A Saccharomyces cerevisiae Assay System to Investigate Ligand/AdipoR1 Interactions That Lead to Cellular Signaling
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Type
ArticleAuthors
Aouida, Mustapha
Kim, Kangchang
Shaikh, Abdul Rajjak
Pardo, Jose M.
Eppinger, Jörg

Yun, Dae-Jin
Bressan, Ray Anthony
Narasimhan, Meena L.
KAUST Department
Academic AffairsBiological & 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
2013-06-07Permanent link to this record
http://hdl.handle.net/10754/325317
Metadata
Show full item recordAbstract
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 ONEPubMed ID
23762377PubMed Central ID
PMC3676391ae974a485f413a2113503eed53cd6c53
10.1371/journal.pone.0065454
Scopus Count
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