1887

Abstract

The anti‐diabetic and insulin‐sensitizing effects of adiponectin, a hormone secreted by adipose tissue, are mediated by its molecular interactions with the plasma membrane receptors AdipoR1 and AdipoR2. Adiponectin occurs in several molecular forms and each of these forms has a specific signaling outcome that also depends on the relative abundance of the two receptors. The complex interactions between adiponectin and its receptors are poorly understood. A detailed characterization of how adiponectin interacts with its receptors and, in turn, how these receptors transmit specific downstream signals is critical for the development of meaningful therapeutic interventions for a wide spectrum of metabolic disorders (including type 2 diabetes, obesity, and inflammatory bowel disease) and cancers (of the breast, colon and prostate), all of which are known to be associated with adiponectin function. 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 promises to be an effective tool for the identification of novel pharmaceutically active AdipoR agonists and should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.

Loading

Article metrics loading...

/content/papers/10.5339/qfarf.2013.BIOP-050
2013-11-20
2020-10-21
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/papers/10.5339/qfarf.2013.BIOP-050
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error