Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

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THE KARNIKLABORATORYRESEARCH ASSOCIATESupriya Patil, Ph.D.POSTDOCTORAL FELLOWSAditi Bandyopadhyay, Ph.D.Camelia Gogonea, Ph.D.Yasser Saad, Ph.D.Takanobu Takazako, M.D., Ph.D.TECHNICAL ASSISTANTAnita Shukla, M.S.COLLABORATORSChristine Schomisch Moravec, Ph.D. 1Walter G. Thomas, Ph.D. 21Dept. of Cardiology and Mol.Cardiology2Baker Medical Research Inst.,Melbourne, AustraliaOur research focuses on analyzing thestructure-function and signal transductionmechanisms of angiotensin II (Ang II)receptors. Ang II, an octapeptide hormone, mediatesblood-pressure regulation, salt-water balance, steroidgenesis, reactive oxygen production, proliferativeresponse, and matrix deposition. The AT 1and AT 2isoforms of Ang II receptors, members of the G-protein-coupled receptor (GPCR) superfamily,mediate these responses. In addition to G-proteincoupledreceptor responses, the AT 1receptor initiatesintracellular signal transduction pathways that areusually activated by cytokine and growth-factorreceptors. The AT 2receptor mediates differentiation,inhibition of growth and cell apoptosis.The AT 1receptor is the prototype for peptidehormone GPCRs. Its cytoplasmic domain binds andactivates the G-protein and interacts with differentkinases and adapter proteins that act as signalingplatforms. The extracellular domain, in combinationwith the seven transmembrane (7TM) helical bundle,binds the hormone and selective drugs that areclinically important. The ligand/G-protein interactionconforms to allosteric regulation across the membranebarrier. This general mechanism for transduction ofsignals by the 7TM structural motif is important forthe actions of therapeutic drugs and pathologic agentstargeting GPCRs. Therefore, elucidating theintramolecular communication between the twofunctional domains situated on opposite sides of themembrane is vital.Our current studies of the AT 1receptor areaimed at elucidating, at the molecular level, thefollowing: (i) the receptor’s interactions with Ang II,(ii) the mechanism of receptor inhibition by differentclasses of Ang II-receptor blockers, (iii) specificconformational changes that govern AT 1receptoractivation,and (iv) interactions of G-protein withthe activated AT 1receptor. To understand the in vivoconsequences of “constitutive activation” of the AT 1receptor, we have developed transgenic mousemodels expressing wild-type and constitutivelyThe Department of Molecular CardiologyAnalogs, Mutant Receptors Help DefineAng II Activation Mechanismsactivated mutant AT 1receptors. These models are usedfor: (i) evaluation of inverse agonists and antagonistsof the AT 1receptors, (ii) study of growth-factor andcytokine changes induced during AT 1receptormediatedhypertrophy, (iii) profiling of gene expressionchanges associated with hypertrophy, and (v)evaluation of dominant-negative AT 1receptormutants.The AT 2receptor is an important regulator ofphysiological ontogenesis in the developing fetus and inadult tissue-remodeling. Familial mutations in the AT 2receptor gene cause congenital abnormality of kidneyand urinary tract in humans. The AT 2receptor can beupregulated by physiological and pathological stimuli infailing and infarcted hearts, in neointima formationafter vascular injury, in atretic ovarian follicles, inuterine endometrium and in healing skin wounds. Mostoften, the high levels of AT 2receptor re-expression arelocalized to remodeling sites. We have shown that denovo overexpression of the AT 2receptor inducesapoptosis through activation of caspase and p38MAPK. The AT 2receptor cytoplasmic domaininteracts with novel adapter molecules to induceapoptosis. The goal of AT 2receptor research is toidentify the components and signal transductionmechanisms leading to apoptosis. We will use theprotein-interaction analysis by mass spectrometry toaccomplish this goal.Molecular variants of the AT 1and AT 2receptors and their role in the human cardiovasculardisease process are not fully defined. We haveundertaken a genetic analysis of the AT 1and AT 2receptor loci. Direct sequencing and genotyping of AT 1and AT 2receptor genes, in diseased and non-diseasedhuman subjects, was undertaken. The genomicsequence provided the genotypic and allelic frequencydata for the single-nucleotide polymorphisms that arenecessary for association studies. Functional studies todetermine the identity of the genetic variantsassociated with disease states and the molecularmechanisms by which genetic variants influencereceptor expression and function are in progress.Miura, S., and S.S. Karnik (2000) Ligand-independent signals from the angiotensin II type-2 receptor induce apoptosis.EMBO J. 19:4026-4035.Karnik, S.S. (2002) Analysis of structure-function from expression of G protein-coupled receptor fragments. Meth.Enzymol. 343:248-259.Sadashiva S. Karnik, Ph.D.Holloway, A.C., Qian, H., Pipolo, L., Ziogas, J., Miura, S., Southwell, B.R., Lew, M.J., Thomas, W.G., and S.S. Karnik(2002) Side-chain substitution within angiotensin II reveal different requirements for signaling, internalization andphosphorylation of type 1A angiotensin receptors. Mol. Pharmacol. 