We report the development of a method to analyze receptor and β-arrestin2 mobilization between Class A and B GPCRs via time-resolved fluorescent microscopy coupled with semiautomated high-content multiparametric analysis. Using transiently expressed, tagged β2-adrenergic receptor (β2-AR) or parathyroid hormone receptor type 1 (PTH1R), we quantified trafficking of the receptors along with the mobilization and colocalization of coexpressed tagged β-arrestin2. This classification system allows for exclusion of cells with nonoptimal characteristics and calculation of multiple morphological and spatial parameters including receptor endosome formation, β-arrestin mobilization, colocalization, areas, and shape. Stimulated Class A and B receptors demonstrate dramatically different patterns with regard to β-arrestin interactions. The method provides high kinetic resolution measurement of receptor translocation, which allows for the identification of the fleeting β-arrestin interaction found with β2-AR agonist stimulation, in contrast to stronger mobilization and receptor colocalization with agonist stimulation of the PTH1R. Though especially appropriate for receptor kinetic studies, this method is generalizable to any dual fluorescence probe system in which quantification of object formation and movement is desired. These methodologies allow for quantitative, unbiased measurement of microscopy data and are further enhanced by providing real-time kinetics.

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, Go, reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). Go antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, Go is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

Deactivation of light-activated squid rhodopsin was studied in vitro using GTPγS binding by G-protein as a direct measure of rhodopsin activity. Deactivation was inhibited by dilution of the retinal suspension or by removal of soluble components. Deactivation could be restored by addition of soluble material to washed membranes. These results indicate that the deactivation is not due entirely to a conformational transition within rhodopsin itself, but depends on the interaction with other molecules. The possibility that phosphorylation is involved in the deactivation was studied. Deactivation occurred in the presence and absence of added ATP. Deactivation also occurred in the presence of kinase inhibitors and after addition of apyrase, which reduced residual ATP levels to below 1μM. These results indicate that light-induced phosphorylation is not required for deactivation of squid rhodopsin. In this regard deactivation of squid rhodopsin is different from that of vertebrate rhodopsin, which requires phosphorylation.

The immunocytochemical labeling of arrestin (S-antigen) in photoreceptors of the ovine retina was examined following incubation of the retina with hydroxylamine (NH2OH), an agent known to inhibit the phosphorylation of photoactivated rhodopsin. Intact, isolated retinas bathed in medium containing 20 mM NH2OH, or in control medium lacking NH2OH, were maintained in darkness or exposed to bright light for 3 min (dark-adapted and light-adapted conditions, respectively); further incubated in darkness for 10 min; and then fixed and prepared for cryosectioning. Cryosections were incubated with anti-S-antigen monoclonal antibody MAb A2G5; with secondary antibodies that were conjugated with horseradish peroxidase; and with either 3–amino-9–ethyl carbazole or diaminobenzidine as chromogen. Anti-arrestin labeling in cryosections was then analyzed densitometrically using a light-microscopic image processing system. In dark-adapted control retinas, labeling density of the photoreceptor outer segment (OS) layer (0.061 ± 0.004; average ± S.e.m.) was less than that of the inner segment (IS) layer (0.138 ± 0.011). In light-adapted control retinas, OS labeling density (0.139 ± 0.007) exceeded IS labeling density (0.095 ± 0.005). Incubation with NH2OH eliminated this light-dependent increase in labeling of the OS relative to that of the IS, i.e. eliminated the increase in relative OS/IS labeling. Densities of labeling were 0.110 ± 0.006 (OS) and 0.183 ± 0.006 (IS) in NH2OH-treated dark-adapted retinas vs. 0.078 ± 0.004 (OS) and 0.182 ± 0.008 (IS) in NH2OH-treated light-adapted retinas. Anti-arrestin labeling was also examined in retinas that were exposed to 3 min or 13 min of bright light and then immediately fixed. Among retinas incubated in the absence of NH2OH, an increase in OS/IS labeling density was evident after 3 min of illumination, and retinas illuminated for 13 min exhibited an even larger increase in OS/IS labeling. An increase in OS/IS labeling was also exhibited by NH2OH-treated retinas that had been illuminated for 3 min; by comparison with dark-adapted NH2OH-treated controls (average value of OS/IS labeling: 0.60), OS/IS labeling in these illuminated retinas was 0.97. However, OS/IS labeling in NH2OH-treated retinas that had been illuminated for 13 min (average value: 0.35) was lower than that of the dark-adapted controls. The results indicate that, within intact rods, NH2OH inhibits the light-dependent increase in OS/IS anti-arrestin labeling that is ordinarily expressed at long times (~10 min) after major bleaching of the visual pigment. Among the possible bases for the effect of NH2OH are a reduction in the driving force for the movement of arrestin from the inner to the outer segment and/or a facilitation of the degradation of arrestin in the outer segment.

