Abstract
Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.
Highlights
Key aspects of the neuronal activity from neurotransmission to gene expression are regulated by changes in the intracellular free calcium concentration
For this purpose we applied the in vitro phosphorylation assay in the reconstituted system consisting of 0.3–0.5 units of rhodopsin kinase (RK) purified from ROS, 10 μM dark-adapted rhodopsin in the content of urea-washed ROS membranes, and various concentrations of calmodulin ranging from 0.01 to 100 μM (Figure 1A)
In the present work we have demonstrated that the ubiquitous Ca2+-sensor calmodulin is able to function as an inhibitor of RK
Summary
Key aspects of the neuronal activity from neurotransmission to gene expression are regulated by changes in the intracellular free calcium concentration. Effects of calcium on various processes in the cell are mediated by Ca2+ sensor proteins. The effects of calcium in different cells of the most tissues are mediated primarily by ubiquitous calcium sensor calmodulin (Hoeflich and Ikura, 2002), belonging to the large group of EFhand calcium-binding proteins. The other EF-hand calcium-sensitive proteins of the neuronal calcium sensor (NCS) and caldendrin/calneuron families are expressed in neurons (McCue et al, 2010). These proteins regulate different aspects of neuronal function including neurotransmitter release, channel and receptor regulation, control of gene transcription, neuronal growth, and survival. Some of the protein targets are common for both calmodulin and NCS proteins and in a number of cases they were shown to compete for the same binding sites in target molecules (Schaad et al, 1996; Haynes et al, 2006; Few et al, 2011)
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