The molecular details of phototransduction have been intensively studied over the last ten years and many features of the processes underlying activation have been well characterized. In contrast, the details of the mechanism underlying inactivation and light-activation are not as well understood . The broad aim of the research outlined in this proposal is to elucidate the molecular mechanism of photopigment deactivation by determining the precise role of rhodopsin phosphorylation in this process and identifying the binding sites on the rhodopsin molecule for both rhodopsin kinase and arrestin. The experimental approach will be to use site-directed mutagenesis of rhodopsin in conjunction with an in vitro reconstitution assay system. There are three specific aims of this proposal. First, to identify the amino acid residues whose phosphorylations are essential for deactivation. Second, to determine the amino acid residues that affect the binding of arrestin and rhodopsin kinase to rhodopsin and to determine if these residues also alter the binding of transducin. Third, to determine if constitutively active rhodopsin mutants [opsin molecules that in vitro activate transducin in the absence of chromophore and the absence of light] are inactivated by the same mechanisms as wild type rhodopsin. The experiments described in this grant are significant for they are aimed at understanding the molecular details that underlie photopigment deactivation. In addition, it appears that some forms of retinal degeneration maybe caused by persistent activation of the visual transduction cascade. Therefore, insights into the molecular details of rhodopsin deactivation will certainly lead to an understanding of pathologies that result in constitutive activity and retinal degeneration.
| McDowell, J H; Robinson, P R; Miller, R L et al. (2001) Activation of arrestin: requirement of phosphorylation as the negative charge on residues in synthetic peptides from the carboxyl-terminal region of rhodopsin. Invest Ophthalmol Vis Sci 42:1439-43 |
| Robinson, P R (2000) Assays for detection of constitutively active opsins. Methods Enzymol 315:207-18 |
| Weng, K; Lu, C; Daggett, L P et al. (1997) Functional coupling of a human retinal metabotropic glutamate receptor (hmGluR6) to bovine rod transducin and rat Go in an in vitro reconstitution system. J Biol Chem 272:33100-4 |
| Robinson, P R; Buczylko, J; Ohguro, H et al. (1994) Opsins with mutations at the site of chromophore attachment constitutively activate transducin but are not phosphorylated by rhodopsin kinase. Proc Natl Acad Sci U S A 91:5411-5 |
