Soluble guanylyl cyclase (sGC), the nitric oxide (NO) receptor, is a 150 kDa heterodimeric multi-domain protein that contains heme in the β subunit. Binding of NO to heme leads to rupture of the proximal histidine bond, increased catalytic conversion of GTP to cGMP at a distant guanylyl cyclase catalytic domain, and vasodilation through cGMP signaling. The structure of sGC has not been determined, and little is known about the mechanism by which NO binding to heme leads to increased catalysis. The small molecule YC-1 is known to stimulate sGC activity, but the exact YC-1 binding site and mechanism of action are unknown. Using truncated constructs of Manduca sexta (Ms) sGC lacking the catalytic domain, conformational changes upon YC-1 and NO-binding were characterized using analytical ultracentrifugation and small-angle X-ray scattering. Chemical cross-linking and high-resolution mass spectrometry was used to obtain distance restraints which, when combined with homology models, have provided the first model of sGC domain arrangement and revealed important information about domain-domain interactions. Truncated Ms sGC is highly elongated, contains a coiled-coil in a parallel arrangement, and contains a direct interface between the β H-NOX (Heme Nitric oxide/Oxygen binding domain) and the coiled-coil, and between the β H-NOX and α PAS (Per-arnt-sim) domains. Experiments using analytical ultracentrifugation, fluorescence anisotropy and native mass spectrometry have revealed the YC-1 binding site to be located within the α PAS domain. Additionally, measurement of the kinetics of heme loss and the heme reduction potential were performed to investigate the instability of oxidized sGC heme.

It has been well-known that VILIP-1 but not VILIP-3 regulates the activity of guanylyl cyclase-B. In order to identify the modulated region within VILIP-1 on regulating guanylyl cyclase-B activity, the recombinant myristoylated and nonmyristoylated VILIPs (VILIP-1, VILIP-3, chimeric VILIPs, and mutant VILIP-1) were prepared in the present study. The recombinant proteins were purified using ion-exchanger chromatography followed by gel filtration. CD spectra indicated that the secondary structure of VILIPs was dominant with £\-helix, reflecting a well-conserved EF-hand structure. Tryptic digestion assay and the fluorescence measurement showed that myristoylation, Ca2+ and Mg2+ differently induced the conformational changes of VILIPs. The results of gel filtration chromatography reflected that the EF-3&4 of VILIP-1 and myristoylation were involved in the dimerization of VILIP-1, and the dimer and monomer were converted each other in a dynamic manner. The porcine brain membrane binding assay and liposome binding assay showed that the binding capability of VILIPs were markedly enhanced by myristoylation, Mg2+ and Ca2+. Myristoylation and the intact EF-1 of VILIP-1 were found to essential for the regulation of guanylyl cyclase activity in the presence of Mg2+ and Ca2+. Taken together, theses results suggest that myristoylation and EF hand-1 of VILIP-1 are the structural elements crucial for regulating the guanylyl cyclase activity. In contrast to oligomerization of VILIP-1, Mg2+ and Ca2+ -induced conformational changes of VILIP-1 and enhancement of the binding of VILIP-1 with membrane by Mg2+ and Ca2+ partly but not heavily involve in the action.

No description available.

