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.

The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca 2؉ regulation of guanylate cyclase (GC) by guanylate cyclaseactivating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs؊͞؊ retinas showed no Ca 2؉ dependence, indicating that Ca 2؉ regulation of GCs had indeed been abolished. Flash responses from darkadapted GCAPs؊͞؊ rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs؊͞؊ rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs؊͞؊ rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range.

Timely deactivation of the alpha-subunit of the rod G-protein transducin (Galphat) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the alpha-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Galphat. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Galphat GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gbeta5-L protein that forms a complex with RGS9-1 was absent from RGS9-/- retinas, although Gbeta5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gbeta5-L, is essential for acceleration of hydrolysis of GTP by Galphat and for normal recovery of the photoresponse.

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK؊͞؊ rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK؊͞؊ mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK؉͞؉) rods or rods from RK؊͞؊ mice raised in constant darkness. One day of constant light caused the rods in the RK؊͞؊ mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.

In retinal rods, Ca(2+) exerts negative feedback control on cGMP synthesis by guanylate cyclase (GC). This feedback loop was disrupted in mouse rods lacking guanylate cyclase activating proteins GCAP1 and GCAP2 (GCAPs(-/-)). Comparison of the behavior of wild-type and GCAPs(-/-) rods allowed us to investigate the role of the feedback loop in normal rod function. We have found that regulation of GC is apparently the only Ca(2+) feedback loop operating during the single photon response. Analysis of the rods' light responses and cellular dark noise suggests that GC normally responds to light-driven changes in [Ca(2+)] rapidly and highly cooperatively. Rapid feedback to GC speeds the rod's temporal responsiveness and improves its signal-to-noise ratio by minimizing fluctuations in cGMP.

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

Visual transduction captures widespread interest because its G-protein signaling motif recurs throughout nature yet is uniquely accessible for study in the photoreceptor cells. The light-activated currents generated at the photoreceptor outer segment provide an easily observed real-time measure of the output of the signaling cascade, and the ease of obtaining pure samples of outer segments in reasonable quantity facilitates biochemical experiments. A quiet revolution in the study of the mechanism has occurred during the past decade with the advent of gene-targeting techniques. These have made it possible to observe how transduction is perturbed by the deletion, overexpression, or mutation of specific components of the transduction apparatus.

For over 30 years, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and G protein signaling. Recently, photoreceptors have become an equally attractive model for studying many facets of neuronal cell biology. The primary goal of this review is to illustrate this rapidly growing trend. We will highlight the areas of active research in photoreceptor biology that reveal how different specialized compartments of the cell cooperate in fulfilling its overall function: converting photon absorption into changes in neurotransmitter release. The same trend brings us closer to understanding how defects in photoreceptor signaling can lead to cell death and retinal degeneration.