Molecular Biology of Light Transduction by the Mammalian Photoreceptor, Rhodopsin

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Abstract
Rhodopsin, the vertebrate photoreceptor, is a prototypic molecule in the largest family of G- protein coupled receptors (GPCR). Like all receptors of this family, it contains three distinct domains: the cytoplasmic (intracellular) domain that is involved in all the protein-protein interactions; the transmembrane (TM) domain where the signal transduction begins, by light- catalysed isomerization of 11-cis-retinal to all trans-retinal, and the intradiscal domain which appears to be involved in a specific tertiary structure. The main focus of this talk is to describe efforts to understand specific structure and function in each domain. The main findings to be presented are as follows: 1. Intradiscal domain contains a globular tertiary structure. A central feature is a disulfide bond (Cys110-Cys187) which is conserved in most of the known GPCR. 2. The correct folding in vivo requires the formation of the above disulfide bond. Misfolding resulting in non-retinal binding is frequently caused by Retinitis Pigmentosa (RP) point mutations in the intradiscal and the TM domain. 3. In vivo folding studies, using RP mutations in every one of the seven helices, have shown that the packing of the helices in the TM domain and folding to form the intradiscal tertiary structure are coupled. 4. Cysteine mutagenesis has been used systematically to study the tertiary structure and light-dependent changes throughout the cytoplasmic face by combination of biochemical and biophysical studies. In particular, EPR spectroscopy following spin labeling of selected double cysteine mutants has shown movements in helices, including tilting, following retinal isomerization. 5. Large scale expression of mutants has allowed application of both 19F-NMR (solution) and MAS solid state NMR (in collaboration with Dr. Steve Smith's group, SUNY, Stony Brook). Results of current work are promising for detailed study of the conformational change. Finally, a unifying hypothesis, which is termed the central dogma in the GPCR field, will be proposed. This states that despite the enormous variation in “accessory” structural details, the principal mechanism of signal transduction starting with pertubation in the seven helical bundle is fundamentally the same in all GPCRs. Experiments to test helix movements, the first step in signal transduction following ligand binding in two adrenergic receptors are now feasible. The patterns of helix movements in them will be compared with the pattern demonstrated for rhodopsin and its mutants.

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