61:768-777.Miura, S., and S.S. Karnik (2002) Constitutive activation of angiotensin II type 1 receptor alters the orientation oftransmembrane Helix-2. J. Biol. Chem. 277:24299-24305.Saad, Y., Durkin, S., Hwang, J.Y., Boros, J., Moravec, C.S., and S.S. Karnik (2002) Haplotype variation at the humanangiotensin II receptor loci. In: Stillman, B., ed. Abstracts of papers presented at the LXVII Cold Spring HarborSymposium on Quantitative Biology: May 29-June 3, 2002. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory,p. 105.Miura, S., Zhang, J., Boros, J., and S.S. Karnik (2003) TM2-TM7 interaction in coupling movement of transmembranehelices to activation of the angiotensin II type-1 receptor. J. Biol. Chem. 278:3720-3725.124
Atrial natriuretic peptide (ANP) is a hormonesecreted by the heart in response toelevation in blood pressure and volume.ANP stimulates excretion of salt (natriuresis) andfluid (diuresis) and dilates arterial blood vessels,thereby exerting hypotensive effects. ANP alsosuppresses actions of neuronal and hormonalfactors that cause high bloodpressure. Thereby, ANP plays amajor role in regulating bloodpressure and body-fluid volumehomeostasis.ANP’s hormonal actionsare mediated by the cell surfacereceptor that carries intrinsicguanylyl cyclase (GCase)activity. Binding of ANPactivates GCase catalysis andelevates intracellular cGMPlevels. In turn, cGMP mediateshormonal actions of ANPthrough cGMP-regulated ionchannels,protein kinases, andphosphodiesterases. The ANPreceptor is a single-spantransmembrane receptor andoccurs as a dimer of atransmembrane polypeptidecontaining an extracellularANP-binding domain and anintracellular domain consistingof an ATP-binding regulatorydomain and a GCase catalyticdomain. ANP binding to the extracellular domainactivates GCase catalysis by an as yet unknownmechanism. The ANP receptor belongs to thefamily of membrane-bound, GCase-coupledreceptors having a similar molecular topology and,presumably, a similar signaling mechanism. TheseGCase-coupled receptors belong to the superfamilyof single-span transmembrane receptors, for whichthe mechanism of signal transduction has not beenwell defined.Our goal is to determine the ANPreceptor’s structure and to elucidate the mechanismsof hormone-binding and transmembranesignaling at atomic resolutions. We applystructural biology (X-ray crystallography), proteinbiochemistry, molecular biology, and biophysicsThe Department of Molecular CardiologyStructure and Transmembrane SignalingMechanism of the ANP Receptor:Development of Drugsand More Effective Treatmentstechniques to pursue our research goals.Our recent studies include identification of aconserved structural motif in the ANP receptor,termed GCase-signaling motif; expression andpurification of the hormone-binding domain(ANPR); determination of its complete covalentstructure by LC/MS; discovery of the chloridedependenceof ANPbinding; and determinationof ANP binding andreceptor dimerizationequilibria. We have alsodetermined the X-raystructures of the apo- andANP-bound ANPRcomplex and haveproposed a novel structuralmechanism for transmembranesignal transductionby the ANP receptor(submitted).Besides its majorrole in blood pressure andvolume regulation, ANP isinvolved in the pathogenesisof hypertension, heartfailure, and othercardiovascular diseases.Thus, the ANP receptorpresents an ideal target fordrugs used in treating suchdiseases. Elucidation ofthe ANP receptor’sstructure and mechanism of signaling will allowrational drug design. These studies will also give usbetter understanding of the pathophysiologicalroles of ANP in the cardiovascular system andpromote development of more effective diagnosisand treatment protocols.Kunio S. Misono, Ph.D.THE MISONOLABORATORYINVESTIGATORSP. Haruo Ogawa, Ph.D.Yue Sally Qiu, M.D.Xiaolun Zhang, M.S.COLLABORATORSEwa Folta-Stogniew, Ph.D. 1Earnest Freire, Ph.D. 2Craig Ogata, Ph.D. 3John Philo, Ph.D. 4Kenneth Williams, Ph.D. 11Dept. of Biochem. andBiophys., Yale Univ., NewHaven, CT2Dept. of Biology, JohnsHopkins Univ., Baltimore, MD3Argonne Natl. Laboratory,Chicago, IL4Alliance Protein Laboratories,Inc., Thousand Oaks, CAMisono, K.S., Sivasubramanian, N., Berkner, K., and X. Zhang, (1999) Expression andpurification of the extracellular ligand-binding domain of the atrial natriuretic peptide(ANP) receptor. Biochemistry, 38:516-523.Misono, K.S. (2000) Atrial natriuretic factor binding to its receptor is dependent onchloride concentration: A possible feedback-control mechanism in renal salt regulation.Circ. Res. 86:1135-1139.van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K.S., and V.C. Yee (2000)Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupledreceptor. Nature 406:101-104.Misono, K.S. (2002) Natriuretic peptide receptor: structure and signaling (Review). Mol.Cell. Biochem. 230:49-60.Ogawa, H., Zhang, X., Qiu, Y., Ogata, C., and K.S. Misono (2003). Structuralmechanism for transmembrane signaling by the atrial natriuretic peptide receptor(Submitted).125
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