DOCSLIB.ORG

Mutagenesis of the Cyclic AMP Receptor Protein of Cyclic Nucleotide

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mutagenesis of the Cyclic AMP Receptor Protein of Cyclic Nucleotide Q--DI 1993 Oxford University Press Nucleic Acids Research, 1993, Vol. 21, No. 8 1827-1835 Mutagenesis of the cyclic AMP receptor protein of Escherichia cofi: targeting positions 72 and 82 of the cyclic nucleotide binding pocket Ali O.Belduz+, Eun Ju Lee and James G.Harman* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA Received January 11, 1993; Revised and Accepted March 12, 1993 ABSTRACT The 3', 5' cyclic adenosine monophosphate (cAMP) Three conformational states of CRP are known: native CRP binding pocket of the cAMP receptor protein (CRP) of and complexes having either one or both of the cAMP binding Escherichia coli was mutagenized to substitute leucine, sites occupied by cAMP (6). The CRP:(cAMP), conformer glutamine, or aspartate for glutamate 72; and lysine, appears to be the relevant active form in vivo, considering both histidine, leucine, isoleucine, or glutamine for arginine the intracellular cAMP concentrations in bacteria and the 1:1 82. Substitutions were made in wild-type CRP and in stoichiometry of CRP to cAMP observed in the CRP:cAMP:gal a CRP*, or cAMP-independent, form of the protein to DNA and CRP:cAMP:lac DNA complexes formed in vitro assess the effects of the amino acid substitutions on (7-9). Details of the changes in CRP structure that result upon CRP structure. Cells containing the binding pocket binding one or two molecules of cAMP are incomplete; crystals residue-substituted forms of CRP were characterized of the CRP and the CRP:(cAMP)1 conformers have not yet been through ,B-galactosidase activity and by measurement obtained. Detailed structure analysis information available for of cAMP binding activity. This study confirms a role for the CRP:(cAMP)2 conformer (10) has provided a basis for both glutamate 72 and arginine 82 in cAMP binding and understanding both cAMP-mediated and mutational activation of activation of CRP. Glutamine or leucine substitution of CRP (11-14), and proposals have been advanced on the probable glutamate 72 produced forms of CRP having low affinity effects of cAMP on wild-type CRP. for the cAMP and unresponsive to the nucleotide. Analysis of the CRP:(cAMP)2 crystal structure identified five Aspartate substituted for glutamate 72 produced a low specific amino acid-ligand contacts that are considered important affinity cAMP-responsive form of CRP. CRP has a in cAMP binding to CRP and/or in mediating cAMP activation stringent requirement for the positioning of the position of CRP (13). The authors suggested that the charged phosphate 72 glutamate carboxyl group within the cyclic of cAMP interacts with arginine 82 to form a salt bridge, and nucleotide binding pocket. Results of this study also that specific hydrogen bond interactions occur between a) the indicate that there are differences in the binding axial phosphate oxygen atom of the 3' :5' cyclic phosphate ring requirements of cAMP and cGMP, a competitive and serine 83 b) the 2' hydroxyl of ribose and glutamate 72, and inhibitor of cAMP binding to CRP. c) the N6 amino group of adenine and threonine 127 of one subunit and serine 128 of the other subunit. We have undertaken a study designed to assess the effects of specific amino acid INTRODUCTION substitutions at these positions in CRP to test the predictions of The cyclic 3':5'-adenosine monophosphate (cAMP) receptor the CRP:(cAMP)2 crystal structure analysis for CRP in solution. protein (CRP) ofEscherichia coli is a dimeric protein that binds This study includes analysis of substitutions contained in both to both cyclic nucleotides and, in the presence of cAMP, to wild-type CRP and in a cAMP-independent form of CRP (CRP*) specific DNA sequences located in a number of E. coli promoter that mediates CRP-dependent gene expression in the absence of regions (1). Binding of CRP:cAMP to a promoter region either cAMP (15). stimulates or represses the activity of that promoter depending Many crp* alleles have amino acid substitutions located outside upon the relative locations of the CRP and RNA polymerase of the cAMP binding pocket (12, 14-17). One such mutant, 91 binding sites. The activation of CRP as a transcription control CRP (reference 15, hereafter referred to as CRP*[A14T]), element is initiated by the binding of cAMP. Biochemical contains threonine in place of alanine 144 located in the DNA evidence