The survival of the any living organism depends on its availability to communicate, and a breakdown of cellular signaling can have dire consequences such as uncontrolled cellular proliferations or even cell death. Environmental cues or ligands are perceived by cognate receptors, expressed primarily on the cell surface, and transmitted to the interior of the cell to elicit a response. This universal phenomenon is termed as signal transduction. During this process, second messengers such as cyclic nucleotides, cAMP and cGMP, are produced which serve to amplify the signal. Cyclic GMP is emerging as a universal second messenger, and is found in both prokaryotes and eukaryotes. It is synthesized from GTP by the action of guanylyl cyclases. Vertebrate guanylyl cyclases are of two forms, soluble and membrane-associated. Soluble guanylyl cyclases are heterodimeric enzymes which are activated by nitic oxide. Membrane-associated guanylyl cyclases on the other hand are homodimeric enzymes that act as receptors for divers polypeptide ligands. In mammals, there are seven isoforms of receptors guanylyl cyclase, GC-A through GC-G. These recptors have a highly conseved modular domin organization with an N-terminal extracellular domain, a single transmembrane domain and a C- terminal intra cellular regions. The intracellular region contains a juxtamembrane domain followed by a protein-kinase domain, a linker region and a catalytic guanylyl cyclase domain. The coordinated actions of these domains ensure fine tuned-regulations of the catalytic domain. Guanylyl cyclase-C (GC-C) is a member of the membrane-bound guanylyl cyclases. GC-C is predominantely present in the intestine, on the apical surface of epithelial cells, but has also been detected in the rat epididymis. In the intestine it serves as the guanylin, uroguanylin and lymphoguanylin which are the endogenous peptide ligands, while heat- stable entrotoxins (ST) peptides secreted by enterotoxigenic E.coil, are exogenic ligands. Activation of GC-C by these ligands results in an increase in intracellular cGMP levels, which then activates cGMP-dependent protein kinase and cross-activates protein kinase A. In turn, these activated kinases phosphorylate and active the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in chloride and water secretion into the intestine lumen, thus regulating salt and water homeostasis in the intestine. ST peptide has a greater affinity for GC-C than the endogenous ligands and activation of GC-C by ST results in masiive fluid and ion efflux from the intestine cells from which manifests as Travelers’ Diarrhea. The GC-C mediated cGMP signal transduction pathway also maintains intestinal crypt-villus axis homeostatis by exerting a cytostatic effect on the epithelial cells, there by regulating their turn over. Over the years multiple mechanisms of regulation of GC-C activity has been identified including allosteric controlled by various domains in the receptor and phosphorylation-mediated regulation of guanylyl cyclase activity. The current study describes aspects of the regulation of GC-C by gycosylation, and also reports the molecular phenotypes of a naturally occurring mutation in GC-C causes sever diarrhea in affected individuals. GC-C is expressed as a differentially glycosylated protein (130k Da and 145kDa). While both forms bind with equal affinity, only one the 145 kDa form is activated by its ligands. It is this higher glycosylated form which is selectively downregulated in the process of decensitization of GC-C in colomn carcinoma cells (Caco2). Give the critical role gycosylation plays in protein folding, trafficking, receptor activity and mediating protein inter actions, its influence on GC-C was analysed.

Guanylyl cyclase C (GC-C) is a member of particulate guanylyl cyclases, discovered primarily as the target of a family of heat stable enterotoxins (ST), produced by enterotoxigenic Escherichia coli (ETEC). ST is acknowledged as a prime cause of traveller’s diarrhea and the leading cause of child mortality under the age of 5 years in developing nations. The bacterial expression of ST peptides represents molecular mimicry where the pathogen has exploited a gastrointestinal tract-signaling pathway to disperse and propagate. GC-C is primarily expressed on the apical or the brush border membranes of intestinal epithelial cells. GC-C agonists elaborated in the gastrointestinal tract are a family of guanylin peptides, which are responsible for maintaining fluid-ion homeostasis, essential for normal gut physiology. The signal of liigand binding to the extracellular domain of GC-C is transduced to the catalytic guanylyl cyclase domain, which results in production of intracellular cGMP. The elevated levels of cGMP influence multiple downstream targets, which finally regulate ion-flux through the transporters present on the membrane of an enterocyte. The ST peptide, a GC-C superagonist, produces physiologically abnormal levels of cGMP that manifest as secretory diarrhea. The purview of GC-C misregulation was confined to the notion of its hyperactivation caused by ETEC infection and the ensuing diarrhea. Recently, two seminal studies widened the scope of pathologies associated with GC-C. Studies described point mutations in GUCY2C, which were associated with human disease. One study identified a Norwegian family whose members demonstrated a dominantly inherited syndrome of frequent diarrhea associated with hyperactive GC-C. Following this study, inactivating mutations in GC-C in a small Bedouin population was reported. The current study reports the molecular phenotypes associated with the first germ line mutations in GC-C that result in a severe form of congenital sodium diarrhea. Our collaborators from Austria (Thomas Muller & Andreas Janecke, Department of Pediatrics Innsbruck Medical University) communicated to us their study of patients who had clinical diagnosis of congenital sodium diarrhea, with proportionally high fecal sodium loss, metabolic acidosis and dehydration. Exome sequencing in a cohort of 6 unrelated patients revealed four heterozygous missense mutations in GC-C (R792S, L775P, K507E, N850D). Novel GC-C mutations were de novo spontaneous mutations with the carrier being the only affected family member in contrast to the previous two reports with familial history. Biochemical characterization revealed that the mutants (GC-CR792S, GC-CL775P) were constitutively active with GC-CR792S, GC-CK507E, and GC-CN850D showing further stimulation upon treatment with ST and guanylin family of peptides. Interestingly, there was no change in the binding affinities of the ligands for the mutant receptors compared to wild type. However, a significant decrease (ranging from 10-100 fold) in ligand EC50 for the mutant GC-C receptors was prominent. The in vitro assays suggested that the mutations occupying different domains of GC-C might have resulted in distinct structural consequences reflected in the repertoire of phenotypes that were observed. The results presented in this thesis illustrate the molecular basis of the severe form of congenital diarrhea associated with the GC-C gain-of-function mutations. This study has also elaborated our understanding of the regulation of GC-C activity by its various domains.

