Abstract

This is an amended proposal to understand the structural basis of G protein-coupled receptor activation. Specifically proposed is the analysis of constitutive activity exhibited by the yeast alpha-pheromone receptor, and subsequently the mammalian adenosine receptor, in yeast using a novel, lacZ-based reporter assay.
The first aim outlines strategies to determine which residues, when mutated, cause constitutive activity. Both directed (TM5 and 6) and random mutagenesis will be used, with the idea that the mapping will provide the material for subsequently proposed assays of ligand binding and structural changes.
The second aim addresses quantification of constitutive activity to provide a relative ranking of activity among the mutants and an assessment of ligand binding.
The third aim deals with the analysis of receptor structure using a method that examines spatial proximity of selected residues. The method is based on the insertion of cysteine residues to form intramolecular disulfide bonds. While the first three aims deal with the alpha-pheromone receptor, the fourth aim represents an extension of the developed techniques to the mammalian adenosine receptor in order to determine common principles in GPCR activation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055107-04
Application #
6180640
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Program Officer
Shapiro, Bert I
Project Start
1997-09-01
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
4
Fiscal Year
2000
Total Cost
$208,732
Indirect Cost

  Institution

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

  Publications

Ballon, Daniel R; Flanary, Paul L; Gladue, Douglas P et al. (2006) DEP-domain-mediated regulation of GPCR signaling responses. Cell 126:1079-93
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

Showing the most recent 10 out of 13 publications