Ca(2+)-dependent interaction of recoverin with rhodopsin kinase.
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.
Chen
C K
CK
Department of Biochemistry, University of Washington, Seattle 98195, USA.
Inglese
J
J
Lefkowitz
R J
RJ
Hurley
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D051597:Hippocalcin; D008074:Lipoproteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D010786:Photoreceptor Cells / Q000201:enzymology / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051603:Recoverin; D012374:Rod Cell Outer Segment / Q000201:enzymology; D014508:Urea / Q000737:chemistry
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; D051603:Recoverin; D051597:Hippocalcin; D014508:Urea; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium