Molecular Mechanism of Photoreceptor G Protein Signaling

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The long-term goal of this research program is to elucidate the molecular mechanisms underlying transducin signaling in rod and cone photoreceptors. Although a remarkable level of understanding of how transducin functions in the phototransduction cascade has been achieved, the mechanisms underlying the folding of transducin-α (Gαt1) in rod photoreceptors (RPs) remains poorly understood. Defects in protein folding are a common cause of retinal degeneration and blindness; this underscores the need to investigate the folding mechanisms of key photoreceptor proteins including transducin. Evidence has emerged that the protein known as resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) is a chaperone of G-protein α-subunits of the Gαi/o family, to which transducin belongs. Based on our finding that Ric8A is expressed in RPs, we hypothesize that Ric8A is a chaperone for newly synthesized and/or light-translocated Gαt1. In order to elucidate the mechanism of Ric8A chaperone activity, we will investigate the structure and properties of the complex between Gαt1 and Ric8A. The structure of the Gαt1-Ric8A complex in solution will be determined by small angle X-ray scattering (SAXS), using atomic models of Gαt1 and Ric8A as a framework, and distance constraints derived from cross-linking experiments. In parallel, X-ray crystallography will be used to determine high-resolution structures of Ric8A, alone and in complex with Gαt1. Structural information on the Gαt1-Ric8A complex will serve as a starting point for mutational and biochemical analyses of both the protein interface and the chaperone activity of Ric8A. We developed a mouse model in which Ric8A is knocked out specifically in RPs (Ric8AF/FCre+), and our preliminarily data support a role for Ric8A as a Gαt1 chaperone. In parallel with the structural studies, we will investigate the functional significance of Ric8A in RPs by conducting comprehensive examination of this mouse model. In addition, we will examine the potential role of Ric8A as a chaperone of Gαo in rod bipolar cells (RBCs). Elucidation of molecular details of Gαt1 folding by Ric8A is expected to have important implications for retinal diseases and to deepen our understanding of the G-protein chaperone machinery more generally. A second major focus of the proposed research is on the mechanisms whereby synaptic transmission between RPs and RBCs is modulated by light- translocated transducin. Based on our initial finding of Cav1.4 Ca2+ channel activation by transducin- (Gβ1γ1) in HEK293T cells, we hypothesize that Gβ1γ1 modulates signaling at the RP-RBC synapse. The mechanisms underlying the modulation of RP output by Gβ1γ1 will be investigated in biochemical and electrophysiological experiments using in vitro and in vivo approaches. The proposed analysis of synapse modulation by transducin is expected to cause a profound paradigm shift, expanding the role of transducin from the generation of visual signals to the modulation of the RP output.