The long-term objective of this program is to investigate molecular mechanisms of transducin (Gt) signaling in the vertebrate photoreceptor cells. The properties and regulation of rod transducin Gt1 during phototransduction are understood to a far greater extent than those of cone transducin Gt2. It remains unknown whether the conserved sequence differences in rod and cone transducin-a (Gat1) subunits translate into the functional differences that contribute the remarkably distinct physiology of rods and cones. We hypothesize that because of conserved differences within the Gat N-termini and the interdomain interfaces, cone Gt2 is activated by photoexcited pigment with a lower efficiency than rod Gt1,and the Gat2-mediated stimulation of cGMP-phoshodiesterase 6 is less potent in comparison to the effector enzyme activation in rods. In order to test this hypothesis, we will carry out detailed characterization and mutational analysis of Gat2 and heterotrimeric cone transducin Gat2?3?8. The proposed analysis will be based on a newly established procedure for bacterial expression of human Gat2. These studies will advance our understanding of the molecular mechanisms that distinguish phototransduction in cones and rods. Another obscure aspect of transducin biology and signaling involves the mechanisms of its bi-directional transport between the inner and outer segments in rods, the determinants of light-dependent compartmentalization, and mobility on photoreceptor membranes. We will explore the roles of transducin/rhodopsin interactions and lipid modifications in transducin targeting, membrane mobility and interdisc transfer using transgenic Xenopus laevis expressing mutant EGFP-fused Gat1 subunits in rod photoreceptors. The mutant Gat1 models will be examined with EGFP imaging, immunofluorescence, and Fluorescence Recovery After Photobleaching (FRAP) analysis of lateral and longitudinal diffusion. The proposed research will provide important insights into transport and mobility of peripheral membrane proteins in photoreceptor cells.

Public Health Relevance

Photoreceptor GTP-binding proteins, transducins, are the key signaling molecules in vision. Functional properties of cone transducin and the differences in signaling of cone and rod transducins are largely unknown. The proposed studies will yield a new level of understanding the function and regulation of cone transducin and advance our understanding of the molecular mechanisms responsible for the markedly distinct physiology of cones and rods. This research will also provide important insights into the transport and mobility of transducin in photoreceptor cells.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012682-13
Application #
8317673
Study Section
Biology and Diseases of the Posterior Eye Study Section (BDPE)
Program Officer
Neuhold, Lisa
Project Start
2000-05-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
13
Fiscal Year
2012
Total Cost
$348,334
Indirect Cost
$110,734
Name
University of Iowa
Department
Physiology
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Cheguru, Pallavi; Majumder, Anurima; Yadav, Ravi et al. (2015) The solution structure of the transducin-?-uncoordinated 119 protein complex suggests occlusion of the G????-binding sites. FEBS J 282:550-61
Manes, Gaƫl; Cheguru, Pallavi; Majumder, Anurima et al. (2014) A truncated form of rod photoreceptor PDE6 ?-subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the ?-subunit. PLoS One 9:e95768
Kuburas, Adisa; Thompson, Stewart; Artemyev, Nikolai O et al. (2014) Photophobia and abnormally sustained pupil responses in a mouse model of bradyopsia. Invest Ophthalmol Vis Sci 55:6878-85
Majumder, Anurima; Gopalakrishna, Kota N; Cheguru, Pallavi et al. (2013) Interaction of aryl hydrocarbon receptor-interacting protein-like 1 with the farnesyl moiety. J Biol Chem 288:21320-8
Sinha, Satyabrata; Majumder, Anurima; Belcastro, Marycharmain et al. (2013) Expression and subcellular distribution of UNC119a, a protein partner of transducin * subunit in rod photoreceptors. Cell Signal 25:341-8
Majumder, Anurima; Pahlberg, Johan; Boyd, Kimberly K et al. (2013) Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells. Proc Natl Acad Sci U S A 110:12468-73
Gopalakrishna, Kota N; Boyd, Kimberly K; Artemyev, Nikolai O (2012) Comparative analysis of cone and rod transducins using chimeric G? subunits. Biochemistry 51:1617-24
Kerov, Vasily; Artemyev, Nikolai O (2011) Diffusion and light-dependent compartmentalization of transducin. Mol Cell Neurosci 46:340-6
Gopalakrishna, Kota N; Doddapuneni, Krishnarao; Boyd, Kimberly K et al. (2011) Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors. J Biol Chem 286:28954-62
Muradov, Hakim; Boyd, Kimberly K; Artemyev, Nikolai O (2010) Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent. J Biol Chem 285:39828-34

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