Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis.

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis(Iseki et al., Nature, 2002, 415, 1047--1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACalpha and PACbeta). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD"(BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACalpha by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28--0.32, and the half-life was 34--44 s at 25 degrees C for the recombinant PACalpha F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACalpha F2 and the F2 domain of the PACalpha homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.     

Photoactivated adenylyl cyclase (PAC) genes in the flagellate Euglena gracilis mutant strains.

The unicellular, green flagellate wild-type Euglena gracilis(strain Z) and its colorless phototaxis-mutant strains as well as the non-photosynthetic close relative, Astasia longa, possess several genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha(M(r) 105 kDa) and two PACbeta(90 kDa) subunits. While the proteins were first believed all to be located in the paraxonemal body (PAB), confocal microscopy revealed that Astasia longa as well as some of the mutant strains do not contain a PAB. Immunofluorescence using PAC antibodies showed that the PAC proteins are also located along the total length of the flagellum at least in some of the strains. In order to determine if the genes responsible for the PAC proteins in the PAB and flagella are identical, sequences of all PAC proteins were analyzed in the Euglena and Astasia strains studied for PAC protein location. Full sequence analysis using PCR and 3' and 5' RACE indicated a substantial divergence between strains with a homology between strains of between 45 and 100%. Sequence alignment and sequence tree construction for the main functional groups (BLUF domain, which binds FAD, and adenylyl cyclase) showed that the pacalpha and the pacbeta gene products form clusters each with some of the mutants being closely related while others show a substantial degree of genetic diversity. The conclusion of these results is that there is a family of very dissimilar PAC proteins located in the PAB and the flagellum where they serve different functions in phototaxis and step-up photophobic reactions.     

Kinetic and thermodynamic characterization of adenylyl cyclase from Euglena gracilis

Some kinetic and thermodynamic properties of the plasma membrane adenylyl cyclase (AC) from the protist Euglena gracilis were examined. The AC kinetics for Mg-ATP was hyperbolic with a K(m) value of 0.33-0.43 mM, whereas the inhibition exerted by 2('),5(')-dideoxyadenosine was of the mixed type with a K(i) of 80-147 microM. The V(m) value (0.9 or 1.8 nmol(mg protein)(-1)min(-1)) changed, depending upon the carbon source in the growth medium (lactic acid or glutamate plus malate). Lactic acid membrane AC was slightly more thermolabile (from 28 to 40 degrees C) and showed higher activation energy (range 15-25 degrees C). With lactate, the total and saturated fatty acid percentage content in the plasma membrane was significantly greater than with glutamate plus malate, whereas the percentage content of polyunsaturated (n-3) fatty acids was lower. The data suggest that the fatty acid composition, as changed by the carbon source in the growth medium, may modulate the AC activity in Euglena.     

Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit

XLalpha(s), the large variant of the stimulatory G protein alpha subunit (Gsalpha), is derived from GNAS1 through the use of an alternative first exon and promoter. Gs(alpha) and XLalpha(s) have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2-13. XLalpha(s) can mimic some functions of Gs(alpha), including betagamma interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XLalpha(s) to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XLalpha(s) and Gs(alpha) (Gnas(E2-/E2-)). Gnas(E2-/E2-) cells transfected with plasmids encoding XLalpha(s) and different heptahelical receptors, including the beta2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gs(alpha) and these receptors. Our findings thus indicate that XLalpha(s) is capable of functionally coupling to receptors that normally act via Gs(alpha).     

Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa

The recent success of channelrhodopsin in optogenetics has also caused increasing interest in enzymes that are directly activated by light. We have identified in the genome of the bacterium Beggiatoa a DNA sequence encoding an adenylyl cyclase directly linked to a BLUF (blue light receptor using FAD) type light sensor domain. In Escherichia coli and Xenopus oocytes, this photoactivated adenylyl cyclase (bPAC) showed cyclase activity that is low in darkness but increased 300-fold in the light. This enzymatic activity decays thermally within 20 s in parallel with the red-shifted BLUF photointermediate. bPAC is well expressed in pyramidal neurons and, in combination with cyclic nucleotide gated channels, causes efficient light-induced depolarization. In the Drosophila central nervous system, bPAC mediates light-dependent cAMP increase and behavioral changes in freely moving animals. bPAC seems a perfect optogenetic tool for light modulation of cAMP in neuronal cells and tissues and for studying cAMP-dependent processes in live animals.     

Sonication as a tool for the study of adenylyl cyclase activity.

