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Abstract
Recoverin (Rv) is a myristoylated Ca(2+)-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca(2+)-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca(2+)-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca(2+)-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as "high gain" phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 microM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca(2+)-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as beta-adrenergic receptor kinase 1 nor inhibits beta-adrenergic receptor kinase 1 activity. The specific and Ca(2+)-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation.
Highlights
Teins that participate in several biochemical reactions
Identification ofRetinal Proteins That Bind to Recoverin in a Ca2 + -dependent Manner-To identify proteins that bind Rv, either C14:0-Rv or Nonacylated Rv (NA-Rv) was linked to CNBr-activated Sepharose to make an immobilized Rv column
The column was rinsed with 1 mM Ca2+, and Ca2 + -dependent Rv-binding proteins were eluted with 5 mM EGTA
Summary
Teins that participate in several biochemical reactions. Low Ca2 + concentrations stimulate resynthesis ofcGMP by the action of Ca2+ -binding proteins that stimulate guanylyl cyclase (Koch and Stryer, 1988; Gorczyca et al, 1994; Dizhoor et al, 1994). When internally dialyzed into rod outer segments (ROS), Rv prolongs the photoresponse (Gray-Keller et al, 1993) This in vivo effect ofRv is consistent with the in vitro observation that S-modulin, a frog homologue of Rv, enhances the effect of light on cGMP phosphodiesterase and inhibits rhodopsin phosphorylation (Kawamura, 1993). Ca2 + -dependent Recoverin I Rhodopsin Kinase Interaction dopsin phosphorylation (Chen and Hurley, 1994; Kawamura et al, 1994) This suggests that binding ofRv to ROS membranes is not necessary for its inhibitory effect. When Rv is added to our reconstituted system, high gain phosphorylation is reduced in a Ca2 +-titratable manner
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