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Biochem Biophys Res Commun. 2014 Mar 21;445(4):746-56. doi: 10.1016/j.bbrc.2014.02.033. Epub 2014 Feb 19.

CHIP-MYTH: a novel interactive proteomics method for the assessment of agonist-dependent interactions of the human β₂-adrenergic receptor.

Author information

1
Donnelly Centre, University of Toronto, Ontario, Canada.
2
Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
3
Princess Margaret Cancer Centre, University Health Network, University of Toronto, Ontario, Canada.
4
Princess Margaret Cancer Centre, University Health Network, University of Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Ontario, Canada; Department of Computer Science, University of Toronto, Ontario, Canada.
5
Department of Pharmaceutical Sciences, 2405 Wesbrook Mall, University of British Columbia, Vancouver, Canada.
6
Donnelly Centre, University of Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Ontario, Canada; Department of Biochemistry, University of Toronto, Ontario, Canada. Electronic address: igor.stagljar@utoronto.ca.

Abstract

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human β2-adrenergic receptor (β2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting β2AR-agonist. Our results suggest that β2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαβγ, into Gα and Gβγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel β2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.

KEYWORDS:

G-protein coupled receptors; LUMIER assay; drug-dependent protein interactions; high-throughput screening; membrane two-hybrid assay; protein-protein interactions

PMID:
24561123
DOI:
10.1016/j.bbrc.2014.02.033
[Indexed for MEDLINE]
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