Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.
Although experience-dependent changes in neural circuits are commonly assumed to be mediated by synaptic plasticity, modifications of intrinsic excitability may serve as a complementary mechanism. In whole-cell recordings from spontaneously firing vestibular nucleus neurons, brief periods of inhibitory synaptic stimulation or direct membrane hyperpolarization triggered long-lasting increases in spontaneous firing rates and firing responses to intracellular depolarization. These increases in excitability, termed firing rate potentiation, were induced by decreases in intracellular calcium and expressed as reductions in the sensitivity to the BK-type calcium-activated potassium channel blocker iberiotoxin. Firing rate potentiation is a novel form of cellular plasticity that could contribute to motor learning in the vestibulo-ocular reflex.
Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein  subunit (G5). RGS9⅐G5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or G5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9⅐G5 complex, and therefore all three proteins, RGS9, G5, and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.Timely termination of the light response in retinal photoreceptors is essential for normal vision (reviewed in Refs. 1 and 2). On the molecular level, the normal time course of the light response requires rapid deactivation of the G protein transducin, which relays the visual signal to the effector, cyclic GMP phosphodiesterase. Deactivation of transducin occurs when the transducin ␣ subunit hydrolyzes its bound GTP. In normal rods, GTP hydrolysis is catalyzed by the complex of the regulator of G protein signaling protein (RGS9) 1 with type 5 G protein  subunit (G5) (reviewed in Refs. 2 and 3). Recent studies have demonstrated that photoreceptors lacking RGS9 or G5 produce light responses that recover at an abnormally slow rate (4, 5).In photoreceptors, the RGS9⅐G5 complex is tightly associated with the transmembrane protein R9AP (RGS9 anchor protein), which anchors RGS9⅐G5 on the surface of the disc membranes of the outer segment, which is the subcellular compartment where visual transduction occurs (6 -8). R9AP is a 25-kDa protein structurally related to members of the SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) protein family, which are involved in vesicular trafficking and exocytosis (8 -10). In mammals, R9AP is expressed predominantly in the retina (6, 9), whereas in chicken it is also present in cochlear hair cells and dorsal root ganglion neurons (9). R9AP dramatically enhances the ability of RGS9⅐G5 to stimulate transducin GTPase (7,8,10) and participates in the delivery of RGS9⅐G5 to photoreceptor outer segment (10).In this study, we analyzed visual signaling in rods of R9AP knockout mice. The knockout did not affect the overall retinal morphology or photoreceptor development. However, light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or G5 knockout rods. The effect of the R9AP knockout on the photoresponse recovery was explained by a...
Timely deactivation of G-protein signaling is essential for the proper function of many cells, particularly neurons. Termination of the light response of retinal rods requires GTP hydrolysis by the G-protein transducin, which is catalyzed by a protein complex that includes regulator of G-protein signaling RGS9-1 and the G-protein beta subunit Gbeta5-L. Disruption of the Gbeta5 gene in mice (Gbeta5-/-) abolishes the expression of Gbeta5-L in the retina and also greatly reduces the expression level of RGS9-1. We examined transduction in dark- and light-adapted rods from wild-type and Gbeta5-/- mice. Responses of Gbeta5-/- rods were indistinguishable in all respects from those of RGS9-/- rods. Loss of Gbeta5-L (and RGS9-1) had no effect on the activation of the G-protein cascade, but profoundly slowed its deactivation and interfered with the speeding of incremental dim flashes during light adaptation. Both RGS9-/- and Gbeta5-/- responses were consistent with another factor weakly regulating GTP hydrolysis by transducin in a manner proportional to the inward current. Our results indicate that a complex containing RGS9-1-Gbeta5-L is essential for normal G-protein deactivation and rod function. In addition, our light adaptation studies support the notion than an additional weak GTPase-accelerating factor in rods is regulated by intracellular calcium and/or cGMP.
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