The objective of this research is to elucidate the biochemical mechanism by which cGMP influences visual transduction in vertebrate rod photoreceptors. Both biochemical studies of the cGMP pathway (rhodopsin to GTP-bining protein to phosphodiesterase) and electrophysiological effects of cGMP on whole cell recordings and isolated membrane patches demonstrate the importance of cGMP in modulating the light-dependent permeability mechanism in the plasma membrane. The molecular mechanism by which cGMP acts has not, however, been determined.
The aims of the proposed research are as follows: (1) To evaluate whether small light-induced decreases in total cGMP concentration reflect larger changes in cGMP concentration in the cytoplasm. The observed decrease in total cGMP concentration is too small to exert a significant effect on the permeability mechanism by a simple binding model. However, subcellular partitioning of cGMP into free and bound pools may occur, and will be examined by fractionating total cellular cGMP to determine whether a significant portion of total cGMP is bound and hence not sensitive to rapid light-induced hydrolysis by phosphodiesterse. (2) To identify possible sites of action of cGMP in the rod. cGMP is known to have effects on ion permeability of the plasma membrane, Ca2+ release from disc membranes, protein kinase activity, and GTP-binding protein. Identification and characterization of cGMP binding sites will lead to purification of protein components that may regulate, via changes in cGMP concentration and/or binding, the ionic permeability of the rod. These experiments should permit more definitive tests of various models for cGMP action in visual transduction, and lead to increased understanding of why defects in cGMP meatabolism lead to photoreceptor cell degeneration and death.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY005798-02
Application #
3261361
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1986-05-01
Project End
1988-02-29
Budget Start
1987-05-01
Budget End
1988-02-29
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Graduate Schools
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Zeng-Elmore, Xiaohui; Gao, Xiong-Zhuo; Pellarin, Riccardo et al. (2014) Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling. J Mol Biol 426:3713-3728
Zhang, Xiu-Jun; Gao, Xiong-Zhuo; Yao, Wei et al. (2012) Functional mapping of interacting regions of the photoreceptor phosphodiesterase (PDE6) ýý-subunit with PDE6 catalytic dimer, transducin, and regulator of G-protein signaling9-1 (RGS9-1). J Biol Chem 287:26312-20
Cahill, Karyn B; Quade, Jonathan H; Carleton, Karen L et al. (2012) Identification of amino acid residues responsible for the selectivity of tadalafil binding to two closely related phosphodiesterases, PDE5 and PDE6. J Biol Chem 287:41406-16
Matte, Suzanne L; Laue, Thomas M; Cote, Rick H (2012) Characterization of conformational changes and protein-protein interactions of rod photoreceptor phosphodiesterase (PDE6). J Biol Chem 287:20111-21
Cahill, Karyn B; Cote, Rick H (2011) Phosphodiesterase 6C, cGMP-specific cone alpha'. AFCS Nat Mol Pages 2011:
Gitschier, Hannah J; Cote, Rick H (2011) Phosphodiesterase 6D, cGMP-specific rod delta. AFCS Nat Mol Pages 2011:
Zhang, Xiu-Jun; Skiba, Nikolai P; Cote, Rick H (2010) Structural requirements of the photoreceptor phosphodiesterase gamma-subunit for inhibition of rod PDE6 holoenzyme and for its activation by transducin. J Biol Chem 285:4455-63
Liu, Yu-Ting; Matte, Suzanne L; Corbin, Jackie D et al. (2009) Probing the catalytic sites and activation mechanism of photoreceptor phosphodiesterase using radiolabeled phosphodiesterase inhibitors. J Biol Chem 284:31541-7
Zhang, Xiu-Jun; Cahill, Karyn B; Elfenbein, Arye et al. (2008) Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6. J Biol Chem 283:29699-705
Pentia, Dana C; Hosier, Suzanne; Cote, Rick H (2006) The glutamic acid-rich protein-2 (GARP2) is a high affinity rod photoreceptor phosphodiesterase (PDE6)-binding protein that modulates its catalytic properties. J Biol Chem 281:5500-5

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