The long-term goal of our research is to understand the molecular mechanisms regulating the G-protein coupled signaling cascade critical for vision in the fruitfly, Drosophila melanogaster. The rapid activation and feedback regulation of many signaling cascades may be tightly coupled. Recently, many of the components required for Drosophila phototransduction have been found to be linked into a supramolecular signaling complex (signalplex). The coordinator is INAD, a protein with five tandem protein interaction motifs, referred to as PDZ domains. The identifications of the signalplex permits a reevaluation of the modes by which fly vision is regulated. The goal of the current project is to investigate the role of the INAD signalplex in activation and termination the Drosophila photoresponse. Several strategies to study the function of the INAD signalplex are proposed. The first will employ in vitro techniques to characterize the interactions of INAD with various target proteins as well as sites important for homomultimerization and studies to probe the role of the signalplex in vivo. This will be accomplished by constructing site- specific mutations that disrupt specific protein interactions and introducing the altered genes into the fly using P-element mediated germ-line transformation. The effects of these alterations will be assessed using a combination of electrophysiological, immunocytochemical and biochemical techniques.
The specific aims are to test the hypotheses that: 1) association of signaling proteins with INAD is critical for activation and termination, 2) interaction of calmodulin with the signalplex functions in termination of the photoresponse, 3) the association of signaling proteins with INAD is regulated dynamically by PKC, and 4) homomultimerization of INAD is required in vivo for termination and/or activation of the photoresponse. The fundamental importance of activating and switching signals off with normal kinetics is illustrated by the profound retinal degeneration resulting from defects in activation and termination of phototransduction in Drosophila photoreceptor cells. Mutations in many signaling proteins central to Drosophila phototransduction result in retinal degeneration. In most cases, homologs of these Drosophila proteins are known to be expressed in the vertebrate retina. A long range goal of the current research will be to ascertain whether similar retinal dystrophies result from mutations in these vertebrate homologs.
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