No description available.

Nitric oxide (NO), a reactive diatomic gas and a potent signaling molecule, is required for proper cardiovascular functioning. Soluble guanylate cyclase (sGC), a heterodimeric heme protein, is the key intracellular NO receptor protein which, upon NO binding, undergoes conformational changes leading to catalysis and the cGMP signaling cascade. Several small molecules that allosterically stimulate sGC have been developed for treatment of pulmonary hypertension, but little is known about their binding site or how they stimulate activity. This dissertation describes experiments designed to uncover the molecular basis for signal transduction in sGC by NO and small molecule stimulators. The crystal structure of the α-subunit PAS domain from Manduca sexta (Ms) sGC was solved at 1.8 Å resolution revealing the expected PAS fold but with an additional β strand and a shorter Fα helix. CO binding measurements on different Ms sGC N-terminal constructs and the β₁ (1-380) construct revealed that the α-subunit keeps the β₁ H-NOX domain in an inhibited conformation and this inhibition is relieved by removal of the α-subunit or by addition of stimulatory compounds such as compound YC-1. Linked-equilibria measurements on the N-terminal constructs show that YC-1 binding affinity is increased in the presence of CO. Surface plasmon resonance (SPR) studies on the in-vitro biotinylated constructs showed that YC-1 binds near or directly to the β₁ H-NOX domain. Computational and mutational analysis of the β₁ H-NOX domain revealed a pocket important in allostery and drug action. Finally, we show that the coiled coil domain plays an important role in allosteric regulation of the β₁ H-NOX domain and possibly in signal transduction. Our data are consistent with a model of allosteric activation in which the α-subunit and the coiled coil domains function to keep heme in a low affinity conformation while YC-1 binding to the β₁ H-NOX domain switches heme to a high affinity conformation, and sGC to its high activity form.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19964号 / 医博第4154号 / 新制||医||1017(附属図書館) / 33060 / 京都大学大学院医学研究科医学専攻 / (主査)教授 柳田 素子, 教授 横出 正之, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

All cells are equipped to sense changes in their environment and make adaptive responses according to the stimuli. Signal recognition usually occurs at the cell membrane (with the exception of steroid signalling) where the ligand, which can be a small molecule, a peptide or a protein, binds its cognate receptor. This results in a change in the conformation of the receptor which in turn can regulate the production of second messengers. Second messengers can now modulate specific pathways which control gene expression and modify various aspects of cell behaviour. The signalling cascade is terminated by the removal of second messenger and/or by desensitisation of the receptor to the extracellular signal. Cyclic guanosine monophosphate (cGMP) was first identified in the rat urine and since then has emerged as an important second messenger regulating diverse cell processes. Subsequent to its discovery in mammalian cells, enzymes responsible for its synthesis (guanylyl cyclases), hydrolysis (phosphodiesterases) and its most common effectors (cGMP-dependent protein kinases) were identified. Guanylyl cyclases exist in two forms, cytosolic and membrane bound. Both have a conserved guanylyl cyclase domain, but differ in their choice of ligands, overall structure and tissue localization. It is now known that cytosolic and the membrane-bound forms are involved in eliciting distinct cellular responses. Receptor guanylyl cyclase C (GC-C) was identified as the target for a family of heat-stable enterotoxin toxins (ST) produced by enterotoxigenic E.coli. Stable toxin-mediated diarrhoeas are observed frequently in infants and contribute significantly to the incidence of Travellers’ Diarrhea. Early studies demonstrated that the effects of ST were mediated by an increase in intracellular cGMP levels in intestinal cells, and the receptor for ST was almost exclusively expressed in the apical microvilli of the intestinal brush-border epithelia. Effectors of cGMP in intestinal cells include protein kinase G (PKG), cyclic nucleotide gated ion channel 3 (CNG), and the cystic fibrosis transmembrane conductance regulator (CFTR). ST is an