Peptide hormones control a myriad of important and essential biological processes in all organisms and tissues. Mutations in genes encoding hormone receptors lead to many pathological conditions in humans, and hormone receptors are the target of a majority of pharmacological agents used in medicine today. Despite major efforts by many laboratories, there remains a lack of understanding of the mode of action of peptide hormones. We propose to continue to use the powerful model system of alpha-factor, a tridecapeptide Saccharomyces cerevisiae pheromone, interacting with its receptor Ste2p, a member of the ubiquitous seven transmembrane domain, G protein-coupled receptor (GPCR) family to address three major aspects of hormone-receptor biochemistry: (i) the makeup of a peptide binding site within a GPCR, (it) the structure of the receptor as deduced from its synthetic fragments, and (iii) the conformational changes in a receptor initiated by activation due to ligand binding or mutation to constitutive activity.
These aims will be accomplished using chemical, biochemical, biophysical, and molecular biological approaches. For example, we propose to synthesize free, cyclized, and anchorable extracellular domains of Ste2p and to biosynthesize receptor fragments containing multiple transmembrane domains and loops. The conformation of these receptor domains will be studied using circular dichroism (CD), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and nuclear magnetic resonance spectroscopy (NMR). We plan to investigate the driving forces for transmembrane domain-domain interactions and discern the role of loop residues on tilt and insertion of transmembrane domains in bilayers. Simultaneously, site-directed mutations will be made to interrogate and test the results from these studies. We propose to construct a model of Ste2p and refine this model using biological and biophysical data. Overall, these studies should provide fundamental information concerning the structure and function of peptide-activated GPCRs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM022087-27
Application #
6580547
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Chin, Jean
Project Start
1978-05-01
Project End
2006-12-31
Budget Start
2003-01-01
Budget End
2003-12-31
Support Year
27
Fiscal Year
2003
Total Cost
$297,303
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
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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
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Moseri, Adi; Biron, Zohar; Arshava, Boris et al. (2015) The C4 region as a target for HIV entry inhibitors--NMR mapping of the interacting segments of T20 and gp120. FEBS J 282:4643-57
Zuber, Jeffrey; Danial, Shairy Azmy; Connelly, Sara M et al. (2015) Identification of destabilizing and stabilizing mutations of Ste2p, a G protein-coupled receptor in Saccharomyces cerevisiae. Biochemistry 54:1787-806
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
Abayev, Meital; Moseri, Adi; Tchaicheeyan, Oren et al. (2015) An extended CCR5 ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions. FEBS J 282:1906-1921

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