clearly indicates that cAMP binding alters CRP binding domain of CRP. This substitution renders CRP conformation and induces DNA sequence specific recognition conformation similar to that of the active cAMP-modified (reviewed in 1 and 2). Cyclic nucleotides other than cAMP (i.e., conformation of the wild type CRP:cAMP complex (15). The cGMP) bind CRP but fail to activate the protein (3-5). inherent transcription control activity of many forms of CRP* * To whom correspondence should be addressed + Present address: Karadeniz Technical University, Department of Biology, 61080-Trabzon, Turkey 1828 Nucleic Acids Research, 1993, Vol. 21, No. 8 (including CRP*[AI44T]) is stimulated by both cAMP and cGMP. from Difco Laboratories. 3'-5' cAMP, 3'-5' cGMP and subtilisin CRP* forms of CRP are thought to be intermediate in both BPN' (type XXVII, 7.9 units/mg) were purchased from Sigma conformation and activity between the active and inactive Chemical Company. 3H (2'-8' labeled) 3'-5' cAMP (31.3 conformations of wild type CRP; they are capable of undergoing Ci/mol), C-35 S-labeled ATP (500 Ci/mol) and a-32 P-labeled a conformation change upon binding of cyclic nucleotide, and ATP (3000 Ci/mol) were purchased from New England Nuclear. have lost the capacity to discriminate between cAMP and cGMP. XAR-5 X-ray film was obtained from Eastman Kodak. Synthetic We report here the results of our analysis of CRP mutants with oligonucleotides were purchased from Midland Certified Reagent amino acid substitutions at positions 72 and 82 of the cyclic Company. Qiagen DNA isolation kits were purchased from nucleotide binding pocket. The effects of these substitutions on Qiagen, Inc. Common salts and buffer components were reagent CRP function were assessed by measuring the capacity of mutant grade or better. CRP, expressed in a Acya Acrp strain of E.coli, to support f- galactosidase synthesis in the absence of cAMP, in the presence Construction of M13crp of cAMP or in the presence of cGMP. Partially purified CRP The 1321 bp Alu I DNA fragment of pKC30crp (15) was cloned preparations were utilized to measure mutant CRP affinity for into Sma I-digested, alkaline phosphatase-treated M13mp 18 cAMP at physiologically relevant salt and cAMP concentrations. DNA. The ligation mixture was used to transform competent Parallel cAMP binding pocket mutants contained in the E. coli TG- 1 cells. Single stranded DNA was isolated and CRP*[Al44T] background provided a measure of whether specific sequenced to confirm that the DNA from one of the resultant amino acid substitutions resulted in only local perturbations or plaques, designated M 13crp, contained the cmp gene. in global changes in CRP*[Al44T] structure. The results of this study confirm the importance of both glutamate 72 and arginine Site directed mutagenesis of the E.coli cmp gene 82 in cAMP binding and in cAMP-mediated activation of CRP. Introduction ofa Nde I site. Mutagenesis of cmp was accomplished using the method of Kunkel (22) as outlined in the protocol MATERIALS AND METHODS provided by Bio-Rad Laboratories. Uracil-containing M13cmp DNA was annealed to a mutagenic primer (pGATAA- Bacterial strains and plasmid DNA CCGCATATGGTGCTTGGCoH) and replicated in vitro to E.coli strains CA8445/pRK248 (Acya 845, Acrp 45, thilXclts, introduce a Nde I restriction endonuclease site that utilized the tetr) (15) and D1210HP [hsd S20 (rB; mB), sup E44, ara 14, ATG start codon in the cmp structural gene. Mutagenized DNA gal K2, lac Y1, pro A2, rps L20, xyl -5, mtl -1, rec A13, (rB-; was used to transform competent E. coli MV 1190 cells. The Nde mBO), mcr A+, mcr B-, XcIts, xis-, kil-, int+] (Stratagene) I/Sac I fragment containing the crp structural gene of one mutant were used as hosts for recombinant crp plasmids. E. coli strains (M13cmp NdeI) was subcloned into Nde I/,Sac I-digested, alkaline TG-1 [A (lac -pro) thi, sup E, hsd D5, fF':tra D36, pro AB, phosphatase-treated pRE2. The ligation mixture was used to lac Iq, lac ZAM15}] (18) and XL-1 Blue [X-, rec A1, end A 1, transform competent E.coli D121OHP. Supercoiled plasmid DNA gyr A96, thi, hsd 17, sup E44, rel Al, (lac), [F', pro AB, lac isolated from from one ampicillin resistant isolate, designated Iq, lac ZAM15, TnlO (tetr)j] (Stratagene) were used to propagate pRE2cmp, was sequenced using cmp sequencing primers (15) to M13mpl 8 (19) and its crp-containing derivatives. E. coli strains confirm the sequence of the construct. DNA sequencing reactions MV1 190 [,A(lac -pro) thi, sup E, A(srl-rec A0306: :TnlO were run as outlined in the protocols provided by New England tetr)[F':tra D36, pro AB, lac Iq, lac ZAM15a] (Bio-Rad Biolabs and by United States Biochemical Corporation, Inc. Laboratories) and CJ236 [dut, ung, thi, rel AfpCJ105 (cmr)J] (Bio-Rad Laboratories) were used for mutagenesis of cmp. Plasmid Introduction ofcyclic nucleotide binding pocket mutations. The was of California pRK248 (20) the gift of D.Helinski, University cmp gene was mutagenized as described above using uracil- at San Diego, La Jolla, CA. pKC30crp (15) containing wild type containing Ml13cmp NdeI DNA as template and the following crp cloned downstream of the XPL promoter was used as the mutagenic primers. Position 72; ATTGGCGACCTGGGCC (G- source of the cmp structural gene.
Load more

Recommended publications
  • Molecular Targets and Biological Functions of Camp Signaling in Arabidopsis
    biomolecules Article Molecular Targets and Biological Functions of cAMP Signaling in Arabidopsis Ruqiang Xu 1,2,*, Yanhui Guo 1, Song Peng 1, Jinrui Liu 1, Panyu Li 1, Wenjing Jia 1 and Junheng Zhao 1 1 School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China; [email protected] (Y.G.); [email protected] (S.P.); [email protected] (J.L.); [email protected] (P.L.); [email protected] (W.J.); [email protected] (J.Z.) 2 Zhengzhou Research Base, State Key Laboratory of Cotton Biology, Zhengzhou University, Zhengzhou 450001, China * Correspondence: [email protected]; Tel.: +86-0371-6778-5095 Abstract: Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. However, the mechanism of cAMP signaling in plants remains very poorly understood. Here, we employ the engineered activity of soluble adenylate cyclase to induce cellular cAMP elevation in Arabidopsis thaliana plants and identify 427 cAMP-responsive genes (CRGs) through RNA-seq analysis. Induction of cellular cAMP elevation inhibits seed germination, disturbs phytohormone contents, promotes leaf senescence, impairs ethylene response, and compromises salt stress tolerance and pathogen resistance. A set of 62 transcription factors are among the CRGs, supporting a prominent role of cAMP in transcriptional regulation. The CRGs are significantly overrepresented in the pathways of plant hormone signal transduction, MAPK signaling, and diterpenoid biosynthesis, but + they are also implicated in lipid, sugar, K , nitrate signaling, and beyond. Our results provide a basic framework of cAMP signaling for the community to explore. The regulatory roles of cAMP signaling Citation: Xu, R.; Guo, Y.; Peng, S.; in plant plasticity are discussed.
    [Show full text]
  • Mechanisms Whereby Extracellular Adenosine 3',5'- Monophosphate Inhibits Phosphate Transport in Cultured Opossum Kidney Cells and in Rat Kidney
    Mechanisms whereby extracellular adenosine 3',5'- monophosphate inhibits phosphate transport in cultured opossum kidney cells and in rat kidney. Physiological implication. G Friedlander, … , C Coureau, C Amiel J Clin Invest. 1992;90(3):848-858. https://doi.org/10.1172/JCI115960. Research Article The mechanism of phosphaturia induced by cAMP infusion and the physiological role of extracellular cAMP in modulation of renal phosphate transport were examined. In cultured opossum kidney cells, extracellular cAMP (10-1,000 microM) inhibited Na-dependent phosphate uptake in a time- and concentration-dependent manner. The effect of cAMP was reproduced by ATP, AMP, and adenosine, and was blunted by phosphodiesterase inhibitors or by dipyridamole which inhibits adenosine uptake. [3H]cAMP was degraded extracellularly into AMP and adenosine, and radioactivity accumulated in the cells as labeled adenosine and, subsequently, as adenine nucleotides including cAMP. Radioactivity accumulation was decreased by dipyridamole and by inhibitors of phosphodiesterases and ecto-5'-nucleotidase, assessing the existence of stepwise hydrolysis of extracellular cAMP and intracellular processing of taken up adenosine. In vivo, dipyridamole abolished the phosphaturia induced by exogenous cAMP infusion in acutely parathyroidectomized (APTX) rats, decreased phosphate excretion in intact rats, and blunted phosphaturia induced by PTH infusion in APTX rats. These results indicate that luminal degradation of cAMP into adenosine, followed by cellular uptake of the nucleoside by tubular cells, is a key event which accounts for the phosphaturic effect of exogenous cAMP and for the part of the phosphaturic effect of PTH which is mediated by cAMP added to the tubular lumen under the influence of the hormone.
