title: Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells.
abstract: The endothelial isoform of nitric oxide synthase (eNOS), a key regulator of vascular tone, is activated in endothelial cells by diverse Ca(2+)-mobilizing agonists, including vascular endothelial growth factor (VEGF). Although the activation state of eNOS and the subcellular localization of the enzyme are both highly regulated, the relationship between enzyme activity and subcellular targeting remains obscure. We aim here to elucidate this relationship by direct dynamic imaging analysis of Ca(2+)/CaM-dependent eNOS activation in living endothelial cells, using high-resolution confocal microscopy and donor dequenching fluorescence resonance energy transfer (FRET) techniques. Confocal images show a complex pattern of eNOS subcellular distribution; the enzyme is concentrated in both the plasma membrane and internal membranes, with robust expression in the perinuclear region. We construct a fusion protein between eNOS and the FRET-based calcium sensor cameleon, and analyze the temporal and spatial pattern of VEGF-mediated calcium mobilization using donor dequenching FRET methods. We find that VEGF promotes rapid mobilization of intracellular calcium throughout the regions of the cell in which eNOS is distributed. We further create a series of fusion proteins and use FRET imaging methods to study the interactions between eNOS and its obligate allosteric activator protein calmodulin. We clone the FRET acceptor EYFP (enhanced yellow fluorescent protein) at the C-terminus of calmodulin, and the FRET donor ECFP (enhanced cyan fluorescent protein) into eNOS at a site adjacent to its calmodulin-binding domain. FRET imaging analysis of individual endothelial cells cotransfected with eNOS-ECFP and calmodulin-EYFP shows that VEGF induces interactions between eNOS and calmodulin wherever both are present in the cell. Our studies provide evidence that the pool of rapidly responsive receptor-activated eNOS is distributed throughout endothelial cells in both plasma membrane and internal membrane structures, and that this distribution parallels the localization of agonist-induced intracellular Ca(2+) changes in the vicinity of eNOS.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Jobin
authors.forename: Hongjie
authors.initials: H
authors.lastname: Chen
authors.forename: Alison J
authors.initials: AJ
authors.lastname: Lin
authors.forename: Patrick W
authors.initials: PW
authors.lastname: Yacono
authors.forename: Junsuke
authors.initials: J
authors.lastname: Igarashi
authors.forename: Thomas
authors.initials: T
authors.lastname: Michel
authors.forename: David E
authors.initials: DE
authors.lastname: Golan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D019556:COS Cells; D020013:Calcium Signaling / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D029721:Drosophila Proteins / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D049452:Green Fluorescent Proteins; D007424:Intracellular Fluid / Q000201:enzymology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011956:Receptors, Cell Surface / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D029721:Drosophila Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C473067:cameleon 2.1 protein, Drosophila; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III