title: CHIP-MYTH: a novel interactive proteomics method for the assessment of agonist-dependent interactions of the human β₂-adrenergic receptor.
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.
authors.affiliation: Donnelly Centre, University of Toronto, Ontario, Canada.
authors.forename: Saranya
authors.initials: S
authors.lastname: Kittanakom
authors.affiliation: Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Miriam
authors.initials: M
authors.lastname: Barrios-Rodiles
authors.affiliation: Donnelly Centre, University of Toronto, Ontario, Canada.
authors.forename: Julia
authors.initials: J
authors.lastname: Petschnigg
authors.affiliation: Donnelly Centre, University of Toronto, Ontario, Canada.
authors.forename: Anthony
authors.initials: A
authors.lastname: Arnoldo
authors.affiliation: Donnelly Centre, University of Toronto, Ontario, Canada.
authors.forename: Victoria
authors.initials: V
authors.lastname: Wong
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, University of Toronto, Ontario, Canada.
authors.forename: Max
authors.initials: M
authors.lastname: Kotlyar
authors.affiliation: Donnelly Centre, University of Toronto, Ontario, Canada.
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Heisler
authors.affiliation: 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.
authors.forename: Igor
authors.initials: I
authors.lastname: Jurisica
authors.affiliation: Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Wrana
authors.affiliation: Department of Pharmaceutical Sciences, 2405 Wesbrook Mall, University of British Columbia, Vancouver, Canada.
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
authors.affiliation: 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.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D000420:Albuterol / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D000068299:Salmeterol Xinafoate; D015398:Signal Transduction / Q000187:drug effects; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D025801:Ubiquitin; D000068299:Salmeterol Xinafoate; D000420:Albuterol
keywords: G-protein coupled receptors; LUMIER assay; drug-dependent protein interactions; high-throughput screening; membrane two-hybrid assay; protein-protein interactions