The investigator will use phototransduction in Drosophila as a model system to understand the coordination of the activation and deactivation processes of signal transduction. The experimental strategy is to study a protein named INAD, to understand how it regulates phototransduction. The investigator has shown that INAD interacts with two components involved in the activation and deactivation of the visual cascade. INAD interacts with TRP and a loss of interaction leads to a slow recovery of the visual response. INAD associates with NORPA, the effector of the visual cascade. NORPA flies lack any light-triggered receptor potential. Knowledge gained may help identify INAD homologues in vertebrates. The investigator proposes to : 1) investigate the INAD-TRP interaction; 2) investigate the INAD-NORPA association; 3) study the structure-function relationship of INAD in vivo; 4) generate null alleles (loss-of-function) of InaD and examine the electrophysiological defects of the mutants. This application proposes to study visual transduction in Drosophila. The focus is on a gene called InaD. In the 1970's, Bill Pak and his colleagues isolated a large collection of Drosophila visual mutants that were defective in the ERG. InaD was part of this collection; it was not one of the remarkable mutants. Its basic ERG defect was called inactivation-no-afterpotential, hence its name. The world of phototransduction has changed considerably since the 1970's. there is now have a fairly solid description of vertebrate visual transduction and the G-protein coupled, cGMP-mediated cascade that generates it. The situation for Drosophila is somewhat less advanced, but clearly goes through a different pathway with Phospholipase C (NORPA) being a major player via IP-3 and DAG (diacylglycerol). More recently, the field has been investigating mechanisms of adaptation: how photo responses are modulated appropriately for background light conditions. This is clearly a complicated process and involves control at many levels along the transduction cascade. [Vertebrate inactivation and adaptation: Ca involved - regulates photoreceptor sensitivity. Ca (dark) enters via cGMP-gated channels inhibits guanylate cyclase via guanylate cyclase activating protein (Ca binding protein). Second pathway: regulation of amplification by photoactivated opsins. Ca inhibits phosphorylation of opsin. Mediated by Ca binding protein S-modulin (recoverin). Ca modulates cGMP-gated channel by decreasing affinity of cGMP.] Presently, the main issues are: how are the photoresponse and adaptation responses integrated?; what are the coupling mechanisms?; and' how are they controlled? Preliminary work generated by the investigator indicates that for Drosophila, INAD may be a central player, hence, the main intellectual interest in the present proposal. The study of INAD is timely and addresses the main concerns of the field, at present. The investigator has shown that INAD protein interacts physically with 2 others: TRP and NORPA. Phenotypically, InaD and trp mutants are somewhat similar: they are defective in deactivation and adaptation of the photoresponse. TRP appears to be a Ca channel that may mediate ionic responses underlying adaptation. The most interesting finding is the interaction with NORPA. NORPA stands for no receptor potential. It is a Phospholipase C that has always been believed to be the major player in the activation cascade. Thus, the placement of InaD in the phototransduction cascade is very intriguing: early in the link between the activation cascade and the adaptation response. In this proposal the investigator focuses on the functional consequences of eliminating protein-protein interactions between INAD, TRP, and NORPA. She will generate mutations that alter or destroy the interaction between INAD and TRP, and between INAD and NORPA. The appropriate transgenics will be produced. The investigator will then examine electrophysiological consequences. Expression will assessed to control for appropriate levels and subcellular localization.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY009743-08
Application #
6180014
Study Section
Neurology C Study Section (NEUC)
Program Officer
Mariani, Andrew P
Project Start
1993-04-01
Project End
2002-03-31
Budget Start
2000-04-01
Budget End
2002-03-31
Support Year
8
Fiscal Year
2000
Total Cost
$275,108
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Lu, Haiqin; Leung, Hung-Tat; Wang, Ning et al. (2009) Role of Ca2+/calmodulin-dependent protein kinase II in Drosophila photoreceptors. J Biol Chem 284:11100-9
Peng, Li; Popescu, Daniela C; Wang, Ning et al. (2008) Anchoring TRP to the INAD macromolecular complex requires the last 14 residues in its carboxyl terminus. J Neurochem 104:1526-35
Wang, Ning; Leung, Hung-Tat; Pak, William L et al. (2008) Role of protein phosphatase 2A in regulating the visual signaling in Drosophila. J Neurosci 28:1444-51
Popescu, Daniela C; Ham, Amy-Joan L; Shieh, Bih-Hwa (2006) Scaffolding protein INAD regulates deactivation of vision by promoting phosphorylation of transient receptor potential by eye protein kinase C in Drosophila. J Neurosci 26:8570-7
Parker, Lisan L; Backstrom, Jon R; Sanders-Bush, Elaine et al. (2003) Agonist-induced phosphorylation of the serotonin 5-HT2C receptor regulates its interaction with multiple PDZ protein 1. J Biol Chem 278:21576-83
Shieh, Bih-Hwa; Parker, Lisan; Popescu, Daniela (2002) Protein kinase C (PKC) isoforms in Drosophila. J Biochem 132:523-7
Kumar, R; Shieh, B H (2001) The second PDZ domain of INAD is a type I domain involved in binding to eye protein kinase C. Mutational analysis and naturally occurring variants. J Biol Chem 276:24971-7
Liu, M; Parker, L L; Wadzinski, B E et al. (2000) Reversible phosphorylation of the signal transduction complex in Drosophila photoreceptors. J Biol Chem 275:12194-9
Adamski, F M; Zhu, M Y; Bahiraei, F et al. (1998) Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies. J Biol Chem 273:17713-9
Shieh, B H; Zhu, M Y; Lee, J K et al. (1997) Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo. Proc Natl Acad Sci U S A 94:12682-7

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