A number of biological activities are regulated by the interactions of hormones, polypeptide growth factors, or other agents (including light) with specific cell surface receptors. Many of these receptor-coupled signal transduction pathways are compressed of at least three types of components: the receptor protein, itself, a GTP-binding protein (G protein) which serves as a transducer in mediating receptor-regulation and an effector enzyme or an ion channel which is responsible for maintaining the level of a specific cellular second messenger. The studies outlined in this proposal will use the retinal visual transduction system as a model for addressing a number of issues relevant to receptor-G protein- effector interactions. Each of the primary components of the vision system (i.e. the photoreceptor, rhodopsin, the G protein, transducin, and the effector enzyme, the cyclic GMP phosphodiesterase (PDE)) will be purified and their functional interactions reconstituted in well defined phospholipid vesicle systems, fluorescence spectroscopic techniques, together with a variety of biochemical approaches, will be used to probe receptor- G protein and G protein-effector coupling in these systems. This proposal is divided into four specific aims: 1) the characterization of the individual steps of the receptor (rhodopsin)-stimulated activation-deactivation cycle of a G protein (transducin), 2) structure-function characterization of the phosphorylation of the alpha subunit of transducin, 3) structure- function studies of the G protein-beta gamma subunit complexes, and 4) the characterization of the molecular mechanisms by which G proteins regulate the activities of effector proteins. Among the specific issues which will be addressed include: a) the detailed sequence of events resulting in receptor-stimulated guanine nucleotide exchange, G protein-subunit dissociation, G protein- effector interactions, and the determination of the rates for these steps, using recently developed fluorescence approaches, b) whether the phosphorylation of G proteins serves as a means to regulate the function of these transducers, c) the individual roles of the beta and gamma subunits of G proteins in promoting the receptor- stimulated activation of G-alpha subunits, d) whether the receptor protein directly accentuates (via a receptor-G protein complex) G protein-effector interactions and e) the mechanism by which the GDP-bound form of the G protein-alpha subunit results in an immediate deactivation of effector activity. In addition, the reconstitution of a G protein-mediated inhibition of a verapamil- sensitive Ca2+ channel has recently been achieved and this system will be used to examine the similarities between G protein-channel interactions and transducin-PDE interactions. It is expected that the results of these studies will be relevant to understanding the molecular mechanism underlying visual transduction as well as other receptor-coupled signaling systems such as those responsible for the regulation of adenylate cyclase, phosphoinositide lipid turnover, ion channels and growth factor action.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Physiological Chemistry Study Section (PC)
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Cornell University
Schools of Veterinary Medicine
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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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