Mechanism of rhodopsin kinase activation.
The role of the cytoplasmic loops and C-terminal region of bovine rhodopsin (Rho) in binding and activating rhodopsin kinase was investigated. The ability of various enzymatically truncated forms of photolyzed rhodopsin (Rho*) to stimulate rhodopsin kinase activity was quantified. Following endopeptidase Asp-N cleavage of all phosphorylation sites on the C-terminal, the resulting truncated Rho* (329G-Rho*) was not phosphorylated by rhodopsin kinase. This suggests that rhodopsin kinase only phosphorylates C-terminal sites of Rho*. However 329G-Rho* could bind rhodopsin kinase and stimulate phosphorylation of exogenous peptide. Kinase stimulation was investigated for other truncated forms of Rho* in which the C-terminal region was either partially or completely eliminated, and the V-VI loop was either cleaved or left intact (339K-Rho*, 341E239E-Rho*, 329G239E-Rho*, 327P240S-Rho*). Results suggest that the V-VI loop is crucial for kinase binding (similar to the binding of GT). Mastoparan, a model peptide for G-protein-coupled receptors, was found to stimulate rhodopsin kinase in a mechanism similar to that of truncated Rho*. We conclude that rhodopsin kinase binds to the cytoplasmic loops of Rho* to cause a stimulation of its catalytic activity.
Palczewski
K
K
R.S. Dow Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209.
Buczyłko
J
J
Kaplan
M W
MW
Polans
A S
AS
Crabb
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism
D005136:Eye Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D015293:Transducin