Mechanistic studies on rhodopsin kinase : a farnesylated protein
Mechanistic studies on rhodopsin kinase : a farnesylated protein
At least 3 proteins involved in visual signalling are isoprenylated. In this thesis the role of the farnesyl group in the interaction of rhodopsin kinase with Rho* was investigated. An efficient method was devised for the synthesis of farnesyl peptides whose sequences corresponded to 12 to 24 C-terminal amino acids of bovine rhodopsin kinase and the γ-subunit of transduction. The farnesyl peptides, but not their non-farnesyl counterparts, inhibited the phosphorylation of Rho* by rhodopsin kinase with IC50 values of 500 μM for the farnesyl derivative of residues 544 to 558 of rhodopsin kinase (Far-15-mer RK) and 800 μM for the farnesyl derivative of residues 60 to 71 of the γ-subunit of transducin (Far-12-mer Tγ). The mechanism of this inhibition was studied by spectroscopic methods since the spectral intermediate metarhodopsin II is believed to be equivalent to Rho*. Using ovine rod outer segment membranes a half-life for the decay of metarhodopsin II (λmax 389 nm) into its product, metarhodopsin III (λmax 463 nm), of 3.2 minutes (20oC pH 8.0) was regularly obtained. The farnesyl peptides increased this value; Far-15-mer RK to 5.21 minutes and Far-21-mer Tγ to 8.02 minutes, with non-farnesyl peptides having no effect. Therefore, the farnesyl group of the farnesyl peptides is required for stabilisation of metarhodopsin II, as measured by its half-life, as it is for inhibition of Rho* phosphorylation by rhodopsin kinase. The specificity of these interactions was investigated using a range of detergents (500 μM final concentration). Although detergents had a drastic effect on the stability of metarhodopsin II, these effects were found to be non-specific and were attributed to a general membrane disruption effect. In addition, generally no significant inhibition by detergents of the phosphorylation of Rho* by rhodopsin kinase was observed. These results suggest that farnesyl peptides inhibit the phosphorylation of Rho* by rhodopsin kinase by binding to Rho*.
University of Southampton
McCarthy, Nina Elizabeth Mariam
1998
McCarthy, Nina Elizabeth Mariam
McCarthy, Nina Elizabeth Mariam
(1998)
Mechanistic studies on rhodopsin kinase : a farnesylated protein.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
At least 3 proteins involved in visual signalling are isoprenylated. In this thesis the role of the farnesyl group in the interaction of rhodopsin kinase with Rho* was investigated. An efficient method was devised for the synthesis of farnesyl peptides whose sequences corresponded to 12 to 24 C-terminal amino acids of bovine rhodopsin kinase and the γ-subunit of transduction. The farnesyl peptides, but not their non-farnesyl counterparts, inhibited the phosphorylation of Rho* by rhodopsin kinase with IC50 values of 500 μM for the farnesyl derivative of residues 544 to 558 of rhodopsin kinase (Far-15-mer RK) and 800 μM for the farnesyl derivative of residues 60 to 71 of the γ-subunit of transducin (Far-12-mer Tγ). The mechanism of this inhibition was studied by spectroscopic methods since the spectral intermediate metarhodopsin II is believed to be equivalent to Rho*. Using ovine rod outer segment membranes a half-life for the decay of metarhodopsin II (λmax 389 nm) into its product, metarhodopsin III (λmax 463 nm), of 3.2 minutes (20oC pH 8.0) was regularly obtained. The farnesyl peptides increased this value; Far-15-mer RK to 5.21 minutes and Far-21-mer Tγ to 8.02 minutes, with non-farnesyl peptides having no effect. Therefore, the farnesyl group of the farnesyl peptides is required for stabilisation of metarhodopsin II, as measured by its half-life, as it is for inhibition of Rho* phosphorylation by rhodopsin kinase. The specificity of these interactions was investigated using a range of detergents (500 μM final concentration). Although detergents had a drastic effect on the stability of metarhodopsin II, these effects were found to be non-specific and were attributed to a general membrane disruption effect. In addition, generally no significant inhibition by detergents of the phosphorylation of Rho* by rhodopsin kinase was observed. These results suggest that farnesyl peptides inhibit the phosphorylation of Rho* by rhodopsin kinase by binding to Rho*.
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Published date: 1998
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Local EPrints ID: 463531
URI: http://eprints.soton.ac.uk/id/eprint/463531
PURE UUID: 1b3d4988-d653-4f3a-acd9-9256c9d167f2
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Date deposited: 04 Jul 2022 20:53
Last modified: 04 Jul 2022 20:53
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Author:
Nina Elizabeth Mariam McCarthy
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