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    A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling

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    Type
    Article
    Authors
    Aouida, Mustapha cc
    Kim, Kangchang
    Shaikh, Abdul Rajjak
    Pardo, Jose M.
    Eppinger, Jörg cc
    Yun, Dae-Jin
    Bressan, Ray Anthony
    Narasimhan, Meena L.
    KAUST Department
    Academic Affairs
    Biological & Organometallic Catalysis Laboratories
    Biological and Environmental Science and Engineering (BESE) Division
    Center for Desert Agriculture
    Chemical Science Program
    KAUST Catalysis Center (KCC)
    Office of the VP
    Physical Science and Engineering (PSE) Division
    Plant Stress Genomics Research Lab
    Date
    2013-06-07
    Permanent link to this record
    http://hdl.handle.net/10754/325317
    
    Metadata
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    Abstract
    Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides' ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology. 2013 Aouida et al.
    Citation
    Aouida M, Kim K, Shaikh AR, Pardo JM, Eppinger J, et al. (2013) A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling. PLoS ONE 8: e65454. doi:10.1371/journal.pone.0065454.
    Publisher
    Public Library of Science (PLoS)
    Journal
    PLoS ONE
    DOI
    10.1371/journal.pone.0065454
    PubMed ID
    23762377
    PubMed Central ID
    PMC3676391
    ae974a485f413a2113503eed53cd6c53
    10.1371/journal.pone.0065454
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Physical Science and Engineering (PSE) Division; Chemical Science Program; KAUST Catalysis Center (KCC); Center for Desert Agriculture

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