Identification of the N-terminal region in rhodopsin kinase involved in its interaction with rhodopsin.
Upon illumination rhodopsin kinase (RK) phosphorylates the visual pigment, rhodopsin, in a reaction that is thought to terminate in part the biochemical events that follow photon absorption. In this paper, RK was studied to assign functional regions to the primary structure of the enzyme. Peptides derived from the sequence of RK were used to prepare site-specific anti-peptide antibodies against: 1) the N-terminal region located between residues 17 and 34, which contains an autophosphorylation site; 2) the Lys/Arg-rich region corresponding to residues 216-237 near the catalytic domain; 3) the region located between residues 483 and 497, which encompasses the major autophosphorylation sites; and 4) the C-terminal region located between residues 539 and 556, close to the isoprenylation site of RK. Antibodies also were raised against purified RK. Application of the antibodies directed against the N-terminal domain blocks RK activity toward Rho*, but has no affect on the phosphorylation of a synthetic peptide substrate. Additionally, a significant portion of the inhibitory effect seen with an antibody directed against whole RK could be reversed by the peptide derived from the N-terminal region. We conclude that the N-terminal region of RK contains a sequence involved in the recognition of photolyzed Rho. Furthermore, the inhibition of RK activity eliminates the effect of ATP during the inactivation of cGMP phosphodiesterase, implying that RK is a necessary component of a cascade of reactions involved in the quenching of phototransduction. Light microscopic immunocytochemistry using these antibodies revealed that RK was localized to the rod and cone outer segments of human and bovine retinas.
Department of Ophthalmology, University of Washington, Seattle 98195.
K
K
Palczewski
J
J
Buczyłko
L
L
Lebioda
J W
JW
Crabb
A S
AS
Polans
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001120:Arginine / Q000737:chemistry; D002417:Cattle; D002853:Chromatography, Liquid; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010766:Phosphorylation; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000906:Antibodies; D005136:Eye Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D012243:Rhodopsin; D001120:Arginine; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D008239:Lysine