The technique of sonication was applied in studying adenylyl cyclase activity of cultured fibroblasts. Exposure of BHK 21 c/13 to brief periods of low power sonication gives cell preparations with greater basal, fluoride and hormone sensitive adenylyl cyclase activites than those of broken cell preparations of homogenized cells. The sonicated cells provide a convenient method to study adenylyl cyclase since they are added directly to the adenylyl cyclase reaction vessels without further processing. Maximal epinephrine stimulated activity in sonicated cells is nearly equivalent to that activated by sodium fluoride, but the apparent affinity of the enzyme system is similar to that of broken cell preparations. Furthermore, broken cell preparations of sonicated cells possess greater adenylyl cyclase activity than broken cell preparations of unsonicated cells. This procedure may provide a useful tool for the analysis of the hormonal regulation of adenylyl cyclase activity of isolated cells.     

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

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.     

Thyroliberin receptor binding and adenylyl cyclase activation in cultured prolactin-producing rat pituitary tumor cells (GH cells)

A thyroliberin (TRH)-responsive particulate bound adenylyl cyclase is present in two rat anterior pituitary tumor cell strains (GH4C1 and GH3) which synthesize and secrete prolactin. At a given Mg2+ concentration, ATP and the guanyl nucleotides GTP and guanyl 5'-yl-imidodiphosphate (GMP-P(NH)P) caused a dose-dependent increase in adenylyl cyclase activity. The maximum response to thyroliberin occurred with ATP and GTP at concentrations above 0.30 mM and 2 microM, respectively. The maximal stimulatory effect of thyroliberin on adenylyl cyclase activity was 2-fold in the presence of GTP. GMP-P(NH)P increased the basal enzyme activity 4- to 10-fold over and above that of equimolar concentrations of GTP but supported poorly the TRH-induced response. Mg2+ caused a dose-dependent increase in the basal enzyme activity and reduced TRH and fluoride-induced responses. Also, Mn2+ and Co2+ stimulated the basal adenylyl cyclase activity while Zn2+, Ca2+, and Cu2+ inhibited the enzyme, and neither cations supported the TRH response. Half-maximal stimulation of the adenylyl cyclase by TRH and half-maximum binding of [3H]TRH to membranes at 35 degrees C were 102 and 56 nM, respectively. Pretreatment with TRH decreased the apparent Vmax of the enzyme and the maximal binding of [3H]TRH. Of 6 TRH analogs tested, only one was able to displace [3H]TRH from its receptor and to increase the adenylyl cyclase activity. We suggest that adenylyl cyclase activation is an early event in the stimulus secretion coupling between TRH and prolactin-producing GH cells.     

Role of 5'-guanylylimidodiphosphate in the activation of adenylyl cyclase by parathyroid hormone.

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.     

Kinetic properties of "soluble" adenylyl cyclase. Synergism between calcium and bicarbonate

"Soluble" adenylyl cyclase (sAC) is a widely expressed source of cAMP in mammalian cells that is evolutionarily, structurally, and biochemically distinct from the G protein-responsive transmembrane adenylyl cyclases. In contrast to transmembrane adenylyl cyclases, sAC is insensitive to heterotrimeric G protein regulation and forskolin stimulation and is uniquely modulated by bicarbonate ions. Here we present the first report detailing kinetic analysis and biochemical properties of purified recombinant sAC. We confirm that bicarbonate regulation is conserved among mammalian sAC orthologs and demonstrate that bicarbonate stimulation is consistent with an increase in the V(max) of the enzyme with little effect on the apparent K(m) for substrate, ATP-Mg(2+). Bicarbonate can further increase sAC activity by relieving substrate inhibition. We also identify calcium as a direct modulator of sAC activity. In contrast to bicarbonate, calcium stimulates sAC activity by decreasing its apparent K(m) for ATP-Mg(2+). Because of their different mechanisms, calcium and bicarbonate synergistically activate sAC; therefore, small changes of either calcium or bicarbonate will lead to significant changes in cellular cAMP levels.     

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.     

Genetic and biochemical analysis of the adenylyl cyclase of Schizosaccharomyces pombe.

The adenylyl cyclase gene, cyr1, of Schizosaccharomyces pombe has been cloned. We have begun an analysis of the function and regulation of adenylyl cyclase by disrupting this gene and by over-expressing all or parts of this gene in various strains. cyr1- strains are viable and contain no measurable cyclic AMP. They conjugate and sporulate under conditions that normally inhibit wild-type strains. Strains containing the cyr1 coding sequences transcribed from the strong adh1 promoter contain greatly elevated adenylyl cyclase activity, as measured in vitro, but only modestly elevated cAMP levels. Such strains conjugate and sporulate less frequently than wild-type cells upon nutrient limitation. Strains which carry the wild-type cyr1 gene but that also express high levels of the amino terminal domain of adenylyl cyclase behave much like cyr1-strains, suggesting that the amino terminal domain can bind a positive regulator. A protein that copurifies with the adenylyl cyclase of S. pombe cross-reacts to antiserum raised against the S. cerevisiae adenylyl cyclase-associated regulatory protein, CAP.     

