The overall goal of this research is to elucidate the molecular basis of visual excitation and adaptation in retinal rod cells. We plan to carry out the following enzymatic, spectroscopic, structural, and electrophysiological studies of rod outer segment proteins: (1) The light-triggered amplification cycle in rod outer segments involving photoexcited rhodopsin, transducin, and the cyclic GMP phosphodiesterase (PDE) will be investigated in molecular detail. Fluorescence energy transfer studies will be carried out to establish the location of the subunits of transducin relative to each other, R*, and the disk membrane. (2) The structural differences between membrane-bound and soluble GTP- transducin will be determined and the functional significance of the two forms will be ascertained by reconstitution experiments. (2) Energy transfer studies will also be carried out to determine how activated transducin reverses the inhibition of PDE imposed by its gamma subunit. Specifically, does transducin displace the gamma subunit from the inhibited holoenzyme or does it carry it away? (3) A synthetic gene for the gamma subunit has been prepared for site-specific mutagenesis studies.
The aim i s to pinpoint the region of gamma that binds to the catalytic subunits and blocks their activity It will also be interesting to engineer mutants that irreversibly inhibit PDE. (4) Nonhydrolyzable analogs of cGMP will be used to determine where most of the cGMP in rod outer segments is bound and how its uptake and release are controlled. Defects of cGMP buffer sites may be important in the pathogenesis of some degenerative diseases of the retina. (5) We have recently found that guanylate cyclase is activated by small decreases in the concentration of Ca2+ in the vicinity of 10-7 M. The control of guanylate cyclase by Ca2+ is likely to be important for recovery and adaptation. The calcium-binding protein mediating this highly cooperative effect will be purified and its mechanism of regulation will be investigated. (6) The molecular architecture of the cGMP- activated channel in the plasma membrane will be probed by fluorescent analogs of cGMP. Energy transfer will be used as a spectroscopic ruler to map the channel. Fluorescence and conductance studies of reconstituted membranes containing purified channel protein will define the allosteric mechanism by which cGMP cooperatively opens the conductance pathway. The conductance properties of channels containing a defined number of covalently attached cGMP analog molecules should be highly informative in revealing how cGMP allosterically opens the channel. A detailed understanding of the cyclic GMP cascade of vision will be highly rewarding in understanding signal transduction processes generally and molecular diseases arising from defective coupling.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY002005-14
Application #
3256381
Study Section
Visual Sciences B Study Section (VISB)
Project Start
1978-12-01
Project End
1993-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
14
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Medicine
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
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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