    [Show full text]
  • Challenges on Cyclic Nucleotide Phosphodiesterases Imaging with Positron Emission Tomography: Novel Radioligands and (Pre-)Clinical Insights Since 2016
    International Journal of Molecular Sciences Review Challenges on Cyclic Nucleotide Phosphodiesterases Imaging with Positron Emission Tomography: Novel Radioligands and (Pre-)Clinical Insights since 2016 Susann Schröder 1,2,* , Matthias Scheunemann 2, Barbara Wenzel 2 and Peter Brust 2 1 Department of Research and Development, ROTOP Pharmaka Ltd., 01328 Dresden, Germany 2 Department of Neuroradiopharmaceuticals, Institute of Radiopharmaceutical Cancer Research, Research Site Leipzig, Helmholtz-Zentrum Dresden-Rossendorf (HZDR), 04318 Leipzig, Germany; [email protected] (M.S.); [email protected] (B.W.); [email protected] (P.B.) * Correspondence: [email protected]; Tel.: +49-341-234-179-4631 Abstract: Cyclic nucleotide phosphodiesterases (PDEs) represent one of the key targets in the research field of intracellular signaling related to the second messenger molecules cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). Hence, non-invasive imaging of this enzyme class by positron emission tomography (PET) using appropriate isoform- selective PDE radioligands is gaining importance. This methodology enables the in vivo diagnosis and staging of numerous diseases associated with altered PDE density or activity in the periphery and the central nervous system as well as the translational evaluation of novel PDE inhibitors as therapeutics. In this follow-up review, we summarize the efforts in the development of novel PDE radioligands and highlight (pre-)clinical insights from PET studies using already known PDE Citation: Schröder, S.; Scheunemann, radioligands since 2016. M.; Wenzel, B.; Brust, P. Challenges on Cyclic Nucleotide Keywords: positron emission tomography; cyclic nucleotide phosphodiesterases; PDE inhibitors; Phosphodiesterases Imaging with PDE radioligands; radiochemistry; imaging; recent (pre-)clinical insights Positron Emission Tomography: Novel Radioligands and (Pre-)Clinical Insights since 2016.
    [Show full text]
  • Geochemical Influences on Nonenzymatic Oligomerization Of
    bioRxiv preprint doi: https://doi.org/10.1101/872234; this version posted December 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Geochemical influences on nonenzymatic oligomerization of prebiotically relevant cyclic nucleotides Authors: Shikha Dagar‡, Susovan Sarkar‡, Sudha Rajamani‡* ‡ Department of Biology, Indian Institute of Science Education and Research, Pune 411008, India Correspondence: [email protected]; Tel.: +91-20-2590-8061 Running title: Cyclic nucleotides and emergence of an RNA World Key words: Dehydration-rehydration cycles, lipid-assisted oligomerization, cyclic nucleotides, analogue environments Dagar, S. 1 bioRxiv preprint doi: https://doi.org/10.1101/872234; this version posted December 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract The spontaneous emergence of RNA on the early Earth continues to remain an enigma in the field of origins of life. Few studies have looked at the nonenzymatic oligomerization of cyclic nucleotides under neutral to alkaline conditions, in fully dehydrated state. Herein, we systematically investigated the oligomerization of cyclic nucleotides under prebiotically relevant conditions, where starting reactants were subjected to repeated dehydration-rehydration (DH- RH) regimes, like they would have been on an early Earth. DH-RH conditions, a recurring geological theme, are driven by naturally occurring processes including diurnal cycles and tidal pool activity. These conditions have been shown to facilitate uphill oligomerization reactions in terrestrial geothermal niches, which are hypothesized to be pertinent sites for the emergence of life.
