No description available.

Adenosine plays a critical role in the heart signalling while affecting heart rate, contractility or coronary flow. Nowadays, four adenosine receptor subtypes are distinguished which are present in most of tissues and cells: A1, A2A, A2B and A3. All these receptors belong to the family of G protein-coupled receptors. Upon activation, their main target is an enzyme adenylyl cyclase which produces an important second messenger cAMP. The main goal of this thesis was characterization of adenosine receptors in the rat myocardium, assessment of their distribution, binding properties and signalling. We examined a possible disparity in receptors distribution between the left and right ventricles using SDS-PAGE electrophoresis and Western blotting. The same methods have been used in studies of adenosine receptor distribution in lipid rafts. Samples of lipid rafts and soluble fraction were prepared using a nonionic detergent Triton X-100. We did not find any evidence of different distribution between the left and right ventricles and our results did not confirm compartmentation of the receptors either. For determination of binding properties of the receptors we used radioligand binding assays with the A1 selective radioligand [H3 ]DPCPX. We did not observe any significant difference between the receptor...

Adenylyl Cyclases (ACs) catalyze the formation of the key universal second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) from adenosine 5’- triphosphate. Cyclic AMP participates in several signal transduction pathways and is present in bacteria and higher and lower eukaryotes including higher plants. Previous studies in plants have shown a role for cAMP in signal transduction during e.g. the cell cycle, elongation of the pollen tube and stimulation of protein kinase activity. More recently cAMP has been shown to play a role in stress responses. Interestingly, cAMP has also been shown to regulate ion transport in plant cells. Here we used a similar strategy that led to the discovery of the first guanylyl cyclase in plants that was based on the alignment of conserved and functionally assigned amino acids in the catalytic centre of annotated nucleotide cyclases from lower and higher eukaryotes, to identify a novel candidate ACs in Arabidopsis (Arabidopsis thaliana K+ Uptake 5 and 7). ATKUP5 and 7 are homologous to K+ uptake permeases (KUPs) from bacteria and high-affinity K+ transporters (HAKs) from fungi. The AC activity was investigated by recombinantly expressing the ATKUP5 and 7 AC domain in vitro and by complementation of an E. coli AC mutant (cyaA). Furthermore, ATKUP5 was tested for its ability to functionally complement a yeast mutant deficient in Trk1 and Trk2 high affinity potassium uptake transporters. Site-mutagenesis in the AC domain was used to test the effect of both functions in each other. Furthermore, ATKUP5 was characterized electrophysiologically in HEK-293 cells to characterize the nature of this transporter. The localization of the ATKUP5 in Arabidopsis was examined using a Green Fluorescent Protein (GFP) fusion with the ATKUP5 to determine whether ATKUP5 is expressed at the plasma or tonoplast membrane. Arabiodpsis thaliana of the wild type, overexpressing ATKUP5 and atkup5 mutant lines were used to examine phenotypic differences.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第17931号 / 生博第294号 / 新制||生||38(附属図書館) / 30751 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 渡邉 大, 教授 松崎 文雄 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

Injury causes increased pain sensation in humans and animals but the mechanisms underlying the emergence of persistent pathological pain states, which arise in the absence of on-going physical damage, are unclear. Therefore, elucidating the physiological regulation of such intractable pain is of exceptional biomedical importance. It is well known that endogenous activation of µ-opioid receptors (MORs) provides relief from acute pain but the consequences of prolonged endogenous opioidergic signaling have not been considered. Here we test the hypothesis that the intrinsic mechanisms of MOR signaling promote pathological sensitization of pain circuits in the spinal cord. We found that tissue inflammation produces agonist-independent MOR signaling in the dorsal horn of the spinal cord, which tonically represses hyperalgesia for months, even after complete recovery from injury and re-established normal pain thresholds. Disruption of this constitutive activity with MOR inverse agonists reinstated pain and precipitated cellular, somatic and aversive signs of physical withdrawal. This phenomenon required N-methyl-D-aspartate receptor activation of calcium-sensitive adenylyl cyclase type 1. Thus, we present a novel mechanism of long-lasting opioid analgesia that regulates the transition from acute to chronic pain while, in parallel, generates physical dependence. In conclusion we propose that the prevalence of chronic pain syndromes may result from a failure in constitutive signaling of spinal MORs and a loss of endogenous analgesic control.

Adenylyl cyclases are key points for the concurrent integration of diverse signaling pathways. Controlling production of the second messenger cAMP, adenylyl cyclases provide an important mechanism for the regulation of physiological functions by amplifying signaling events to stimulate downstream effectors. While different isoforms of adenylyl cyclase exhibit distinct patterns of expression and regulation, of particular interest are two groups of Ca2+ regulated isoforms that are highly expressed in the central nervous system. Adenylyl cyclase type 5 (AC5) is a Ca2+ inhibited isoform that is highly expressed in the striatum, and whose activity is involved in the regulation of movement, pain, and metabolism. Adenylyl cyclase type 8 (AC8) is stimulated by Ca2+ in a calmodulin dependent manner, and appears to be involved with long-term memory, anxiety, and reward pathways. Studying the signaling characteristics of these adenylyl cyclase isoforms is necessary for improving our scientific understanding of biological pathways, as well identifying therapeutic targets that can be exploited for treatment of disease. In this work, we investigated changes in the protein interaction network of AC5 following prolonged Gi/o-mediated inhibition that results in heterologous sensitization. The diversity of signaling pathways and multitude of protein interactions that have been implicated in the development of the heterologous sensitization response prompted the development of a novel screening strategy to capture and identify AC5-protein interactions which occur following prolonged Gi/o-mediated inhibition. We utilized bimolecular fluorescence complementation (BiFC) in conjunction with fluorescence activated cell sorting (FACS) and Next Generation sequencing to capture, identify, and characterize novel AC5 interacting partners. We further studied the effects of increased AC5 activity by functionally characterizing a series of gain-of-function mutations that have been identified in patients diagnosed with Familial Dyskinesia and Facial Myokymia (FDFM). Our results demonstrate that the AC5 mutants exhibit enhanced activity to Gs-mediated stimulation and reduced inhibition by Gi/o-coupled receptors. We further suggest that this dysregulation of AC5 in striatal medium spiny neurons likely results in an imbalance in the direct and indirect striatal signaling pathways that coordinate the initiation and maintenance of movement. Genetic models of AC8 regulation have implicated its activity in signaling pathways that may regulate comorbid long-term anxiety and ethanol consumption. Therefore, we developed and conducted a high-throughput screen and validation paradigm of small molecules for the discovery of AC8 selective inhibitors. The screening effort identified two lead compounds that demonstrate enhanced efficacy and selectivity over AC1 compared to currently available adenylyl cyclase inhibitors.

