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
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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