The long-term goals of this proposal are to determine the mechanisms of G protein-coupled receptor (GPCR) activation. GPCRs are characterized structurally by the presence of seven transmembrane domains and functionally by the ability to activate a heterotrimeric guanine-nucleotide binding protein (G protein). Receptors in this family transduce the signals for many medically important hormones and neurotransmitters. GPCRs have also been implicated in the pathogenesis of a broad range of diseases including inflammatory, cardiovascular, and infectious diseases. However, their mechanism of activation is not well understood due to the technical difficulties in studying membrane bound receptors in complex multicellular organisms. Therefore, our work has taken advantage of the genetic accessibility of the yeast S. cerevisiae to study the G protein-coupled alpha-factor pheromone receptor. Preliminary studies based on the analysis of dominant-negative and constitutively-active receptor mutants identified the ends of the transmembrane segments as playing a key role in receptor function. The extracellular ends function in ligand binding and the intracellular ends function in G protein activation. Therefore, the specific aims of this proposal will be to define the roles of the ends of the transmembrane domains in receptor function. The experimental procedures will employ mutational analysis to identify the functionally important residues at the ends of the transmembrane segments, and biochemical approaches to map the residues near the binding sites of the ligand and G protein on the receptor. Biochemical crosslinking experiments and genetic suppressor analysis will then be used in a targeted fashion to establish specific points of contact in the receptor that form the binding pockets for the ligand and G protein. Data from this complementary mix of genetics and biochemistry will be integrated to form a molecular model for the mechanisms of receptor activation. The results of these studies will contribute to greater understanding of GPCR signaling and are expected to have important application to the development of new therapeutic approaches for a wide range of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055107-08
Application #
6797135
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
1997-09-01
Project End
2006-08-31
Budget Start
2004-09-01
Budget End
2006-08-31
Support Year
8
Fiscal Year
2004
Total Cost
$255,573
Indirect Cost
Name
State University New York Stony Brook
Department
Genetics
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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Choi, Yunsook; Konopka, James B (2006) Accessibility of cysteine residues substituted into the cytoplasmic regions of the alpha-factor receptor identifies the intracellular residues that are available for G protein interaction. Biochemistry 45:15310-7
Eilers, Markus; Hornak, Viktor; Smith, Steven O et al. (2005) Comparison of class A and D G protein-coupled receptors: common features in structure and activation. Biochemistry 44:8959-75
Lin, Jennifer C; Duell, Ken; Saracino, Misty et al. (2005) Identification of residues that contribute to receptor activation through the analysis of compensatory mutations in the G protein-coupled alpha-factor receptor. Biochemistry 44:1278-87
Lin, Jennifer C; Duell, Ken; Konopka, James B (2004) A microdomain formed by the extracellular ends of the transmembrane domains promotes activation of the G protein-coupled alpha-factor receptor. Mol Cell Biol 24:2041-51
Lin, Jennifer C; Parrish, William; Eilers, Markus et al. (2003) Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor. Biochemistry 42:293-301
Mentesana, Pamela E; Dosil, Mercedes; Konopka, James B (2002) Functional assays for mammalian G-protein-coupled receptors in yeast. Methods Enzymol 344:92-111
Parrish, William; Eilers, Markus; Ying, Weiwen et al. (2002) The cytoplasmic end of transmembrane domain 3 regulates the activity of the Saccharomyces cerevisiae G-protein-coupled alpha-factor receptor. Genetics 160:429-43
Mentesana, P E; Konopka, J B (2001) Mutational analysis of the role of N-glycosylation in alpha-factor receptor function. Biochemistry 40:9685-94
Dosil, M; Schandel, K A; Gupta, E et al. (2000) The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein. Mol Cell Biol 20:5321-9

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