Magister Scientiae - MSc (Biotechnology) / It has been estimated that the world population will reach about 10 billion by the year 2050 and in order to accommodate the increased demand of food, the world agricultural production needs to rise by 70 % in the year 2030. However, the realisation of the goal in food production is hindered by limited arable land caused by urbanisation, salinisation, desertification and environmental degradation. Furthermore, abiotic and biotic stresses affect plant growth and development, which lead to major crop losses. The long term goal of this study is to improve food security by producing genetically engineered agricultural crops that will be tolerant to diverse stresses. This research aims at developing stress tolerant crops through the determination of important signalling molecules and second messengers, such as nitric oxide (NO), carbon monoxide (CO) and calcium ion (Ca2+), which can bind to plant proteins such as AtNOGC1 in order to induce stress tolerance in plants.

No description available.

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20275号 / 医博第4234号 / 新制||医||1021(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 小西 靖彦, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’hypertension artérielle et l’obésité sont deux composantes conjointement reliées du syndrome métabolique. Les récepteurs de l’ANP (GCA) et de l’oxyde nitrique (GCs) ont des propriétés diurétiques, natriurétiques, vasodilatatrices et sont liés au contrôle de la pression. Des études récentes ont démontré leur implication dans l’obésité. Hypothèse : Une différence génétique au niveau du gène GCA pourrait contribuer à des différences physiologiques. La composante lipidique et/ou sodique de la diète pourrait influencer la fonction rénale, cardiaque et les valeurs anthropométriques différemment chez les souches congéniques. Objectifs : (1) Déterminer l’effet de la composante lipidique et sodique des diètes; (2) Évaluer l’influence de GCA sur la réponse physiologique des souches congéniques; (3) Expliquer les mécanismes physiologiques procurant une réduction de la pression artérielle chez la souche SM9. Méthodologie : Des modèles congéniques du rat Dahl (DSS) hypertendu, nourri avec une diète riche en gras (HF) ou normale (NF), ont été utilisés pour démontrer l’impact d’un segment chromosomique d’origine normotendue. Résultats : La souche SM9 a une prise de poids plus importante que SM12 et DSS sur diète HF malgré un apport alimentaire équivalent. La souche SM9 présente également un ratio masse adipeuse/masse maigre plus élevé que SM12 et DSS. Nous n’avons observé aucune augmentation de la pression artérielle en réponse à la diète HF pour les 3 souches malgré une augmentation du dommage rénal pour les 3 souches. Le dommage rénal est plus important chez DSS que pour les 2 congéniques. La réponse diurétique à l’ANP est plus élevée chez SM9 et est influencée par le contenu en sel dand la diète. La perte glomérulaire plus importante chez le rat DSS semble compensée par une augmentation de la réponse à l’ANP par les glomérules résiduels. Il y a une corrélation entre l’activité de GCA en réponse à l’ANP, les niveaux d’ARNm et le nombre de répétition du dinucléotide TA dans son promoteur. Le rat DSS présente une hypertrophie cardiaque plus importante que les deux souches congénique et ceci n’est pas modifié par la diète HF. Conclusion : Nos études ont permis de mettre en évidence un effet génétique impliquant le segment chromosomique normotendu contenant GCA dans la réponse à une diète HF chez le rat DSS. / Hypertension and obesity are two related components of the metabolic syndrome. The ANP receptor (GCA) and nitric oxide receptor (sGC) have diuretic, natriuretic, vasodilatory properties, and are linked to blood pressure control. Furthermore a recent study has demonstrated the implication of GCA and sGC in the development of obesity. Hypothesis: A genetic difference in GCA gene could contribute to physiological differences. The differencial lipid and/or sodium composition of the diet could influence the renal, cardiac and anthropometric values. Objectives: (1) To determine the effect of fat and sodium on the physiological parameters; (2) To evaluate the influence of GCA on the physiological response of the congenic rat; (3) To explain the mechanisms of the blood pressure reduction in SM9 rats. Methodology: Congenic model of DSS rat, fed with either high fat (HF) or normal (NF) diet, were used to demonstrate the impact of a chromosome segment from normotensive origin on physiological functions. C2SM9 contains GCA and sGC from normotensive origin while C2SM12 harbours only sGC from normotensive origin. Results: HF diet had negative feature on body composition, renal damage, creatinine clearance and inhibited the diuretic/natriuretic effect of ANP. The normotensive segment including GCA and sGC has reduced the blood pressure, improve the renal damage and increased the diuretic/natriuretic capacity of SM9 in response to ANP injection when compared to SM12 and DSS. GCA mRNA and the clearance receptor ratio were reduced in SM9 in the renal cortex and retroperitoneal fat. SM12 and SM9, containing the chromosomal segment that includes sGC, improve their lipid profile compared with DSS. Conclusion: Our results suggested a compensatory increase in the GCA levels for SM12 and DSS that is insufficient to improve their pathophysiologic status as observed in SM9. HF diet increases the metabolic syndrome in those rats.

L’hypertension artérielle et l’obésité sont deux composantes conjointement reliées du syndrome métabolique. Les récepteurs de l’ANP (GCA) et de l’oxyde nitrique (GCs) ont des propriétés diurétiques, natriurétiques, vasodilatatrices et sont liés au contrôle de la pression. Des études récentes ont démontré leur implication dans l’obésité. Hypothèse : Une différence génétique au niveau du gène GCA pourrait contribuer à des différences physiologiques. La composante lipidique et/ou sodique de la diète pourrait influencer la fonction rénale, cardiaque et les valeurs anthropométriques différemment chez les souches congéniques. Objectifs : (1) Déterminer l’effet de la composante lipidique et sodique des diètes; (2) Évaluer l’influence de GCA sur la réponse physiologique des souches congéniques; (3) Expliquer les mécanismes physiologiques procurant une réduction de la pression artérielle chez la souche SM9. Méthodologie : Des modèles congéniques du rat Dahl (DSS) hypertendu, nourri avec une diète riche en gras (HF) ou normale (NF), ont été utilisés pour démontrer l’impact d’un segment chromosomique d’origine normotendue. Résultats : La souche SM9 a une prise de poids plus importante que SM12 et DSS sur diète HF malgré un apport alimentaire équivalent. La souche SM9 présente également un ratio masse adipeuse/masse maigre plus élevé que SM12 et DSS. Nous n’avons observé aucune augmentation de la pression artérielle en réponse à la diète HF pour les 3 souches malgré une augmentation du dommage rénal pour les 3 souches. Le dommage rénal est plus important chez DSS que pour les 2 congéniques. La réponse diurétique à l’ANP est plus élevée chez SM9 et est influencée par le contenu en sel dand la diète. La perte glomérulaire plus importante chez le rat DSS semble compensée par une augmentation de la réponse à l’ANP par les glomérules résiduels. Il y a une corrélation entre l’activité de GCA en réponse à l’ANP, les niveaux d’ARNm et le nombre de répétition du dinucléotide TA dans son promoteur. Le rat DSS présente une hypertrophie cardiaque plus importante que les deux souches congénique et ceci n’est pas modifié par la diète HF. Conclusion : Nos études ont permis de mettre en évidence un effet génétique impliquant le segment chromosomique normotendu contenant GCA dans la réponse à une diète HF chez le rat DSS. / Hypertension and obesity are two related components of the metabolic syndrome. The ANP receptor (GCA) and nitric oxide receptor (sGC) have diuretic, natriuretic, vasodilatory properties, and are linked to blood pressure control. Furthermore a recent study has demonstrated the implication of GCA and sGC in the development of obesity. Hypothesis: A genetic difference in GCA gene could contribute to physiological differences. The differencial lipid and/or sodium composition of the diet could influence the renal, cardiac and anthropometric values. Objectives: (1) To determine the effect of fat and sodium on the physiological parameters; (2) To evaluate the influence of GCA on the physiological response of the congenic rat; (3) To explain the mechanisms of the blood pressure reduction in SM9 rats. Methodology: Congenic model of DSS rat, fed with either high fat (HF) or normal (NF) diet, were used to demonstrate the impact of a chromosome segment from normotensive origin on physiological functions. C2SM9 contains GCA and sGC from normotensive origin while C2SM12 harbours only sGC from normotensive origin. Results: HF diet had negative feature on body composition, renal damage, creatinine clearance and inhibited the diuretic/natriuretic effect of ANP. The normotensive segment including GCA and sGC has reduced the blood pressure, improve the renal damage and increased the diuretic/natriuretic capacity of SM9 in response to ANP injection when compared to SM12 and DSS. GCA mRNA and the clearance receptor ratio were reduced in SM9 in the renal cortex and retroperitoneal fat. SM12 and SM9, containing the chromosomal segment that includes sGC, improve their lipid profile compared with DSS. Conclusion: Our results suggested a compensatory increase in the GCA levels for SM12 and DSS that is insufficient to improve their pathophysiologic status as observed in SM9. HF diet increases the metabolic syndrome in those rats.

