A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

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...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Mustapha Aouida, Kangchang Kim, Abdul Rajjak Shaikh, Jose M Pardo, Jörg Eppinger, Dae-Jin Yun, Ray A Bressan, Meena L Narasimhan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/0186600cc7d64a348ac6ad9fe6edce28
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:0186600cc7d64a348ac6ad9fe6edce28
record_format	dspace
spelling	oai:doaj.org-article:0186600cc7d64a348ac6ad9fe6edce282021-11-18T07:42:37ZA Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.1932-620310.1371/journal.pone.0065454https://doaj.org/article/0186600cc7d64a348ac6ad9fe6edce282013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23762377/?tool=EBIhttps://doaj.org/toc/1932-6203Adiponectin 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.Mustapha AouidaKangchang KimAbdul Rajjak ShaikhJose M PardoJörg EppingerDae-Jin YunRay A BressanMeena L NarasimhanPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e65454 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Mustapha Aouida Kangchang Kim Abdul Rajjak Shaikh Jose M Pardo Jörg Eppinger Dae-Jin Yun Ray A Bressan Meena L Narasimhan A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
description	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.
format	article
author	Mustapha Aouida Kangchang Kim Abdul Rajjak Shaikh Jose M Pardo Jörg Eppinger Dae-Jin Yun Ray A Bressan Meena L Narasimhan
author_facet	Mustapha Aouida Kangchang Kim Abdul Rajjak Shaikh Jose M Pardo Jörg Eppinger Dae-Jin Yun Ray A Bressan Meena L Narasimhan
author_sort	Mustapha Aouida
title	A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
title_short	A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
title_full	A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
title_fullStr	A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
title_full_unstemmed	A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.
title_sort	saccharomyces cerevisiae assay system to investigate ligand/adipor1 interactions that lead to cellular signaling.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/0186600cc7d64a348ac6ad9fe6edce28
work_keys_str_mv	AT mustaphaaouida asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT kangchangkim asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT abdulrajjakshaikh asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT josempardo asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT jorgeppinger asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT daejinyun asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT rayabressan asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT meenalnarasimhan asaccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT mustaphaaouida saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT kangchangkim saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT abdulrajjakshaikh saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT josempardo saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT jorgeppinger saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT daejinyun saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT rayabressan saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling AT meenalnarasimhan saccharomycescerevisiaeassaysystemtoinvestigateligandadipor1interactionsthatleadtocellularsignaling
_version_	1718423075602563072

A Saccharomyces cerevisiae assay system to investigate ligand/AdipoR1 interactions that lead to cellular signaling.

Ejemplares similares