exogenous ligand which serves as a hyperagonist for GC-C, in comparison with the endogenous ligands guanylin and uroguanylin, which maintain fluid-ion homeostasis in the intestinal epithelia. The GC-C/cGMP signal transduction pathway also modulates intestinal cell proliferation along the crypt-villus axis by exerting a cytostatic effect on the epithelial cells, thereby regulating their turnover and neoplastic transformation. The current study describes in molecular detail two signalling pathways, one impinging on and one emerging from GC-C, which regulate colonic cell proliferation. The first part identifies the cross-talk and cross-regulation of GC-C and c-src. The second part delves into the molecular basis of GC-C/cGMP-mediated cytostasis and its effect on colonic tumorigenesis. Cross-talk between signalling pathways is believed to play a key role in regulating cell physiology. Phosphorylation of signalling molecules by protein kinases is frequently used as a means of achieving this cross-regulation. Aberrant hyperactivation of the c-src tyrosine kinase is an early event in the progression of colorectal cancer, and activated c-src specifically phosphorylates a number of proteins in the cell. It was found that c-src can phosphorylate GC-C in T84 colorectal carcinoma cells, as well as in the rat intestinal epithelia. Tyrosine phosphorylation of GC-C resulted in attenuation of ligand-mediated cGMP production; an effect which was reversed by chemical or transcriptional knockdown of c-src. These effects were found to be cell line-independent and relied only on the extent of c-src expression and activation in the cell. Mutational analysis revealed GC-C to be phosphorylated on a conserved tyrosine residue (Y820) in the guanylyl cyclase domain. The sequence of GC-C around Y820 allowed for efficient phosphorylation by c-src, and indeed, kinase assays indicated that the affinity of c-src for the GC-C Y820 peptide was one of the highest reported till date. A phospho-mimetic mutation at this site, which mimics a constitutively phosphorylated receptor, resulted in a sharp reduction of guanylyl cyclase activity of the receptor, reiterating the inhibitory role of Y820 phosphorylation on GC-C activity. Phosphorylation of GC-C at Y820 generated a docking site for the SH2 domain of c-src which could interact and thereby co-localize with GC-C on the cell membrane. Intriguingly, this interaction resulted in activation of c-src, setting-up a feed-forward loop of inhibitory GC-C phosphorylation and c-src activation. Treatment of colorectal carcinoma cells with ligands for GC-C reduces cell proliferation and inhibits tumorigenesis. It was observed that this cytostatic effect can be modulated by the status of c-src activation, and consequently, the fraction of tyrosine phosphorylated GC-C in these cells. Since activation of c-src is a frequent event in intestinal neoplasia, phosphorylation of GC-C by active c-src may be one of the means by which the cytostatic effects of GC-C agonists (guanylin and uroguanylin) in the intestine are bypassed, thereby leading to cancer progression. Colonisation of the gut with enteropathogenic microorganisms induces secretion of IFNγ from the host mucosal immune system, which subsequently activates c-src in intestinal epithelial cells. Ligand-stimulated activity of GC-C was found to be reduced in IFNγ treated cells. This could be one of the host defence mechanisms initiated in response to enterotoxigenic E. coli infection. These results provide the first evidence of cross-talk between a receptor guanylyl cyclase and a tyrosine kinase that results in heterologous desensitisation of the receptor. Populations with a higher incidence of enterotoxigenic E.coli infections appear to be protected from intestinal neoplasia. It was found that mice lacking GC-C, and therefore unable to respond to ST, displayed an increased cell proliferation in colonic crypts and enhanced carcinogen-induced aberrant crypt foci formation, which is a surrogate marker for colorectal carcinogenesis. However, pharmacological elevation of cGMP was able to efficiently induce cytostasis even in GC-C knockout mice, indicating a key role for cGMP in regulating colonic cell proliferation. Through microarray analyses, genes regulated by ST-induced GC-C activation in T84 colorectal carcinoma cells were identified. Genes involved in a number of cellular