptide, which increases the GTPase activity of the protein. The ability of the peptide to stimulate Gs is decreased and the GTPase activity of Gi is increased upon phosphorylation of the protein kinase C site with the 13-aa peptide.

Experimental studies on the influence of weak acids and bases on the activities of ACA and ACG, two adenylylcyclases and cAMP secretion reveal that the pH dependence of ACA and ACG regulate the weak acid influences on their activity. Weak bases such as ammonia do not regulate cAMP concentrations, while they inhibit ACG and ACA activities.

Targeted mutagenetic disruption of the murine Ca2+-stimulated type I adenylyl cyclase gene expressed primarily in brain causes behavioral changes and disturbances in potentiation. The cyclase activity is diminished markedly in the hippocampus, neocortex and cerebellum. Mutant mice also show disturbance in the long-term potentiation in the CA1 region of the hippocampus with decreases in both the initial slope and optimum changes in synaptic responses.