Role of Metallothionein in Nitric Oxide Signaling as Revealed by a Green Fluorescent Fusion Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 1; pp. 477 - 482
• Main Authors: Pearce, Linda L., Gandley, Robin E., Han, Weiping, Wasserloos, Karla, Stitt, Molly, Kanai, Anthony J., McLaughlin, Margaret K., Pitt, Bruce R., Levitan, Edwin S.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 04.01.2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: 3T3 cells; Animals; Arginine - pharmacology; Biological Sciences; Blood vessels; Calcium - metabolism; Complementary DNA; Endothelial cells; Endothelium, Vascular - metabolism; Energy transfer; Fluorescence; Free radicals; Glutathione - analogs & derivatives; Glutathione - metabolism; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Kinetics; Luminescent Proteins - genetics; Male; Mesenteric Arteries; Metallothionein - genetics; Metallothionein - metabolism; Mice; Mice, Inbred Strains; Mice, Knockout; NG-Nitroarginine Methyl Ester - pharmacology; Nitric Oxide - metabolism; Nitric Oxide Synthase - antagonists & inhibitors; Nitroso Compounds - metabolism; Oxides; Pharmacology; Physiological regulation; Protein Conformation; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; S-Nitrosoglutathione; Signal Transduction; Spectrometry, Fluorescence; Zinc
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.1.477

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca2+act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT-/-) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.