pathways were differentially expressed, including those involved in signal transduction, protein and solute secretion, transcriptional regulation and extracellular matrix formation. One of the genes found to be significantly up-regulated was the cell-cycle inhibitor, p21. The increase in p21 expression was validated at both the transcript and protein level. This p53-independent up-regulation of p21 was coupled to the activation of the cGMP-responsive kinase, PKGII, since knockdown of PKGII using specific siRNAs abolished ST-induced p21 induction. Activation of PKGII led to phosphorylation and activation of the stress responsive p38 MAPK. Similar to what was seen following knockdown of PKGII, inhibition of p38 MAPK activity attenuated the up-regulation of p21 in response to cGMP, indicating that PKGII and p38 MAPK could be a part of a pathway regulating p21 expression. It was found that active p38 MAPK phosphorylated the ubiquitous transcription factor SP1, enhancing its occupancy at the proximal p21 promoter. Therefore, SP1 could be one of the factors linking cGMP to transcription of the p21 mRNA. Chronic activation of GC-C led to nuclear accumulation of p21 in colonic cells, which entered a quiescent state. These cells arrested in the G1 phase of the cell cycle, consequent to p21-dependent inhibition of the G1 cyclin-CDK complexes. A fraction of these quiescent cells stochastically initiated a cGMP-dependent senescence programme and displayed all the hallmarks of senescent cells, including flattened cell morphology, expression of SA- galactosidase and formation of senescence-associated heterochromatic foci. Activation of senescence and loss of tumorigenicity in these cells was crucially dependent on the up-regulation of p21. This irreversible exit from the cell cycle due to cGMP-mediated activation of the PKGII/p38/p21 axis was well correlated with reduced colonic polyp formation in mice exposed to ST. In summary, these observations may provide a possible explanation for the low incidence of colorectal carcinoma seen in countries with a high incidence of ST-mediated diarrhoea. Interestingly, c-src mediated tyrosine phosphorylation of GC-C prevented p21 accumulation following ligand application. The findings described in this thesis may have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

O segundo mensageiro cGMP está envolvido em diversas funções celulares incluindo a visão em mamíferos. Embora trabalhos anteriores mostrassem variações nos níveis de cGMP durante o ciclo de vida de Blastocladiela emersonii e evidências da existência de enzimas específicas envolvidas na sua síntese (guanilato ciclase) e degradação (cGMP fosfodiesterase), nenhum genoma de fungo publicado até o momento mostrou a existência de genes codificando estas enzimas. Este fato é atribuído por evolucionistas à completa perda de motilidade dos fungos em geral, já que cGMP está primordialmente associado a células com cílios. Blastocladiomicetos, como Blastocladiella, apresentam células móveis em pelo menos um estágio do seu ciclo de vida, o que poderia explicar a existência dessa via nesses fungos. Uma investigação no banco de ESTs de B. emersonii revelou a existência de cDNAs codificando parte de prováveis guanilato ciclases (BeGC1, BeGC2 e BeGC3) e uma possível cGMP fosfodiesterase (BePDE). Assim, este trabalho buscou confirmar a existência destas enzimas e caracterizar a sinalização por cGMP em B. emersonii. A proteína recombinante selvagem correspondente ao domínio catalítico de BePDE mostrou atividade de degradação sobre cGMP e a mutação E389A foi capaz de alterar a especificidade por cGMP. Com o sequênciamento do genoma de B. emersonii obteve-se as sequências completas das guanilato ciclases. Em BeGC2 não foi possível identificar o ligante responsável por sua ativação. Em BeGC3, a presença de um domínio Heme-Pas sugeriu sua ativação por óxido nítrico. A presença de um domínio rodopsina em BeGC1 sugeriu sua ativação por luz. Experimentos de microscopia por imunofluorescência localizaram BeGC1 no \"eyespot\", BeGC2 no capacete nuclear e BeGC3 no citoplasma de zoósporos de B. emersonii. Verificamos também que zoósporos realizam fototaxia em direção à luz verde e que a adição de hidroxilamina, inibidor de rodopsina, ou do inibidor de guanilato ciclase LY83583 tem efeito negativo na fototaxia, bem como impede o aumento dos níveis de cGMP observado