The role of 48-kDa protein in Visual transduction remains unresolved. Two hypotheses for its role in quenching the light activation of cyclic GMP cascade suggest that the protein binds to either phosphodiesterase or phosphorylated rhodopsin. Since the protein is also reported to bind ATP, we anticipated that the protein may have ATP hydrolyzing activity, and in analogy with the GTP-binding protein of the rod outer segments, such activity may be greatly enhanced by the elements of transduction cyclic GMP cascade, permitting the protein to function cyclically as GTP-binding protein does. We found that purified 48-kDa protein hydrolyzes ATP but at a slow rate of 0.04–0.05 per min. The Km for ATP is about 45–65 μM. The activity is inhibited noncompetitively by ADP with a Ki of about 50 μM. The ATPase activity of 48-kDa protein is not affected by rhodopsin, bleached rhodopsin, phosphorylated rhodopsin, unactivated cyclic GMP phosphodiesterase, or phosphodiesterase (PDE) activated by GMP PNP-bound G-protein. These data show that although 48-kDa protein has ATPase activity, lack of regulation of this activity by the elements of visual transduction makes it unlikely for this activity to have a role in quenching the light activation of cyclic GMP cascade.

The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. We found that light increased the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. We speculate that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including β- and γ-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

Arrestins participate in the termination of phototransduction in both vertebrates and invertebrates. However, the visual arrestins of invertebrates and vertebrates differ significantly from one another in that the invertebrate visual arrestins become phosphorylated rapidly in response to light while those in the photoreceptors of vertebrates do not. In an effort to understand the functional relevance of arrestin phosphorylation, we examined this process in the photoreceptors of the horseshoe crab Limulus polyphemus. We report that Limulus visual arrestin can be phosphorylated at three sites near its C-terminus and show that arrestin molecules phosphorylated on one, two, and three sites are normally present in both light- and dark-adapted photoreceptors. Light adaptation increases the amount of arrestin phosphorylated at three sites.

Most animals experience daily changes in light and darkness. The retinas of many of these animals show concomitant rhythmic changes in the levels of mRNAs that encode proteins involved in the photoresponse. These changes may be circadian and independent of light, independent of circadian clocks and regulated by light, or regulated by a circadian clock and light. We have taken advantage of the organization of the Limulus visual system to examine the separate and combined effects of signals from a circadian clock and light on arrestin mRNA levels in photoreceptors. The clock that regulates photoreceptors in the lateral eye of Limulus is in the brain, and signals from the clock reach the lateral eye via activation of a well-characterized efferent projection in the lateral optic nerve. In the experiments described, clock-driven efferent input to the lateral eye was eliminated by cutting the lateral optic nerve, and light input to the lateral eye was eliminated by placing an opaque patch over the eye. Arrestin mRNA levels were quantified relative to 18s rRNA with a ribonuclease protection assay. We observed the following. In lateral eyes exposed to natural diurnal light and endogenous efferent nerve activity, the level of arrestin mRNA was higher during the day in the light than during the night in the dark. Circadian efferent nerve activity was necessary and sufficient to produce normal daily fluctuations in the level of arrestin mRNA. Light influenced arrestin mRNA levels only in eyes with intact and active efferent projections. We conclude that arrestin mRNA levels in lateral eye photoreceptors are controlled entirely by efferent nerve activity, and that light exerts its effects by modulating this output from the circadian clock. Light-stimulated changes in arrestin mRNA in the vertebrate retina may likewise require interactions between light-driven biochemical cascades and clock output.