G protein-coupled receptors (GPCRs) are the largest integral membrane protein family in the human genome. These receptors function to transduce signals across the plasma membrane allowing cells to respond and adapt to a variety of different stimuli, such as light, odorants, hormones and many other signaling molecules. This diverse family of receptors is intimately associated with a variety of disease states, including cancer and diabetes, making them attractive targets for drug therapy; it is estimated that 30-50% of prescribed medications target pharmaceutically relevant GPCRs.
The aim of this proposal is to identify intracellular proteins that regulate and modulate Ste2p, a yeast receptor that has been used widely as a model for GPCR structure and function. We propose to bridge a large gap in our knowledge of the initial events of GPCR-mediated signal transduction by identifying the quiesome (proteins associated with the receptor in the quiescent or resting state) and the signalosome (intracellular proteins which are recruited and interact with GPCRs upon activation). To accomplish these aims, we will utilize unnatural amino acid replacement to genetically modify Ste2p to contain benzoylphenylalanine (Bpa), a photoactivatable, unnatural amino acid to allow the capture of proteins associated with the intracellular domains of Ste2p in intact, living cells in the presence and absence of its peptide ligand. Our hypothesis is that specific proteins interact with intracellular domains of Ste2p, that these proteins can be captured by the use of laser activation of Bpa in order to generate a time-resolved picture of the intracellular protein partners which will be identified by mass spectrometry, and that our methods using live cells, laser activation will yield a more accurate and dynamic account of GPCR protein partners compared to other methods used to date such as immunoprecipitation. It is expected that the experimental techniques proposed herein will be applicable to medically important GPCRs and that the description of the quiesome and signalosome will provide new targets for drug design leading to new and effective therapies for diseases involving GPCRs.

Public Health Relevance

G protein-coupled receptors (GPCRs) are the target of 30-50% of all currently prescribed drugs and are implicated in various pathologies, including diabetes and cancer. Identification of the intracellular protein partners that interact with GPCRs in the resting and activated state will fill fundamental gaps in understanding GPCR signaling and regulation and will facilitate the design and synthesis of medicines to treat many diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM112496-02
Application #
8898156
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Dunsmore, Sarah
Project Start
2014-09-01
Project End
2018-04-30
Budget Start
2015-05-01
Budget End
2016-04-30
Support Year
2
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Tennessee Knoxville
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
003387891
City
Knoxville
State
TN
Country
United States
Zip Code
37996
Hauser, Melinda; Qian, Chen; King, Steven T et al. (2018) Identification of peptide-binding sites within BSA using rapid, laser-induced covalent cross-linking combined with high-performance mass spectrometry. J Mol Recognit 31:
Uddin, M Seraj; Hauser, Melinda; Naider, Fred et al. (2016) The N-terminus of the yeast G protein-coupled receptor Ste2p plays critical roles in surface expression, signaling, and negative regulation. Biochim Biophys Acta 1858:715-24
Cai, Houjian; Hauser, Melinda; Naider, Fred et al. (2016) Halo Assay for Toxic Peptides and Other Compounds in Microorganisms. Bio Protoc 6:
Rymer, Jeffrey K; Hauser, Melinda; Bourdon, Allen K et al. (2015) Novobiocin and peptide analogs of ?-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p. Biochim Biophys Acta 1848:916-24