em zoósporos expostos à luz verde. O bloqueio da síntese de retinal por Norflurazon também inibiu a fototaxia sendo esta restaurada quando adicionamos retinalA1. Estes dados, juntamente com o fato de o domínio rodopsina de BeGC1 ser a única rodopsina presente no genoma, indicam que BeGC1 é responsável pela fototaxia nos zoósporos de B. emersonii. O genoma do fungo apresenta ainda um possível canal de potássio ativado por cGMP (BeCNG1) localizado na membrana plasmática de zoósporos, similar ao canal regulado por cGMP envolvido na visão em humanos. Ensaios de microfluorimetria também evidenciaram a presença de um canal ativado por cGMP relacionado com o influxo de potássio e a motilidade dos zoósporos. Um modelo para a via de sinalização da fototaxia em B.emersonii foi proposto e comparado com a sinalização presente na visão de mamíferos, destacando a existência de cGMP e rodopsina em ambos os processos e sugerindo uma possível origem comum. Portanto, os resultados obtidos suportam a existência da sinalização por cGMP em B. emersonii, além de indicar o papel dessa sinalização na fototaxia dos zoósporos, sendo esta a primeira via de sinalização por cGMP caracterizada em fungos. / The second messenger cyclic GMP is involved in a wide array of cellular processes including vision in mammals. Although previous studies demonstrated changes in cGMP levels during the life cycle of Blastocladiela emersonii and evidences of specific enzymes involved in its synthesis (guanylyl cyclase) and hydrolysis (cGMP-phosphodiesterase), no fungal genome published so far shows the presence of genes encoding these enzymes. Evolutionists attribute the absence of cGMP signaling pathways in higher fungi to the sedentary life style of these organisms, since cGMP is primarily associated with ciliated cells. However, blastocladiomycetes like Blastocladiella, have motile cells in at least one stage of their life cycle, which could explain the existence of this pathway in these primitive fungi. Inspection of B. emersonii EST data bank, revealed cDNAs encoding part of three putative guanylyl cyclases (BeGC1, BeGC2 e BeGC3) and one possible cGMP phosphodiesterase (BePDE). Thus, the purpose of this study was to confirm the existence of these enzymes and characterize the cGMP signaling pathway in this model. The recombinant protein containing the wild type catalytic domain of BePDE presented activity towards hydrolysis of cGMP and the E389A mutation of this domain changed the cGMP specificity of this enzyme. The complete nucleotide sequence of the guanylyl cyclases were obtained by sequencing of B. emersonii genome. In BeGC2 we were unable identify the ligand responsible for its activation, but in BeGC3, the presence of a Heme-Pas domain suggested its activation by nitric oxide. The presence of a rhodopsin domain in BeGC1 suggested its activation by light. Immunofluorescence microscopy localized BeGC1 in the \"eyespot\" structure, BeGC2 in the nuclear cap and BeGC3 in the cytoplasm of zoospores of B. emersonii. We found that Blastocladiella zoospores performed phototaxis toward green light and photobleaching of rhodopsin function using hydroxylamine prevented both phototaxis and the increased cGMP levels observed when zoospores were exposed to green light. The same effect was observed using the guanylyl cyclase inhibitor LY83583. Inhibition of retinal synthesis using Norflurazon prevented the phototaxis response, which could be restored by zoospore complementation with retinalA1. The BeGC1 gene is the only rhodopsin found in the draft assembly of B. emersonii genome, which indicates that BeGC1 is responsible for phototaxis observed in zoospores. We also found in the genome a possible cGMP-activated potassium channel (BeCNG1), localized in the plasma membrane of the zoospores, which is similar to the cGMP-activated channel involved in human vision. In addition, microfluorimetry assays revealed the presence of a cGMP-activated potassium channel involved in potassium influx and zoospore motility. The signaling model of B. emersonii phototaxis was proposed and compared with the mammalian vision system, with cGMP and rhodopsin acting in both signaling pathways, suggesting a common origin. Altogether our data indicate that Blastocladiella emersonii has a cGMP signaling system involved in phototaxis, being the first cGMP signaling pathway characterized in fungi.