Molecular biology of light transduction by the mammalian photoreceptor, rhodopsin

Rhodopsin, the vertebrate photoreceptor, is a prototypic molecule in the la rgest family of G-protein coupled receptors (GPCR). Like all receptors of t his family, it contains three distinct domains: the cytoplasmic (intracellu lar) domain that is involved in all the protein-protein interactions; the t ransmembrane (TM) domain where the signal transduction begins, by light-cat alysed isomerization of Il-cis-retinal to all irans-retinal, and the intrad iscal 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 ar e as follows: 1. Intradiscal domain contains a globular tertiary structure. A central feature is a disulfide bond (Cys110-Cys187) which is conserved i n most of the known GPCR. 2. The correct folding in vivo requires the forma tion of the above disulfide bond. Misfolding resulting in non-retinal bindi ng is frequently caused by Retinitis Pigmentosa (RP) point mutations in the intradiscal and the TM domain. 3. In vivo folding studies, using RP mutati ons in every one of the seven helices, have shown that the packing of the h elices in the TM domain and folding to form the intradiscal tertiary struct ure are coupled. 4. Cysteine mutagenesis has been used systematically to st udy the tertiary structure and light-dependent changes throughout the cytop lasmic face by combination of biochemical and biophysical studies. In parti cular, EPR spectroscopy following spin labeling of selected double cysteine mutants has shown movements in helices, including tilting, following retin al isomerization. 5. Large scale expression of mutants has allowed applicat ion of both F-19-NMR (solution) and MAS solid state NMR tin collaboration w ith Dr. Steve Smith's group, SUNY, Stony Brook). Results of current work ar e promising for detailed study of the conformational change. Finally, a uni fying 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 starti ng with pertubation in the seven helical bundle is fundamentally the same i n all GPCRs. Experiments to test helix movements, the first step in signal transduction following ligand binding in two adrenergic receptors are now f easible. The patterns of helix movements in them will be compared with the pattern demonstrated for rhodopsin and its mutants.