GTP-binding (G) proteins act as mojecular switches in signal transduction pathways that control a diversity of biological responses. Two families of G proteins have been well characterized, namely the large or heterotrimeric G proteins (subunits designated alpha,beta,gamma) and the small monomeric Ras-related G proteins. We have used the retinal G protein transducin, which is essential for vertebrate vision, as a model for large G proteins, and Cdc42, which stimulates cell-cycle progression and when hyperactive induces malignant transformation, as a model for small G proteins. In the past funding period, we used a combination of fluorescence spectroscopy and X-ray crystallography to study the molecular regulation of the GTP-binding/GTPase cycle of Cdc42, as well as how transducin and Cdc42 bind to their target/effectors. In this renewal application, we will examine two fundamentally important aspects of G protein function. The first concerns the conformational and molecular changes that the a subunit of a large G protein must undergo to exchange GTP for GDP and thus become activated for signal propagation. Using transducin as a model, we are particularly interested in determining whether the large helical domain that is adjacent to the guanine nucleotide-binding (GTPase) domain must move away from the GTPase domain in order for GTP-GDP exchange to occur and/or if other changes in the G protein a (Ga) subunit are necessary. We also want to know how the Gbetagamma subunit complex influences the activation of a Ga subunit. These studies should identify new types of activating mutations in Ga subunits that will serve as important reagents for studying other G protein signaling systems. The second major aim of the proposed studies is to understand how activated Ga subunits stimulate their target/effectors. We will determine why the Switch III domain of the a subunit of transducin (aT), which is conformationally sensitive to GTP-binding and thought to be unique to large G proteins, is essential for stimulating effector activity. We also will examine why the PDE contains two binding sites for activated aT and if there is an important regulatory interplay between these two sites. The results of these studies should raise questions regarding the necessity of a third switch domain on small G proteins like Cdc42 and the general role of target/effector dimerization in G protein signaling. We expect that the proposed work will not only be relevant to vertebrate vision but also to a variety of signaling pathways which when deregulated give rise to a number of disease states including cancer and different neurodegenerative and endocrine disorders.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006429-21
Application #
7087784
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Mariani, Andrew P
Project Start
1986-05-01
Project End
2008-06-30
Budget Start
2006-07-01
Budget End
2008-06-30
Support Year
21
Fiscal Year
2006
Total Cost
$310,527
Indirect Cost
Name
Cornell University
Department
Other Basic Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Phillips, W J; Cerione, R A (1994) A C-terminal peptide of bovine rhodopsin binds to the transducin alpha-subunit and facilitates its activation. Biochem J 299 ( Pt 2):351-7
Mittal, R; Cerione, R A; Erickson, J W (1994) Aluminum fluoride activation of bovine transducin induces two distinct conformational changes in the alpha subunit. Biochemistry 33:10178-84
Erickson, J W; Cerione, R A (1993) Regulation of the cGMP phosphodiesterase in bovine rod outer segments. Use of resonance energy transfer to distinguish between associative and dissociative activation mechanisms. J Biol Chem 268:3328-33
Phillips, W J; Cerione, R A (1992) Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy. J Biol Chem 267:17032-9
Phillips, W J; Wong, S C; Cerione, R A (1992) Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin. J Biol Chem 267:17040-6
Erickson, J W; Cerione, R A (1991) Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade. Biochemistry 30:7112-8
Phillips, W J; Cerione, R A (1991) Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions. J Biol Chem 266:11017-24
Shinjo, K; Koland, J G; Hart, M J et al. (1990) Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc Natl Acad Sci U S A 87:9853-7
Guy, P M; Koland, J G; Cerione, R A (1990) Rhodopsin-stimulated activation-deactivation cycle of transducin: kinetics of the intrinsic fluorescence response of the alpha subunit. Biochemistry 29:6954-64
Phillips, W J; Trukawinski, S; Cerione, R A (1989) An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity. J Biol Chem 264:16679-88

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