A Saccharomyces cerevisiae assay system to investigate ligand/adipoR1 interactions that lead to cellular Signaling
Autor: | Dae-Jin Yun, Ray A. Bressan, Mustapha Aouida, Meena L. Narasimhan, Kangchang Kim, Abdul Rajjak Shaikh, José M. Pardo, Jörg Eppinger |
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Jazyk: | angličtina |
Rok vydání: | 2013 |
Předmět: |
Mitochondrial Diseases
Anatomy and Physiology Yeast and Fungal Models Ligands Biochemistry Endocrinology Genes Reporter Luciferases Firefly Gene Expression Regulation Fungal Drug Discovery Molecular Cell Biology Membrane Receptor Signaling Phosphorylation Promoter Regions Genetic Plant Proteins Multidisciplinary Signal transducing adaptor protein Hormone Receptor Signaling Ligand (biochemistry) Molecular Docking Simulation Pleckstrin homology domain Eukaryotic Cells Medicine Biological Assay Adiponectin Receptors Adiponectin Cellular Types Signal transduction Signal Transduction Research Article Biotechnology Cell signaling Science Endocrine System Saccharomyces cerevisiae Protein Serine-Threonine Kinases Biology Model Organisms Genetics Luciferase Protein kinase A Diabetic Endocrinology Endocrine Physiology Cell Membrane Human Genetics Diabetes Mellitus Type 1 Antigens Plant Diabetes Mellitus Type 2 Fusion protein Hormones Diabetes Mellitus and Deafness Peptides |
Zdroj: | PLoS ONE, Vol 8, Iss 6, p e65454 (2013) Digital.CSIC. Repositorio Institucional del CSIC instname PLoS ONE |
Popis: | 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. |
Databáze: | OpenAIRE |
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