Philosophiae Doctor - PhD / Nitric Oxide (NO) influences a wide range of physiological processes in plants including growth and development, responses to abiotic and biotic stress and pathogen responses. NO binds to the heme group of the mammalian soluble guanylyl cyclase, which activates the enzyme to convert guanosine 5’ triphosphate (GTP) to a second messenger guanosine 3’, 5’ cyclic monophosphate (cGMP). Cyclic GMP further activates other signalling cascades including the regulation of protein kinases, ion gated channels and phosphodiesterases. In plants, a few GCs have been identified and these include AtGC1, AtBRI1, AtWAKL10, and AtPSKR1, however, a GC that contains a heme binding motif that senses NO has yet to be identified. In order to identify such molecules, a search motif based on conserved HNOX domains and the conserved and functionally assigned amino acid residues in the catalytic centres of annotated GCs was designed and used to search the Arabidopsis thaliana proteome. Several candidate molecules were identified including a flavin-containing monooxygenase (FMO)-like protein and the At5g57690 which is currently annotated as a diacylglycerol kinase. FMOs found in bacteria, yeast, and animals are the most important monooxygenases since they are involved in xenobiotic metabolism and variability in drug response. FMOs in plants are implicated in catalysing specific steps in auxin biosynthesis,metabolism of glucosinolates and pathogen defense mechanisms. The human diacylglycerol kinase acts as a lipid kinase that mediates a wide range of biological processes which include cell proliferation, differentiation and turmogenesis. In prokaryotes, the structure of Escherichia coli lipid kinase has been solved however, its function has not yet been demonstrated. So far, the occurrence of the diacylglycerol kinases in plants has not yet been reported, and their structure and function also remain elusive. The domain architecture of the 2 molecules (AtNOGC1 and At5g57690) identified by the HNOX-based search strategy revealed that these molecules contain a GC and a heme-binding motif that is conserved among all known heme-binding proteins.In this study, the role of AtNOGC1, a novel NO binding protein in higher plants was investigated and the results showed that this molecule contains an NO-dependant active GC domain. The sequence was first analysed and the location of the HNOX and the GC motifs highlighted. The protein was then recombinatly expressed as a His-SUMO fusion protein and the purification optimised by a second step of ion exchange chromatography. Electrochemical techniques such as cyclic voltammetry and square wave voltammetry were used to demonstrate the binding of NO and O2 to the AtNOGC1. Electrochemical data revealed that AtNOGC1 has a lower affinity for O2 and a higher affinity for NO, an important signalling molecule in plants.The presence of the GC activity in AtNOGC1 was investigated by conducting GC activity assays in vitro in the presence or absence of NO. The GC activity assays demonstrated that AtNOGC1 can synthesize cGMP from GTP in vitro. It was also noted that NO was required for the maximum activation of AtNOGC1 catalytic activity. NO-activated catalysis resulted in a >2 fold excess of cGMP production compared to an NO-independent GC activity assay. The effect of calcium in regulating the GC activity was also investigated and an increase in cGMP levels was observed however, this was just a preliminary finding that requires further experimentation.3 Homology models for both the FMO-like (AtNOGC1) and the diacylglycerol kinase(At5g57690) were built using Modeller program, and important amino acid residues underlying the heme-binding and GC motifs were identified. Residues corresponding to the motifs, which give signature to AtNOGC1 as an FMO, were also noted. In addition,computational functional prediction also suggested the role of AtNOGC1 in a number of processes which include ion binding and functioning as an FMO.Taken together, these findings suggest that AtNOGC1 is a novel Arabidopsis thaliana hemebinding protein that senses NO with higher affinity than for O2. Though AtNOGC1 is currently annotated as a FMO-like protein, it contains a NO-sensitive GC activity and shares limited sequence similarities with mammalian sGC and the recently identified HNOX domains. Homology modelling strongly suggests that AtNOGC1 and At5g57690 belong to the families of FMOs and diacylglycerol kinases respectively. The domain organisation of AtNOGC1 suggests that more of its functions still remain to be identified. The cloning and characterisation of the At5g57690 gene will provide possible means for further experimentation as well as affording more insights into the exact functions of lipid kinases in plants.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the 1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

During development, neurons must find their way to and make connections with their appropriate targets. Growth cones are dynamic, motile structures that are integral to the establishment of appropriate connectivity during this wiring process. As growth cones migrate through their environment, they encounter guidance cues that direct their migration to their appropriate synaptic targets. The gaseous messenger nitric oxide (NO), which diffuses across the plasma membrane to act on intracellular targets, is a signaling molecule that affects growth cone motility. However, most studies have examined the effects of NO on growth cone morphology when applied in large concentrations and to entire cells. In addition, the intracellular second messenger cascade activated by NO to bring about these changes in growth cone morphology is not well understood. Therefore, this dissertation addresses the effects that a spatially- and temporally-restricted application of physiological amounts of NO can have on individual growth cone morphology, on the second messenger pathway that is activated by this application of NO, and on the calcium cascades that result and ultimately affect growth cone morphology. Helisoma trivolvis, a pond snail, is an excellent model system for this type of research because it has a well-defined nervous system and cultured neurons form large growth cones. In the present study, local application of NO to Helisoma trivolvis B5 neurons results in an increase in filopodial length, a decrease in filopodial number, and an increase in the intracellular calcium concentration ([Ca2+]i). In B5 neurons, the effects of NO on growth cone behavior and [Ca2+]i are mediated via sGC, protein kinase G, cyclic adenosine diphosphate ribose, and ryanodine receptor-mediated intracellular calcium release. This study demonstrates that neuronal growth cone pathfinding in vitro is affected by a single spatially- and temporally-restricted exposure to NO. Furthermore, NO acts via a second messenger cascade, resulting in a calcium increase that leads to cytoskeletal changes. These results suggest that NO may be a signal that promotes appropriate pathfinding and/or target recognition within the developing nervous system. Taken together, these data indicate that NO may be an important messenger during the development of the nervous system in vivo.

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.