Abstract
Upon illumination, rhodopsin kinase (RK) phosphorylates the visual pigment rhodopsin, which is thought to partially terminate the biochemical events that follow photon absorption. RK enzymology was explored by mutagenesis of the residues Ser488, Thr489 (major autophosphorylation sites), and Lys491 (a distal residue). We found the following to be true. (i) Double mutations at residues Ser488 and Thr489 to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser488 influences the affinity of RK for heparin-Sepharose only moderately, whereas Thr489 and Lys491 are important for this interaction. RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the Km for ATP, suggesting that this region is involved in binding of ATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation.
Highlights
From the Departments of :j:Ophthalmology and §Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195 and the IlDepartments of Medicine (Cardiology) and Biochemistry, Howard Hughes Medical Institute, Duke University, Durham, North Carolina 27710
We found the following to be true. (i) Double mutations at residues Ser488 and Thr48 9 to Ala or Asp decrease autophosphorylation to substoichiometrical levels, while single mutations at either residue independently reduce autophosphorylation by half. (ii) Phosphorylation of residue Ser488 influences the affinity of rhodopsin kinase (RK) for heparinSepharose only moderately, whereas Thr4 89 and Ly s49 1 are important for this interaction
RK K491A does not phosphorylate acidic peptides, suggesting that this residue participates in substrate binding. (iii) Mutations in the autophosphorylation region affect the K m for ATP, suggesting that this region is involved in binding ofATP to the catalytic site. (iv) RK mutants S488A or S488D and RK S488A and T489A have an increased ability to phosphorylate Rho in the dark. (v) Mutations at the autophosphorylation region change the initial site of phosphorylation on photolyzed rhodopsin (Rho*), implying that this region may regulate selectivity of the site of phosphorylation
Summary
From the Departments of :j:Ophthalmology and §Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195 and the IlDepartments of Medicine (Cardiology) and Biochemistry, Howard Hughes Medical Institute, Duke University, Durham, North Carolina 27710. RK enzymology was explored by mutagenesis of the residues Ser488, Thr (major autophosphorylation sites), and Ly s4 9 1 (a distal residue). (ii) Phosphorylation of residue Ser488 influences the affinity of RK for heparinSepharose only moderately, whereas Thr and Ly s49 1 are important for this interaction. (iii) Mutations in the autophosphorylation region affect the K m for ATP, suggesting that this region is involved in binding ofATP to the catalytic site. Rhodopsin kinase (RK)l phosphorylates photoactivated rhodopsin (Rho*) in retinal photoreceptor cells. The phosphorylation of Rho* leads to inhibition of G protein binding (Gt , transducin) through increased binding of the regulatory protein arrestin (Wilden et al, 1986). Gt , RK, and arrestin are functional archetypes for related G protein-coupled transduction systems that involve different types of receptors, G proteins, G protein-coupled receptor kinases (GRKs), and arrestin isoforms Gt , RK, and arrestin are functional archetypes for related G protein-coupled transduction systems that involve different types of receptors, G proteins, G protein-coupled receptor kinases (GRKs), and arrestin isoforms (reviewed by Inglese et at. (1993)
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