Direct binding of visual arrestin to a rhodopsin carboxylterminal synthetic phosphopeptide

[摘要] Purpose: The phosphorylated carboxyl terminus of rhodopsin isrequired for the stable binding of visual arrestin to the full lengthrhodopsin molecule. Phosphorylation of the carboxyl terminus has beenshown to induce conformational changes in arrestin, which promote itsbinding to the cytoplasmic loops of rhodopsin. However, it has not beendetermined whether phosphorylation is also responsible for the directbinding of the rhodopsin carboxyl terminus to arrestin. To furtherinvestigate the role of rhodopsin phosphorylation on arrestin binding,surface plasmon resonance was used to measure the interaction between asynthetic phosphopeptide corresponding to the carboxyl terminus ofrhodopsin and visual arrestin in real time.Methods: Synthetic peptides were generated that correspond to thephosphorylated and nonphosphorylated carboxyl terminus of bovinerhodopsin. These peptides were immobilized on a biosensor chip and theirinteraction with purified visual arrestin was monitored by surfaceplasmon resonance on a BIAcore 2000 or 3000.Results: A synthetic peptide phosphorylated on residuescorresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovinerhodopsin was sufficient for direct binding to visual arrestin. Incontrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342and a nonphosphorylated synthetic peptide were not able to bindarrestin. A peptide fully substituted at all serine and threonineresidues with glutamic acid was unable to substitute forphosphorylation.Conclusions: Surface plasmon resonance is a sensitive method fordetecting small differences in affinity. We were successful in usingthis technique to detect differences in the affinity of phosphorylatedand nonphosphorylated rhodopsin peptides for visual arrestin. The datasuggest that these are low-affinity interactions and indicate thatphosphorylation is responsible for the direct binding of the rhodopsincarboxyl terminus to visual arrestin. Four phosphorylated residues aresufficient for this interaction. Because the affinity of the syntheticphosphopeptide for arrestin is substantially lower than the full lengthrhodopsin molecule, the cytoplasmic loops and rhodopsin carboxylterminus appear to interact in a cooperative manner to stably bindarrestin.

[学科分类] 生物化学/生物物理