Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.     

A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination.

Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.     

Phosphorylation of rhodopsin and quenching of cyclic GMP phosphodiesterase activation by ATP at weak bleaches

Light and GTP-dependent cyclic GMP phosphodiesterase activation of rod disk membranes is rapidly quenched by ATP. Maximum speed of this effect occurs only with the weakest bleaches. Though it has been proposed that ATP mediates its effect through rapid phosphorylation of bleached rhodopsin, previous workers have found phosphorylation kinetics too slow by more than an order of magnitude to be causal in quenching of cyclic GMP phosphodiesterase activation. In this report, we use preparations retaining more endogenous rhodopsin kinase, higher specific activity ATP, and cyclic GMP phosphodiesterase quenching conditions to show that ATP-dependent multiple phosphorylation of rhodopsin at very weak bleaches (10(-5)) is complete in less than 2 s, easily compatible with cyclic GMP phosphodiesterase quench times of 4 s measured under identical conditions. Thus, it seems likely that previous efforts to achieve high 32P counts by using large bleaches have produced conditions of substrate saturation where much longer times to completion are caused by a very large ratio of substrate to enzyme velocity. Such conditions are not appropriately compared to those that support rapid quenching. We conclude that the speed of rhodopsin phosphorylation is, in fact, adequate to explain ATP quenching of cyclic GMP phosphodiesterase activation.     

Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system

Rhodopsin kinase was extracted into a buffer containing 200 mM KCl and no MgCl2. The activity of the enzyme was stabilized with the use of a mixture of protease inhibitors, aprotinin, benzamidine, leupeptin, and pepstatin. The extract consisted of three major proteins of molecular weight (Mr) 65,000, 56,000, and 37,000, of which the Mr 65,000 protein was identified with the kinase activity since preparations containing the other proteins had no kinase activity and the Mr 65,000 protein was phosphorylated when the extract was incubated with ATP. A reconstituted cGMP phosphodiesterase (PDE) system consisting of peripheral protein-depleted rod disk membranes (RDM), GTP binding protein (G-protein), and PDE was used to test the effectiveness of the rhodopsin kinase preparation in mediating the ATP-dependent quench of light activation of PDE. In the absence of kinase, light-activated PDE activity lasted several minutes. In its presence, ATP and to a lesser extent GTP quenched the activation about as rapidly as in rod disk membranes. The influence of kinase was unaffected by increasing G-protein or PDE content of the reconstituted system but was slowed down by brighter flashes, showing that quench was caused by the inactivation of bleached rhodopsin and not of PDE or G-protein.     

Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments.

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.     

Cholesterol modulation of photoreceptor function in bovine retinal rod outer segments

Rhodopsin, a prototypical G protein receptor, is found both in the plasma membrane and in discs of bovine rod outer segments. The ability of each of these membranes to activate phosphodiesterase upon stimulation by light in the presence of GTP and cGMP was investigated. The plasma membrane showed little or no activity when compared with disc membranes. The plasma membrane contains approximately 28 mol% cholesterol compared to 8 mol % found in discs. Upon oxidation of at least 70 % of the cholesterol in the plasma membrane to cholestenone, the phosphodiesterase activity in the plasma membrane approached that initiated by the disc membranes. When a 50:50 mixture of disc and plasma membrane rhodopsin was tested for phosphodiesterase activity, the results were found to be additive. Therefore, cholesterol is implicated in regulation of the receptor activity.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

Chemical probing of the light-induced interaction between rhodopsin and G-protein. Near-infrared light-scattering and sulfhydryl modifications

Near-infrared light scattering and gel electrophoresis were used to monitor the interaction between rhodopsin and G-protein at photoreceptor disc membranes. It is found that substitution of one SH hydrogen at the G alpha-subunit by N-ethylmaleimide or thionitrobenzoate still allows dark binding of the G-unit to the membrane but blocks its light binding to rhodopsin. Repair of the modification restores the interaction of the proteins. Evidence is provided that substitution by the less bulky cyanide is also able to remove the hindrance to light binding. The rhodopsin-G interaction is reduced in proportion to bound N-ethylmaleimide, and the interaction kinetics remain constant over the measurable range. GTP/GDP exchange at the G-protein after interaction with rhodopsin does not reduce the accessibility of the relevant SH group. In contrast to the effect of G-protein modification, even exhaustive modification of rhodopsin has no effect on the dark and light binding of G-protein.     

Activation mechanism of rod outer segment cyclic GMP phosphodiesterase. Release of inhibitor by the GTP/GTP-binding protein

The physiological regulation of light-activated cyclic GMP phosphodiesterase (EC 3.1.4.17) in rod outer segments has been shown to depend upon a heat-stable inhibitor and upon the reversal of its effect by a specific GTP/GTP-binding protein complex (Hurley, J. B. (1980) Biochem. Biophys. Res. Commun. 92, 505-510; Yamazaki, A., Bartucca, F., Ting, A., and Bitensky, M. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3702-3706). Washing of illuminated disc membranes with an isotonic buffer released 86% of the peripheral proteins without any release of inhibitor. Subsequent washing with the same isotonic buffer containing GTP released 80% of the inhibitor. When inhibitor was eluted with guanosine-5'-(beta, gamma-imino)triphosphate, it had an apparent molecular weight of 60,000 on Sephadex G-100. The release of inhibitor by guanosine-5'-(beta, gamma-imino)triphosphate was also demonstrated with sucrose density gradient centrifugation. Inhibitor release from the disc membrane by GTP or its analogue was accompanied by the release of the GTP-binding protein and an increased phosphodiesterase activity in the membrane. However, following GTP hydrolysis, both inhibitor and GTP-binding protein returned to the membrane and phosphodiesterase activity in the membrane decreased proportionally. In contrast, incubation of disc membranes with guanosine-5'-(beta, gamma-imino)-triphosphate produced an increase of inhibitor activity in the supernatant and an increase of phosphodiesterase activity in the pellet which remained constant after the initial increase. These data clearly show that the activation of phosphodiesterase by the GTP/GTP-binding protein complex resulted from the release of inhibitor. Hydrolysis of GTP resulted in the reassociation of inhibitor with and concomitant inhibition of disc membrane phosphodiesterase.     

Effect of calmodulin on the structural state of photoreceptor membranes and rhodopsin-containing phospholipid vesicles

The effect of calmodulin on the order of lipids in rhodopsin-free and rhodopsin-containing membranes has been studied using spin-label electron spin resonance methods. Calmodulin, up to 10(-6)M, did not change the measured order of lipids in bilayer membranes containing only rhodopsin. However, for bovine rod outer segment disc membranes, which contain rhodopsin and other proteins, calmodulin induced a significant concentration and temperature dependent increase in the order of the membrane lipids. This suggests that the site of calmodulin binding is remote from rhodopsin itself, and the nature of the binding appears to be a membrane surface phenomenon.     

Cyclic nucleotide dependent protein kinase and the phosphorylation of endogenous proteins of retinal rod outer segments.

Cyclic nucleotide dependent protein kinase has been extracted wiht Tris or Lubrol PX from purified rod outer segments (ROS) of bovine retina. The activity of the enzyme is unaffected by light but is stimulated by either cyclic guanosine 3',5'-monophosphate (cGMP) or cyclic adenosine 3',5'-monophosphate (cAMP). Most of the solubilized enzyme elutes from DEAE-cellulose with about 0.18 M NaCl (type II protein kinase). An endogenous 30,000 molecular weight protein of the soluble fraction of ROS as well as exogenous histone are phosphorylated by the protein kinase in a cyclic nucleotide dependent manner. The Tris-extracted enzyme can be reassociated in the presence of Mg2+ with ROS membranes that are depleted of protein kinase activity. The reassociated protein kinase is insensitive to exogenous cyclic nucleotides, and it catalyzes the phosphorylation of the membrane protein, bleached rhodopsin. While the soluble and membrane-associated protein kinases may be interchangeable, they appear to be modulated by different biological signals; soluble protein kinase activity is increased by cyclic nucleotides whereas membrane-bound activity is enhanced when rhodopsin is bleached by light.     

Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments

Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

cGMP influences guanine nucleotide binding to frog photoreceptor G-protein

A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light-induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light-induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

Mechanism of ATP quench of phosphodiesterase activation in rod disc membranes

GTP-dependent light activation of cyclic GMP phosphodiesterase in bovine rod disc membranes was quenched by ATP. ATP reduced both initial velocity (V0) and turn off time (toff) of phosphodiesterase activated by a flash that bleached 1.5 X 10(-5) of the rhodopsin present. In the absence of rhodopsin kinase, ATP had no effect on either V0 or toff of reconstituted preparations containing phosphodiesterase and GTP*-binding protein. Addition of partially purified rhodopsin kinase to such reconstitutions again permitted ATP to quench both initial velocity and turn off time. It is thus likely that kinase-mediated phosphorylation of bleached rhodopsin reduces and arrests light-induced phosphodiesterase activation. Thermolysin cleavage of rhodopsin's COOH-terminal dodecapeptide eliminated ATP's effect on toff, but did not diminish its effect on V0. Thus, the effects of ATP and kinase on V0 may be mediated by sites proximal to and effects on toff by sites distal to the thermolysin cleavage point at rhodopsin's COOH-terminal end.     

The effect of GDP on rod outer segment G-protein interactions

The role of GDP has heretofore been little studied in the analysis of visual receptor G-protein (G) interactions. Here we use kinetically resolved absorption and light scattering spectroscopy, centrifugation, porous membrane filtration, and enzyme assay to compare the effectiveness of GDP with that of GTP or gamma-thio-guanosine-5'-triphosphate in the modulation of G-protein binding to rod disc membranes and activated receptor (R*). We also compare effectiveness of GDP with that of GTP in the separation of G alpha and G beta gamma subunits and in activation of effector, cGMP phosphodiesterase. We find that when different nucleotide affinities are taken into account, actions such as the release of G from R* binding, earlier ascribed to GTP alone, are also typical of GDP. The principal specific actions of GTP that occur only weakly or undetectably for GDP are, respectively, the release of G-protein subunits from the membrane into solution and activation of phosphodiesterase. While GDP, like GTP, releases G-protein binding to receptor, we argue that GDP cannot mediate G-protein subunit separation, even on the membrane surface. GDP retained on G-protein after GTP hydrolysis may function to prevent tight binding to quiescent receptors in a manner analogous to its action on G-protein binding to activated receptors. Weak binding of G.GDP may function to accelerate receptor catalyzed amplification during transduction.     

Stochastic simulation of activation in the G-protein cascade of phototransduction.

Activation of the G-protein cascade underlying phototransduction has been modeled by simulating the two-dimensional diffusional interactions that occur at the rod disc membrane between the three reacting protein species, which are the activated rhodopsin (R*), the G-protein (G), and the effector protein (E, the phosphodiesterase, PDE). The stochastic simulations confirm the main predictions of a simplified analytical model (Lamb, T. D., and E. N. Pugh, 1992, Journal of Physiology 449:719-758), and extend that treatment to more complicated cases, where there is a finite probability of reaction or a finite time for reaction. The simulations also provide quantitative estimates of the efficiency of coupling from activated G-protein (G*) to activated effector (E*) in terms of the concentrations, lateral diffusion coefficients, and binding rate constants of the participating molecules; the efficiency of coupling from G* to E* is found to be not as high as in the previous simplified analytical theory. The findings can be extended to other G-protein cascades, provided that the physical parameters of those cascades are specified.     

Light-induced dephosphorylation of two proteins in frog rod outer segments: influence of cyclic nucleotides and calcium

Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with 32Pi. The proteins, designated component I (13,000 daltons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains approximately 10(6( molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 x 10(5) rhodopsin molecules/outer segment per second. Light which bleaches 5.0 x 10(3) rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J. Gen Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977. J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the light-induced dephosphorylation is observed only in intact retinas.