title: A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
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.
authors.affiliation: Plant Stress Genomics Research Center, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia.
authors.forename: Mustapha
authors.initials: M
authors.lastname: Aouida
authors.forename: Kangchang
authors.initials: K
authors.lastname: Kim
authors.forename: Abdul Rajjak
authors.initials: AR
authors.lastname: Shaikh
authors.forename: Jose M
authors.initials: JM
authors.lastname: Pardo
authors.forename: Jörg
authors.initials: J
authors.lastname: Eppinger
authors.forename: Dae-Jin
authors.initials: DJ
authors.lastname: Yun
authors.forename: Ray A
authors.initials: RA
authors.lastname: Bressan
authors.forename: Meena L
authors.initials: ML
authors.lastname: Narasimhan
mesh_terms: D052242:Adiponectin / Q000378:metabolism*; D052179:Antigens, Plant / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D017930:Genes, Reporter; D008024:Ligands; D049409:Luciferases, Firefly / Q000378:metabolism; D062105:Molecular Docking Simulation; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010940:Plant Proteins / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D054419:Receptors, Adiponectin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C475418:ADIPOR1 protein, human; D052242:Adiponectin; D052179:Antigens, Plant; D008024:Ligands; D010455:Peptides; D010940:Plant Proteins; D054419:Receptors, Adiponectin; C073139:osmotin protein, Nicotiana tabacum; D049409:Luciferases, Firefly; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases