Abstract

The overall goal of this research program is to elucidate the molecular basis of visual excitation in mammalian retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, and ultrastructural studies of rod outer segments (ROS): (1) The light-activated amplification cycle in ROS involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase will be investigated in detail.
The aim i s to elucidate the conformational transitions, kinetics, and free energy change of each step of this cycle. Fluorescence energy transfer, nanosecond emission anisotropy, and infra-red light scattering studies will be carried out to map subunit structure and to monitor the movement of subunits. The regulatory role of covalent modification will be examined. (2) We plan to obtain two-dimensional crystalline arrays of complexes of rhodopsin with transducin, the phosphodiesterase, rhodopsin kinase, and other ROS proteins. The molecular structure of these complexes will be determined by reconstructing three-dimensional images from series of electron micrographs. (3) Cholera catalyzes the ADP-ribosylation of transducin and blocks its GTPase activity. Peptide mapping will be carried out to determine the degree of homology between transducin and the G-protein of the adenylate cyclase system. We want to ascertain the similarities between the signal-coupling proteins in vision and hormone action. (4) Calmodulin from ROS will be purified and its interactions with other proteins will be investigated to identify calcium-sensitive loci. The effects of calcium calmodulin on the capacity of photoexcited rhodopsin to catalyze GTP-GDP exchange in transducin, on the kinetics of sodium-calcium exchange by membrane vesicles, and on the rates of reactions catalyzed by kinases and phosphatases will be measured. (5) A major effort will be devoted to the purification of the ROS plasma membrane. The ion-transport properties of vesicles formed from purified plasma membrane and the effects of calcium ion, calmodulin, and cyclic GMP on ion fluxes will be measured. These vesicles will be used to determine which constituents of the plasma membrane are altered in response to background illumination and transient light pulses.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY002005-12
Application #
3256380
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1978-12-01
Project End
1988-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
12
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305

  Publications

Tanaka, T; Ames, J B; Kainosho, M et al. (1998) Differential isotype labeling strategy for determining the structure of myristoylated recoverin by NMR spectroscopy. J Biomol NMR 11:135-52
Erickson, M A; Lagnado, L; Zozulya, S et al. (1998) The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors. Proc Natl Acad Sci U S A 95:6474-9
Baldwin, A N; Ames, J B (1998) Core mutations that promote the calcium-induced allosteric transition of bovine recoverin. Biochemistry 37:17408-19
Ames, J B; Tanaka, T; Stryer, L et al. (1996) Portrait of a myristoyl switch protein. Curr Opin Struct Biol 6:432-8
Zozulya, S; Ladant, D; Stryer, L (1995) Expression and characterization of calcium-myristoyl switch proteins. Methods Enzymol 250:383-93
Ames, J B; Porumb, T; Tanaka, T et al. (1995) Amino-terminal myristoylation induces cooperative calcium binding to recoverin. J Biol Chem 270:4526-33
Ames, J B; Tanaka, T; Ikura, M et al. (1995) Nuclear magnetic resonance evidence for Ca(2+)-induced extrusion of the myristoyl group of recoverin. J Biol Chem 270:30909-13
Karpen, J W; Brown, R L; Stryer, L et al. (1993) Interactions between divalent cations and the gating machinery of cyclic GMP-activated channels in salamander retinal rods. J Gen Physiol 101:1-25
Flaherty, K M; Zozulya, S; Stryer, L et al. (1993) Three-dimensional structure of recoverin, a calcium sensor in vision. Cell 75:709-16
Zozulya, S; Stryer, L (1992) Calcium-myristoyl protein switch. Proc Natl Acad Sci U S A 89:11569-73

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