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

Adenylyl cyclase (Adcy) is an important early effector of adrenocorticotrophin (ACTH) on the adrenal cortex; however, this enzyme consists of ten isozymes in mammalian cells and the factors governing the expression of different Adcy isozymes have not been well defined. The aim of this study is to investigate the regulation of mouse Adcy4, one of ten isozymes, in Y1 adrenocortical tumor cells and in mutant subclones derived from the Y1 cells. Adcy4 is expressed at a high level in brain but at lower levels in many other tissues including the Y1 cells. Moreover, this isozyme is specifically deficient in Y1 mutants with impaired steroidogenic factor 1 (SF1) activity. These observations support a hypothesis that Adcy4 expression is influenced by both ubiquitously expressed and tissue-specific transcription factors. My sequencing results indicate that mouse Adcy4 is highly homologous to the human and rat counterparts; its gene is located less than 1 kb downstream of Ripk3 and contains 26 exons. Primer extension and in silico analyses suggest that Adcy4 contains a TATA-less promoter and initiates transcription from multiple sites. Luciferase reporter gene assays indicate that Adcy4 promoter activity is mainly stimulated by the proximal GC-rich region but is inhibited by the first intron. This 124 bp GC-rich region is well conserved among several mammalian species and exhibits strong promoter activity in Y1 cells, which is functionally compromised in the Adcy4-deficient mutant. Within this region, three Sp1/Sp3- and one SF1-binding sites have been identified which bind the corresponding proteins Sp1 and Sp3 or SF1 in electrophoretic mobility shift assays (EMSAs). Site-directed mutagenesis reveals that the 5’-most Sp1/Sp3 site enhances Adcy4 promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress this activity. In Y1 mutant cells, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with shRNA increases Adcy4 expression. All these data demonstrate that Adcy4 expression is under the control of the ubiquitous factors Sp1 and Sp3 and the tissue-specific factor SF1 and establish that SF1 is a repressor for Adcy4 promoter activity. This study is the first to demonstrate a repressor function for SF1 in certain promoter contexts.

Cyclic AMP and cyclic GMP (cAMP and cGMP) are important second messengers in key signal-transduction pathways that mediate various physiological functions in bacteria and eukaryotes. Adenylyl Cyclases (ACs) and Guanylyl Cyclases (GCs) cyclize ATP and GTP to produce cAMP and cGMP, respectively. Though most nucleotide cyclises show exquisite specificity for their substrates, there are instances where ACs were observed to have low GC activity as well, and vice versa. To understand structural basis of substrate (ATP or GTP) recognition, discrimination and binding by an adenylyl cyclase, we have taken up Ma1120, an AC from Mycobacterium avium, for our studies. Work presented in the thesis includes crystal structures of Ma1120 in the presence of substrate (ATP or GTP), by-product pyrophosphate and ATP analogue 2′,5′-dideoxyd-3′-adenosine triphosphate (2′,5′-dd-3′-ATP). A triple mutant of Ma1120 (K101→E, D157→G, A167→Y) was generated to increase specificity of Ma1120 towards GTP by mutation in the substrate specifying residues, but the enzyme showed equal specificity for ATP as well as for GTP. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions. ATP bound to Ma-Cat has two different conformations, one with C2′-endo and the other with C3′-endo puckering for the ribose. C3′-endo conformation is favourable for catalysis as it brings 3′-OH group of ribose and free oxygen of α-phosphate closer to each other. The crystal structure of GTP bound to Ma-Cat showed a novel mode of GTP binding to AC. This is the first report of GTP bound to AC. ATP bound to Ma-Cat-KDA→EGY forms non-cognate substrate complex and ATP is stabilized by stacking of adenines over each other with Tyr167 flanking on both sides of adenines. Ma-Cat-KDA→EGY+GTP complex is the first report of GTP bound to a guanylyl cyclase. GTP is bound in reverse orientation when compared to ATP bound to AC. Reverse orientation of GTP is attained to stabilize the guanine in highly electronegative guanine binding pocket. Also, O3' of GTP is placed in opposite orientation as compared to ATP bound to Ma-Cat. Therefore, during cyclization reaction guanine and ribose changes their orientation to bring O3' atom of ribose closer to α-phosphate, after cleavage of the bond between α- and β-phosphates. Thus, this study has revealed novel modes of binding of ATP and GTP to catalytic domains of Ma1120 and its triple mutant, mechanism of substrate discrimination and residual activity for the non-cognate substrate.