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Control of rhodopsin multiple phosphorylation
H Ohguro1, R S Johnson, L H Ericsson
1Department of Ophthalmology, University of Washington School of Medicine, Seattle 98195-0001.
Abstract:
The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor at multiple sites near the C-terminus by rhodopsin kinase and binding of a regulatory protein, arrestin. In the present study, the phosphorylation sites were examined in a partially reconstituted system under several experimental conditions. Initial phosphorylation sites were found to be 338Ser, 343Ser, and 334Ser based on analysis by mass spectrometry of proteolytic peptides from the C-terminus. The extent of phosphorylation was found to be limited by two mechanisms: (1) binding of arrestin to phosphorylated rhodopsin (one to three phosphate groups) appeared to prevent further phosphorylation (arrestin has also been observed to promote the initial phosphorylation of rhodopsin at 338Ser in rod outer segment homogenates); and (2) reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol prevented phosphorylation at more than three sites. We propose that previous observations of higher levels of rhodopsin phosphorylation may be the result of the removal of endogenous arrestin, or of exceeding the capacity of retinol dehydrogenase activity by intense bleaches (e.g., by exhausting endogenous NADPH).
Insights
Rhodopsin phosphorylation, crucial for visual signaling inactivation, is primarily regulated by arrestin binding and chromophore reduction. These mechanisms limit the extent of receptor phosphorylation under physiological conditions.
Area of Science:
- Biochemistry
- Molecular Biology
- Vision Science
Background:
- Photolyzed rhodopsin inactivation requires C-terminal phosphorylation by rhodopsin kinase and arrestin binding.
- Understanding these phosphorylation sites is key to elucidating visual transduction regulation.
Purpose of the Study:
- To identify specific phosphorylation sites on rhodopsin.
- To investigate the mechanisms limiting the extent of rhodopsin phosphorylation.
Main Methods:
- Mass spectrometry analysis of C-terminal proteolytic peptides.
- Partially reconstituted system under various experimental conditions.
Main Results:
- Identified initial phosphorylation sites at 338Ser, 343Ser, and 334Ser.
- Arrestin binding (1-3 phosphates) limited further phosphorylation.
- All-trans-retinol formation prevented phosphorylation beyond three sites.
Conclusions:
- Arrestin binding and chromophore reduction are key regulatory mechanisms limiting rhodopsin phosphorylation.
- Previous reports of higher phosphorylation may stem from altered arrestin levels or intense bleaching conditions.