Peptide hormones influence the metabolism of virtually every eukaryotic cell by initiating signal transduction cascades following interaction with receptors on cell membranes. A multitude of human diseases are linked to hormone and receptor pathology making an understanding of how the hormone-receptor complex is formed and how the subsequent signal transduction is triggered essential for developing medicines to treat many diseases. We propose to continue our studies on the interaction between alpha-factor, a tridecapeptide Saccharomyces cerevisiae pheromone, and its receptor (Ste2p), a member of the ubiquitous seven transmembrane domain, G protein-coupled receptor family. In recent months we have made exciting progress in the purification and reconstitution of the receptor and in the development of photoactivatable alpha-factor analogs. These tools will be used to crosslink peptide analogs into the receptor to map the specific residues that participate in the binding of the alpha-factor. Concurrently, fluorescent analogs will be used to discern the molecular environment of the peptide-binding pocket, and molecular biological approaches will be used to generate mutant receptors to verify and expand upon the binding studies. Segments of the of Ste2p will be synthesized or biosynthesized and their structure will be studied in lipid-like environments using circular dichroism, high resolution solution NMR spectroscopy and solid-state NMR spectroscopy. Interactions among the transmembrane segments of the receptor will be studied by both biophysical and molecular biological methods. Fluorescently labeled receptors will be used in studies designed to determine the conformational changes in the receptor induced by ligand binding. Overall, these studies will provide the basis for building a testable model revealing the ligand binding site of the receptor, uncovering interactions among receptor transmembrane segments, and demonstrating changes in receptor structure at the molecular level on ligand activation. In general, these studies should provide insights into the function of peptide-activated G-protein-coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM022087-23
Application #
2759792
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1978-05-01
Project End
2002-12-31
Budget Start
1999-01-01
Budget End
1999-12-31
Support Year
23
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Tennessee Knoxville
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
City
Knoxville
State
TN
Country
United States
Zip Code
37996
Uddin, M Seraj; Naider, Fred; Becker, Jeffrey M (2017) Dynamic roles for the N-terminus of the yeast G protein-coupled receptor Ste2p. Biochim Biophys Acta Biomembr 1859:2058-2067
Cai, Houjian; Hauser, Melinda; Naider, Fred et al. (2016) Halo Assay for Toxic Peptides and Other Compounds in Microorganisms. Bio Protoc 6:
Sridharan, Rajashri; Connelly, Sara M; Naider, Fred et al. (2016) Variable Dependence of Signaling Output on Agonist Occupancy of Ste2p, a G Protein-coupled Receptor in Yeast. J Biol Chem 291:24261-24279
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
Hauser, Melinda; Cai, Houjian; Naider, Fred et al. (2016) Uptake Assay for Radiolabeled Peptides in Yeast. Bio Protoc 6:
Diaz-Rodriguez, Veronica; Ganusova, Elena; Rappe, Todd M et al. (2015) Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor. J Org Chem 80:11266-74
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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