    [Show full text]
  • Oxidative Stress and the Guanosine Nucleotide Triphosphate Pool: Implications for a Biomarker and Mechanism of Impaired Cell Function
    University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2008 OXIDATIVE STRESS AND THE GUANOSINE NUCLEOTIDE TRIPHOSPHATE POOL: IMPLICATIONS FOR A BIOMARKER AND MECHANISM OF IMPAIRED CELL FUNCTION Celeste Maree Bolin The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Bolin, Celeste Maree, "OXIDATIVE STRESS AND THE GUANOSINE NUCLEOTIDE TRIPHOSPHATE POOL: IMPLICATIONS FOR A BIOMARKER AND MECHANISM OF IMPAIRED CELL FUNCTION" (2008). Graduate Student Theses, Dissertations, & Professional Papers. 728. https://scholarworks.umt.edu/etd/728 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. OXIDATIVE STRESS AND THE GUANOSINE NUCLEOTIDE TRIPHOSPHATE POOL: IMPLICATIONS FOR A BIOMARKER AND MECHANISM OF IMPAIRED CELL FUNCTION By Celeste Maree Bolin B.A. Chemistry, Whitman College, Walla Walla, WA 2001 Dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Toxicology The University of Montana Missoula, Montana Spring 2008 Approved by: Dr. David A. Strobel, Dean Graduate School Dr. Fernando Cardozo-Pelaez,
    [Show full text]
  • The Metabolic Building Blocks of a Minimal Cell Supplementary
    The metabolic building blocks of a minimal cell Mariana Reyes-Prieto, Rosario Gil, Mercè Llabrés, Pere Palmer and Andrés Moya Supplementary material. Table S1. List of enzymes and reactions modified from Gabaldon et. al. (2007). n.i.: non identified. E.C. Name Reaction Gil et. al. 2004 Glass et. al. 2006 number 2.7.1.69 phosphotransferase system glc + pep → g6p + pyr PTS MG041, 069, 429 5.3.1.9 glucose-6-phosphate isomerase g6p ↔ f6p PGI MG111 2.7.1.11 6-phosphofructokinase f6p + atp → fbp + adp PFK MG215 4.1.2.13 fructose-1,6-bisphosphate aldolase fbp ↔ gdp + dhp FBA MG023 5.3.1.1 triose-phosphate isomerase gdp ↔ dhp TPI MG431 glyceraldehyde-3-phosphate gdp + nad + p ↔ bpg + 1.2.1.12 GAP MG301 dehydrogenase nadh 2.7.2.3 phosphoglycerate kinase bpg + adp ↔ 3pg + atp PGK MG300 5.4.2.1 phosphoglycerate mutase 3pg ↔ 2pg GPM MG430 4.2.1.11 enolase 2pg ↔ pep ENO MG407 2.7.1.40 pyruvate kinase pep + adp → pyr + atp PYK MG216 1.1.1.27 lactate dehydrogenase pyr + nadh ↔ lac + nad LDH MG460 1.1.1.94 sn-glycerol-3-phosphate dehydrogenase dhp + nadh → g3p + nad GPS n.i. 2.3.1.15 sn-glycerol-3-phosphate acyltransferase g3p + pal → mag PLSb n.i. 2.3.1.51 1-acyl-sn-glycerol-3-phosphate mag + pal → dag PLSc MG212 acyltransferase 2.7.7.41 phosphatidate cytidyltransferase dag + ctp → cdp-dag + pp CDS MG437 cdp-dag + ser → pser + 2.7.8.8 phosphatidylserine synthase PSS n.i. cmp 4.1.1.65 phosphatidylserine decarboxylase pser → peta PSD n.i.
