ABSTRACT
N-terminal acylation of the alpha-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein alpha-subunit, transducin (G alpha(t)), we generated transgenic mice that expressed a mutant G alpha(t) lacking N-terminal acylation sequence (G alpha(t)G2A). Rods expressing G alpha(t)G2A showed a severe defect in transducin cellular localization. In contrast to native G alpha(t), which resides in the outer segments of dark-adapted rods, G alpha(t)G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G alpha(t)G2A at 5-6% of the G alpha(t) levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G alpha(t)G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G alpha(t)G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G alpha(t) membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.
Subject(s)
Fatty Acids/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Transducin/metabolism , Vision, Ocular/physiology , Acylation , Animals , Kinetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Retinal Rod Photoreceptor Cells/chemistry , Retinal Rod Photoreceptor Cells/physiology , Transducin/deficiency , Transducin/geneticsABSTRACT
PURPOSE: To examine the biochemical characteristics of rod and cone arrestin with respect to their ability to quench the activity of light-activated rhodopsin in transgenic mice. METHODS: The mouse rod opsin promoter was used to drive expression of mouse cone arrestin in rod photoreceptor cells of rod arrestin knockout (arr1-/-) mice. Suction electrode recordings from single rods were performed to investigate cone arrestin's ability to quench the catalytic activity of light-activated rhodopsin. In addition, the ability of cone arrestin to prevent light-induced retinal damage caused by prolonged activation of the phototransduction cascade was assessed. RESULTS: Two independent lines of transgenic mice were obtained that expressed cone arrestin in rod photoreceptors, and each was bred into the arr1-/- background. Flash responses measured by suction electrode recordings showed that cone arrestin reduced signaling from photolyzed rhodopsin but was unable to quench its activity completely. Consistent with this observation, expression of mouse cone arrestin conferred dose-dependent protection against photoreceptor cell death caused by low light exposure to arr1-/- retinas, but did not appear to be as effective as rod arrestin. CONCLUSIONS: Cone arrestin can partially substitute for rod arrestin in arr1-/- rods, offering a degree of protection from light-induced damage and increasing the extent of rhodopsin deactivation in response to flashes of light. Although earlier work has shown that rod arrestin can bind and deactivate cone pigments efficiently, the results suggest that cone arrestin binds light-activated, phosphorylated rhodopsin less efficiently than does rod arrestin in vivo. These results suggest that the structural requirements for high-affinity binding are fundamentally distinct for rod and cone arrestins.
Subject(s)
Arrestins/physiology , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Animals , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Electrophysiology , Fluorescent Antibody Technique, Indirect , Gene Expression , Light , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Phosphorylation , Photic Stimulation , Retinal Cone Photoreceptor Cells/radiation effects , Retinal Rod Photoreceptor Cells/radiation effects , Reverse Transcriptase Polymerase Chain Reaction , Rhodopsin/metabolism , Vision, Ocular/physiology , beta-Arrestin 1 , beta-ArrestinsABSTRACT
The Nougaret form of dominant stationary night blindness is linked to a G38D mutation in the rod transducin-alpha subunit (Talpha). In this study, we have examined the mechanism of Nougaret night blindness using transgenic mice expressing TalphaG38D. The biochemical, electrophysiological, and vision-dependent behavioral analyses of the mouse model revealed a unique phenotype of reduced rod sensitivity, impaired activation, and slowed recovery of the phototransduction cascade. Two key deficiencies in TalphaG38D function, its poor ability to activate PDE6 (cGMP phosphodiesterase) and decreased GTPase activity, are found to be the major mechanisms altering visual signaling in transgenic mice. Despite these defects, rod-mediated sensitivity in heterozygous mice is not decreased to the extent seen in heterozygous Nougaret patients.
