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.

Retinal photoreceptors are highly differentiated postmitotic neurons that transduce photons into electrical signals. While the functions of many photoreceptor-specific genes can be evaluated by direct gene targeting, here we facilitate the studies of nonphotoreceptor-specific genes in these cells by developing an Opsin-iCre transgenic mouse line, iCre-75, in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage P1 Cre recombinase. Immunohistochemical analysis demonstrated that Cre recombinase is present exclusively in the outer nuclear layer of iCre75 mouse retina. Cre expression is found only in rods and not in cones. The expression level reached 188+/-44 ng per retina at postnatal day (pnd) 11 and increased to 687+/-56 ng at 2 months and older. Cre-mediated excision of floxed genomic DNA was absent at pnd 4, became detectable at pnd 7, and was completed by pnd 18. Retinal morphology and electroretinograms were normal in 8-month-old transgenic animals. The iCre-75 transgenic mice are thus suitable for future genetic studies of essential genes in retinal rod photoreceptors.

The Ca 2ϩ -binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca 2ϩ is high, and to be released from the disk membrane in light when Ca 2ϩ is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca 2ϩ -binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.

Light-dependent redistribution of transducin between the rod outer segments (OS) and other photoreceptor compartments including the inner segments (IS) and synaptic terminals (ST) is recognized as a critical contributing factor to light and dark adaptation. The mechanisms of light-induced transducin translocation to the IS/ST and its return to the OS during dark adaptation are not well understood. We have probed these mechanisms by examining light-dependent localizations of the transducin-␣ subunit (Gt␣) in mice lacking the photoreceptor GAP-protein RGS9, or expressing the GTPase-deficient mutant Gt␣Q200L. An illumination threshold for the Gt␣ movement out of the OS is lower in the RGS9 knockout mice, indicating that the fast inactivation of transducin in the wild-type mice limits its translocation to the IS/ST. Transgenic Gt␣Q200L mice have significantly diminished levels of proteins involved in cGMP metabolism in rods, most notably the PDE6 catalytic subunits, and severely reduced sensitivity to light. Similarly to the native Gt␣, the Gt␣Q200L mutant is localized to the IS/ST compartment in light-adapted transgenic mice. However, the return of Gt␣Q200L to the OS during dark adaptation is markedly slower than normal. Thus, the light-dependent translocations of transducin are controlled by the GTP-hydrolysis on Gt␣, and apparently, do not require Gt␣ interaction with RGS9 and PDE6.Heterotrimeric GTP-binding proteins (G proteins) propagate a variety of hormonal and sensory signals from specific cell surface receptors to intracellular effectors (1-3). The visual transduction cascade in vertebrate photoreceptors has served for many years as a paradigm for G protein signaling. In rod photoreceptor cells, illuminated rhodopsin stimulates GTP-GDP exchange on the retinal G protein, transducin (Gt), 2 resulting in dissociation of Gt␣GTP from Gt␤␥ and rhodopsin. Gt␣ in the active GTP-bound conformation stimulates the effector enzyme, cGMP phosphodiesterase (PDE6), by displacing the inhibitory ␥-subunits (P␥) from the PDE6 catalytic core (PDE6␣␤). cGMP hydrolysis by active PDE6 results in closure of cGMP gated channels in the plasma membrane (4, 5). The turn-off phase of the visual signal is determined by reactions controlling the lifetimes of photoexcited rhodopsin (R*) and activated transducin. The catalytic function of R* is blocked by the rhodopsin-kinase mediated phosphorylation and the binding of arrestin to phosphorylated R* (6 -8). The lifetime of Gt␣GTP is controlled by intrinsic GTPase activity. Hydrolysis of GTP switches the Gt␣ molecule to the inactive GDP-bound conformation and allows reinhibition of PDE␣␤ by P␥. RGS9-1, a photoreceptor-specific member of the RGS (regulators of G protein signaling) family, in the complex with G␤5L acts as a GTPase-activating protein for transducin and thus is a major regulator of the turn-off kinetics of the visual signal (9 -11). The RGS9-1/G␤5L complex is anchored to disc membranes through the interaction with R9AP (RGS-9-1-anchor protein) that enhances the complex GAP acti...