    [Show full text]
  • Cyclic Nucleotide-Independent Forms of a Protein Kinase from Beef Heart
    Proc. Nat. Acad. Sci. USA Vol. 68, No. 4, pp. 731-735, April 1971 Interconversion of Cyclic Nucleotide-Activated and Cyclic Nucleotide-Independent Forms of a Protein Kinase from Beef Heart JACK ERLICHMAN, ALLEN H. HIRSCH, AND ORA M. ROSEN* Departments of Medicine and Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated by B. L. Horecker, January 14, 1971 ABSTRACT A protein kinase activated by cyclic nucle- presence. This observation suggests that cyclic AMP activates otides was purified from beef heart. Upon exposure to the protein kinase by relieving an inhibition brought about adenosine 3': 5'-cyclic monophosphate (cyclic AMP) during kinase with a AMP- gel filtration on Sephadex G-200, the protein kinase dis- by the association of the protein cyclic sociated into a cyclic nucleotide-independent protein binding protein. A similar mechanism for the regulation of kinase and a cyclic nucleotide-binding protein. A similar protein kinases from skeletal muscle and parotid gland has or identical cyclic nucleotide-independent protein kinase also been suggested (Z. Sellinger and M. Schramm, personal could be obtained in highly purified form by elution from communication). Tao et al. (15) reported that a cyclic AMP- a DEAE-cellulose column with 10-6 M cyclic AMP; the cyclic AMP-binding protein was apparently retained by dependent protein kinase from rabbit reticulocytes was dis- the resin. The addition of cyclic nucleotide-binding pro- sociated into two subunits in the presence of cyclic AMP. tein to cyclic nucleotide-independent protein kinas e One subunit bound cyclic AMP and lacked catalytic activity, resulted in the reappearance of cyclic nucleotide-depen- whereas the other subunit possessed kinase activity and dent protein kinase, which could be isolated by filtration on nucleotides.
    [Show full text]
  • New Advances in Cyclic AMP Signalling”—An Editorial Overview
    cells Editorial Special Issue on “New Advances in Cyclic AMP Signalling”—An Editorial Overview Stephen John Yarwood Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Edinburgh Campus, Heriot-Watt University, Edinburgh EH14 4AS, UK; [email protected] Received: 9 October 2020; Accepted: 10 October 2020; Published: 12 October 2020 Abstract: The cyclic nucleotides 30,50-adenosine monophosphate (cyclic AMP) signalling system underlies the control of many biological events and disease processes in man. Cyclic AMP is synthesised by adenylate cyclase (AC) enzymes in order to activate effector proteins and it is then degraded by phosphodiesterase (PDE) enzymes. Research in recent years has identified a range of cell-type-specific cyclic AMP effector proteins, including protein kinase A (PKA), exchange factor directly activated by cyclic AMP (EPAC), cyclic AMP responsive ion channels (CICs), and the Popeye domain containing (POPDC) proteins, which participate in different signalling mechanisms. In addition, recent advances have revealed new mechanisms of action for cyclic AMP signalling, including new effectors and new levels of compartmentalization into nanodomains, involving AKAP proteins and targeted adenylate cyclase and phosphodiesterase enzymes. This Special Issue contains 21 papers that highlight advances in our current understanding of the biology of compartmentlised cyclic AMP signalling. This ranges from issues of pathogenesis and associated molecular pathways, functional assessment of novel nanodomains, to the development of novel tool molecules and new techniques for imaging cyclic AMP compartmentilisation. This editorial aims to summarise these papers within the wider context of cyclic AMP signalling. Keywords: cyclic AMP; EPAC; PKA; POPDC; AKAP; adenylate cyclase; cyclic AMP phosphodiesterases; GPCRs 1.
    [Show full text]
  • Relationship Between the Levels of Cyclic Cytidine 3':5'-Monophosphate
    [CANCER RESEARCH 41, 3222-3227, August 1981] 0008-5472/81 /0041 -OOOOS02.00 Relationship between the Levels of Cyclic cytidine 3':5'-Monophosphate, Cyclic Guanosine 3':5'-Monophosphate, and Cyclic Adenosine 3':5'- Monophosphate in Urines and Leukocytes and the Type of Human Leukemias1 Joëlle Scavennec,2 Yves Carcassonne, Jean-Albert Gastaut, AndréBlanc, and HélèneL.Cailla3 Centre ¿Immunologie INSERM-CNRS de Marsei/le-Luminy. Case 906, 13288 Marseille cédex9[J. S.. H. L. C.], and Clinique des Maladies du Sang, Hôpital de la Conception, 13005 Marseille [A. B., J-A. G., Y. C.], France ABSTRACT Since then, specific cyclic CMP phosphodiesterase has been described (10, 13), but an enzymatic activity converting CTP Cyclic cytidine 3':5'-monophosphate (cyclic CMP), cyclic into cyclic CMP has not yet been clearly established. Little is guaríosme 3':5'-monophosphate (cyclic GMP), and cyclic known about cyclic CMP itself because no quantitative assay adenosine 3':5'-monophosphate (cyclic AMP) contents of leu was available until we developed a sensitive radioimmunoassay kocytes and urines of leukemic patients have been investi for cyclic CMP based on our previous work with cyclic AMP gated. We have studied four types of leukemia: acute myelo- and cyclic GMP radioimmunoassays (6, 7). blastic leukemia; chronic myelocytic leukemia; acute lympho- This new tool enabled us to study the cyclic CMP content in blastic leukemia; and chronic lymphocytic leukemia. As con urine and blood cells of patients with leukemia in an attempt to trols, the cyclic nucleotide content of leukocytes and urines of correlate the cyclic CMP concentration with the type or the healthy volunteers and patients with solid tumors selected for stage of leukemia.
