The approximately 1 ,000 G protein-coupled receptors (GPCRs) of humans mediate key signals - triggered by photons, odorants, hormones, and neurotransmitters - in brain, heart, blood vessels, white blood cells, and virtually every organ and endocrine gland. The fact that most of these signals represent potential targets for drug therapy justifies the broad-based strategy of this proposal, which aims to understand the conserved molecular mechanisms responsible for transmitting G protein-mediated signals between signaling molecules, in vitro and in the context of the cell. The first two aims test relations between structure and function of the GPCR and the G protein trimer at the level of individual molecules. Experiments with GPCRs aim to: a. use molecular probes to determine how the extracellular surface of a GPCR actually binds the activating ligand; b. engineer metal binding sites that activate a GPCR by inducing coordinated movement of its transmembrane helices, allowing us to infer how the natural ligand induces similar movements; c. identify sites on the GPCR's intracellular surface that interact specifically with peptides representing different parts of the trimeric target. To understand the conformational changes in a G protein trimer that mediate its activation by the GPCR, a second set of experiments will: a. test the hypothesis that the GPCR uses the beta-gamma subunit of the G protein trimer as a lever to open a route for bound GDP to exit from its binding pocket in the alpha subunit and thereby activate the trimer; b. determine how key structural elements of the G protein alpha subunit cooperate during the activation process, by constructing metal binding sites that restrict movements of these elements, relative to one another. The third set of experiments uses fluorescent probes and fluorescence energy transfer (FRET) to determine the locations of G protein alpha and beta-gamma subunits in intact cells and ask how hormonal activation affects their interaction. Biochemical experiments with pure G protein subunits indicate that activation in the test tube causes the alpha subunit to dissociate from the beta-gamma heterodimer; it is not known, however, whether such a dissociation accompanies activation in an intact cell responding to a hormone. Investigation of this question begins by constructing functioning G protein alpha and beta-gamma subunits attached to fluorescent tags; the fluorescent subunits are used to assess their subcellular distributions in intact cultured cells, and FRET between co-expressed tagged beta-gamma and alpha subunits will reveal whether hormones cause them to dissociate.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Long, Rochelle M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California San Francisco
Schools of Medicine
San Francisco
United States
Zip Code
Wong, Kit; Van Keymeulen, Alexandra; Bourne, Henry R (2007) PDZRhoGEF and myosin II localize RhoA activity to the back of polarizing neutrophil-like cells. J Cell Biol 179:1141-8
Herzmark, Paul; Campbell, Kyle; Wang, Fei et al. (2007) Bound attractant at the leading vs. the trailing edge determines chemotactic prowess. Proc Natl Acad Sci U S A 104:13349-54
Xu, Jingsong; Van Keymeulen, Alexandra; Wakida, Nicole M et al. (2007) Polarity reveals intrinsic cell chirality. Proc Natl Acad Sci U S A 104:9296-300
Wong, Kit; Pertz, Olivier; Hahn, Klaus et al. (2006) Neutrophil polarization: spatiotemporal dynamics of RhoA activity support a self-organizing mechanism. Proc Natl Acad Sci U S A 103:3639-44
Van Keymeulen, Alexandra; Wong, Kit; Knight, Zachary A et al. (2006) To stabilize neutrophil polarity, PIP3 and Cdc42 augment RhoA activity at the back as well as signals at the front. J Cell Biol 174:437-45
Weiner, Orion D; Rentel, Maike C; Ott, Alex et al. (2006) Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis. PLoS Biol 4:e38
Xu, Jingsong; Wang, Fei; Van Keymeulen, Alexandra et al. (2005) Neutrophil microtubules suppress polarity and enhance directional migration. Proc Natl Acad Sci U S A 102:6884-9
Srinivasan, Supriya; Wang, Fei; Glavas, Suzana et al. (2003) Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J Cell Biol 160:375-85
Yu, Wei; O'Brien, Lucy E; Wang, Fei et al. (2003) Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures. Mol Biol Cell 14:748-63
Weiner, Orion D (2002) Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr Opin Cell Biol 14:196-202

Showing the most recent 10 out of 60 publications