The S-helix determines the signal in a Tsr receptor/adenylyl cyclase reporter

A signaling or S-helix has been identified as a conserved, up to 50-residue-long segment in diverse sensory proteins. It is present in all major bacterial lineages and in euryarchea and eukaryotes. A bioinformatic analysis shows that it connects upstream receiver and downstream output domains, e.g. in histidine kinases and bacterial adenylyl cyclases. The S-helix is modeled as a two-helical parallel coiled coil. It is predicted to prevent constitutive activation of the downstream signaling domains in the absence of ligand-binding. We identified an S-helix of about 25 residues in the adenylyl cyclase CyaG from Arthrospira maxima. Deletion of the 25 residue segment connecting the HAMP and catalytic domains in a chimera with the Escherichia coli Tsr receptor changed the response to serine from inhibition to stimulation. Further examination showed that a deletion of one to three heptads plus a presumed stutter, i.e. 1, 2, or 3 × 7 + 4 amino acids, is required and sufficient for signal reversion. It was not necessary that the deletions be continuous, as removal of separated heptads and presumed stutters also resulted in signal reversion. Furthermore, insertion of the above segments between the HAMP and cyclase catalytic domains similarly resulted in signal reversion. This indicates that the S-helix is an independent, segmented module capable to reverse the receptor signal. Because the S-helix is present in all kingdoms of life, e.g. in human retinal guanylyl cyclase, our findings may be significant for many sensory systems.     

Coupling of glucagon receptor to adenylyl cyclase. Requirement of a receptor-related guanyl nucleotide binding site for coupling of receptor to the enzyme.

Effects of glucagon and guanyl nucleotides on the rat liver plasma membrane adenylyl cyclase were studied. It was established that: 1) glucagon stimulates the fully guanyl-5'-yl imidodiphosphate (GMP-P(NH)P)-activated enzyme between 20 and 70%, provided a guanyl nucleotide is present in the assay; 2) glucagon has no effect on adenylyl cyclase activity in membranes activated fully by GMP-P(NH)P and then washed free of nucleotides. It is concluded that occupancy of the guanyl nucleotide binding site that activates the catalytic moiety of the system is not sufficient to promote hormone-receptor coupling to adenylyl cyclase and that occupancy of a second site by guanyl nucleotides is essential to effect stimulation of adenylyl cyclase by the glucagon-receptor complex. The data presented raise the question whether the guanyl nucleotide site that promotes coupling is distinct from the guanyl nucleotide site that modulates binding of glucagon to receptor and whether the occupancy of the guanyl nucleotide site associated with the catalytic moiety is necessary for coupling.     

Somatostatin-dependent adenylyl cyclase activity in nonactivated and mitogen-activated human T cells: evidence for uncoupling of sst3 receptor from adenylyl cyclase

Neuropeptide somatostatin (SRIF) has been shown to modulate interleukin-2 (IL-2) secretion by mitogen-activated T cells. In this study, we further analyzed the transduction pathways underlying SRIF actions on human Jurkat T cells and compared SRIF signaling between nonactivated and mitogen-activated cells. SRIF effects on adenylyl cyclase activity in the absence and presence of mitogens were addressed by using three different analogs: SRIF14, SRIF28, and SMS 201-995. In semipurified membrane preparations obtained from nonactivated cells, all analogs inhibited adenylyl cyclase. However, in membrane preparations obtained from mitogen-activated cells, the maximal inhibition of adenylyl cyclase mediated by SRIF14 and SRIF28 equaled only one third of that measured in the absence of mitogens, whereas SMS 201-995 was completely inactive. To assess the relevant mechanisms associated with different effects of SRIF on adenylyl cyclase activity in nonactivated and mitogen-activated T cells, we performed binding assays by using iodinated SRIF as a radioligand. These experiments suggested that both the number of receptors and their affinities were almost identical in either nonactivated or activated cells. RT-PCR analysis of the pattern of SRIF receptor expression showed that nonactivated as well as activated Jurkat cells expressed only mRNA corresponding to the sst3 receptor subtype. Altogether, these data point to a functional activation-associated uncoupling of sst3 receptors from adenylyl cyclase in human T cells, indicating a T-cell activation-induced alteration in the sst3 receptor transduction pathway.