    [Show full text]
  • Cyclic Nucleotides As Mediators of Acinar and Ductal Function Maria E
    Cyclic Nucleotides as Mediators of Acinar and Ductal Function Maria E. Sabbatini, PhD Augusta University e-mail: [email protected] Version 1.0, March 17, 2016 [DOI: 10.3998/panc.2016.2] 1. Cyclic Nucleotides and their pyrophosphate. This cyclic conformation allows Biosynthesis cyclic nucleotides to bind to proteins to which other nucleotides cannot. The reaction is an intracellular 2+ Cyclic nucleotides, like other nucleotides, are nucleophilic catalysis and requires Mg as a composed of three functional groups: a ribose cofactor, whose function is to decrease the overall sugar, a nitrogenous base, and a single phosphate negative charge on the ATP by complexing with group. There are two types of nitrogenous bases: two of its negatively charged oxygens. If its purines (adenine and guanine) and pyrimidines negative charge is not reduced, the nucleotide (cytosine, uracil and thymine). A cyclic nucleotide, triphosphate cannot be approached by a unlike other nucleotides, has a cyclic bond nucleophile, which is, in this reaction, the 3’ arrangement between the ribose sugar and the hydroxyl group of the ribose (162). Soluble AC 2+ 2+ phosphate group. There are two main groups of prefers Ca to Mg as the coenzyme to coordinate cyclic nucleotides: the canonical or well-stablished ATP binding and catalysis (135). and the non-canonical or unknown function cyclic nucleotides. The two well-established cyclic 2. Canonical Cyclic Nucleotide nucleotides are adenosine 3’,5’-cyclic Signaling in the Exocrine Pancreas monophosphate (cyclic AMP) and guanine-3’,5’- cyclic monophosphate (cyclic GMP). Both cyclic Cyclic nucleotide signaling can be initiated by two AMP and cyclic GMP are second messengers.
    [Show full text]
  • Molecular Diagnosis for Heterogeneous Genetic Diseases
    Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA sequencing applied to retinitis pigmentosa David Arthur Simpson, Graeme Richard Clark, Sharon Alexander, Giuliana Silvestri, Colin Eric Willoughby To cite this version: David Arthur Simpson, Graeme Richard Clark, Sharon Alexander, Giuliana Silvestri, Colin Eric Willoughby. Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA se- quencing applied to retinitis pigmentosa. Journal of Medical Genetics, BMJ Publishing Group, 2010, 48 (3), pp.145. 10.1136/jmg.2010.083568. hal-00591224 HAL Id: hal-00591224 https://hal.archives-ouvertes.fr/hal-00591224 Submitted on 8 May 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. TITLE PAGE Molecular diagnosis for heterogeneous genetic diseases with targeted high- throughput DNA sequencing applied to retinitis pigmentosa David A. Simpson, Graeme R Clark, Sharon Alexander, Giuliana Silvestri, Colin E. Willoughby Corresponding author: David Arthur Simpson Queen's University Belfast, Centre for Vision & Vascular
    [Show full text]
  • Properties of Cyclic Nucleotide-Gated Channels Mediating Olfactory Transduction
    Published July 1, 1992 Properties of Cyclic Nucleotide-gated Channels Mediating Olfactory Transduction Activation, Selectivity, and Blockage STEPHAN FRINGS, JOSEPH W. LYNCH, and BERND LINDEMANN Downloaded from From the Department of Physiology, Universit~it des Saarlandes, D-6650 Homburg/Saar, Germany ABSTRACT Cyclic nucleotide-gated channels (cng channels) in the sensory mem- brane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical jgp.rupress.org excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 ~M for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior on October 21, 2015 identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage depen- dent. Several blockers of Ca 2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respec- tively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV.
    [Show full text]