G protein coupled receptors (GPCRs) that activate cytoplasmic trimeric G proteins play critical roles in diverse cellular signaling pathways. However, the mechanisms by which ligand binding to receptors effect changes in the G proteins are not understood. This proposal focuses on the alpha-factor receptor, encoded by the yeast STE2 gene as a model for studying G protein activation. The role of this receptor in yeast mating has led to development of sensitive screens and selections both for and against receptor function that allow isolation of receptors with altered function from random mutational libraries. Since the STE2 gene product is similar to, and functionally interchangeable with, other GPCRs, the results and techniques used should be widely applicable. The project has four major aims: I. Identification of interacting surfaces and relative motions of transmembrane segments. The three-dimensional arrangement of the seven predicted transmembrane helices of GPCRs is not known. By combining the isolation of loss-of function and second-site intragenic suppressor mutations with intramolecular disulfide crosslinking, interactions between transmembrane segments will be identified. These interactions will be used as constraints in structural modeling of the alpha-factor receptor. In addition, effects of intramolecular crosslinking on receptor function will be used to detect activation-dependent motions of transmembrane segments. II. Localization of the site of ligand binding. This will be accomplished by screening for receptor mutations that interfere with ligand binding and for receptor mutations that can compensate for alterations in the structure of the ligand. III. Determination of the mechanism of interactions between receptors. Our laboratory has isolated dominant negative mutant receptors that interfere with the function of co-expressed normal receptors. In addition, signaling by hypersensitive and constitutively active mutant receptors can be suppressed by co-expressed normal receptors. Such inter-receptor interactions could result from oligomerization of receptors during signaling or sequestration of G protein by inactive receptors. These possibilities will be distinguished by crosslinking of putative oligomers, by examining the effects of G protein overexpression, and by covalently fusing the G protein alpha subunit to various receptor alleles. IV. Identification of sites of receptor-G protein interactions. To identify particular amino acid residues at sites of contact between the receptor and the G protein, genetic screens will be conducted for G protein mutations that specifically block interactions with the receptor and for receptor mutations that suppress G protein mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059357-04
Application #
6520021
Study Section
Genetics Study Section (GEN)
Program Officer
Anderson, Richard A
Project Start
1999-03-01
Project End
2005-01-31
Budget Start
2002-03-01
Budget End
2005-01-31
Support Year
4
Fiscal Year
2002
Total Cost
$293,927
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
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
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
Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M et al. (2014) Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors. Biochim Biophys Acta 1838:15-33
Gehret, Austin U; Connelly, Sara M; Dumont, Mark E (2012) Functional and physical interactions among Saccharomyces cerevisiae ýý-factor receptors. Eukaryot Cell 11:1276-88
Taslimi, Amir; Mathew, Elizabeth; Celic, Andjelka et al. (2012) Identifying functionally important conformational changes in proteins: activation of the yeast ýý-factor receptor Ste2p. J Mol Biol 418:367-78
Mathew, Elizabeth; Bajaj, Anshika; Connelly, Sara M et al. (2011) Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p. J Mol Biol 409:513-28
Tantry, Subramanyam; Ding, Fa-Xiang; Dumont, Mark et al. (2010) Binding of fluorinated phenylalanine alpha-factor analogues to Ste2p: evidence for a cation-pi binding interaction between a peptide ligand and its cognate G protein-coupled receptor. Biochemistry 49:5007-15
Bajaj, Anshika; Connelly, Sara M; Gehret, Austin U et al. (2007) Role of extracellular charged amino acids in the yeast alpha-factor receptor. Biochim Biophys Acta 1773:707-17
Gehret, Austin U; Bajaj, Anshika; Naider, Fred et al. (2006) Oligomerization of the yeast alpha-factor receptor: implications for dominant negative effects of mutant receptors. J Biol Chem 281:20698-714
Bajaj, Anshika; Celic, Andjelka; Ding, Fa-Xiang et al. (2004) A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast. Biochemistry 43:13564-78

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