GTPASE ACTIVATING COMPLEX FROM ROD PHOTORECEPTORS

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The overall objective of this proposal is to understand the molecular mechanism that governs the duration of G protein signaling in photoreceptor cells. The photoreceptor G protein, transducin, plays a central role in vertebrate phototransduction where it conveys a signal from the activated receptor, photoexcited rhodopsin, to its effector, cGMP phosphodiesterase. Transducin is activated when photoexcited rhodopsin catalyses an exchange of GDP for GTP on the transducin alpha subunit and continues to stimulate PDE activity until the bound GTP is hydrolyzed. Studies conducted by this and other laboratories have shown that photoreceptors contain several proteins which accelerate the slow intrinsic GTPase activity of transducin to a rate sufficient for timely photoresponse recovery. These proteins include the immediate target of transducin, the gamma subunit of cGMP phosphodiesterase and the complex between the ninth member of the Regulators of G protein Signaling protein family (RGS9) and the long splice variant of type 5 G protein beta subunit (Gpbeta5L). Understanding the exact molecular mechanism by which members of this protein ensemble contribute to the activation of transducin GTPase in a coordinated manner is the major goal of this proposal. Aim 1 is to establish a role and mechanism of action for each protein and their functional domains in regulating transducin GTPase. Aim 2 is to understand the nature of an unusually tight association of the RGS9-Gbeta5L complex with photoreceptor membranes and to test possibility that this association is mediated via interactions with an unknown transmembrane protein. Aim 3 will utilize transgenic mouse technology to understand which component of the GTPase activating ensemble acts as the rate-limiting factor in physiologically intact rod photoreceptors. Addressing this problem will not only contribute to the understanding of the molecular mechanism of photoresponse recovery, but also will allow us to establish a general framework for investigating the regulation of signal duration in other intracellular signaling pathways. The work is also relevant to understanding of how biochemical pathways responsible for ensuring normal duration of the photoresponse may be affected in various inherited eye diseases.