Reconstitution of the vertebrate visual cascade using recombinant heterotrimeric transducin purified from Sf9 cells

For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (T alpha beta gamma) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant T alpha with GTP gamma S. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTP gamma S-loaded recombinant transducin (T(*)) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T(*) was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T(*) suggested that only the complex of PDE with two T(*) molecules, PDE. T(2)(*), was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T(*) in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin.     

Quantitative Aspects of cGMP Phosphodiesterase Activation in Carp Rods and Cones

Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5′-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.     

A Truncated Form of Rod Photoreceptor PDE6 β-Subunit Causes Autosomal Dominant Congenital Stationary Night Blindness by Interfering with the Inhibitory Activity of the γ-Subunit

Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the phototransduction thus leading to the absence of rod adaptation.     

Interaction between the main components of the vertebrate retinal rod phototransduction system in solutions of detergent n-nonyl-β-D-glucoside

cGMP-Specific phosphodiesterase (PDE6) is the key enzyme of the phototransduction system of vertebrate retinal rod outer segments (ROS). The properties of PDE in extracts prepared by solubilization of bovine ROS in a high concentration (0.5% w/v) of detergent n-nonyl-β-D-glucoside (NG) and following centrifugation (ROS-NG) have been studied. Basal PDE activity of the preparations was low, but it greatly (>50-fold) increased (up to ∼20 μmol cGMP hydrolyzed/min per mg rhodopsin (R)) in the presence of trypsin. In bleached GTPγS-containing preparations the specific PDE activity was dependent on ROS-NG concentration and was half-maximal at about 0.8 μM of ROS G protein transducin (Gt). In dark-adapted GTPγS-containing ROS-NG preparations bleaching of 0.2% of the rhodopsin resulted in half-maximal PDE activation. The same result was obtained when PDE in dark-adapted ROS-NG preparations was activated by addition of a highly purified bleached rhodopsin solubilized by 0.5% solution of NG. The results demonstrate that the presence of NG has no significant influence either on the properties of the main ROS phototrans-duction system elements (R, Gt and PDE) or on the interaction between photoactivated R and Gt and suggest that the detergent NG can be used for crystallization of the rhodopsin-transducin complex.     

Low Activation and Fast Inactivation of Transducin in Carp Cones

Cone photoreceptors show lower light sensitivity and briefer light responses than rod photoreceptors. The light detection signal in these cells is amplified through a phototransduction cascade. The first step of amplification in the cascade is the activation of a GTP-binding protein, transducin (Tr), by light-activated visual pigment (R*). We quantified transducin activation by measuring the binding of GTPγS in purified carp rod and cone membrane preparations with the use of a rapid quench apparatus and found that transducin activation by an R* molecule is ∼5 times less efficient in cones than in rods. Transducin activation terminated in less than 1 s in cones, more quickly than in rods. The rate of GTP hydrolysis in Tr*, and thus the rate of Tr* inactivation, was ∼25 times higher in cones than in rods. This faster inactivation of Tr* ensures briefer light responses in cones. The expression level of RGS9 was found to be ∼20 times higher in cones than in rods, which explains higher GTP hydrolytic activity and, thus, faster Tr* inactivation in cones than in rods. Although carp rods and cones express rod- or cone-versions of visual pigment and transducin, these molecules themselves do not seem to induce the differences significantly in the transducin activation and Tr* inactivation in rods and cones. Instead, the differences seem to be brought about in a rod or cone cell-type specific manner.     

Functional modifications of transducin induced by cholera or pertussis-toxin-catalyzed ADP-ribosylation.

Transducin (T alpha beta gamma), the heterotrimeric GTP-binding protein that interacts with photoexcited rhodopsin (Rh*) and the cGMP-phosphodiesterase (PDE) in retinal rod cells, is sensitive to cholera (CTx) and pertussis toxins (PTx), which catalyze the binding of an ADP-ribose to the alpha subunit at Arg174 and Cys347, respectively. These two types of ADP-ribosylations are investigated with transducin in vitro or with reconstituted retinal rod outer-segment membranes. Several functional perturbations inflicted on T alpha by the resulting covalent modifications are studied such as: the binding of T alpha to T beta gamma to the membrane and to Rh*; the spontaneous or Rh*-catalysed exchange of GDP for GTP or guanosine 5-[gamma-thio]triphosphate (GTP[gamma S]), the conformational switch and activation undergone by transducin upon this exchange, the activation of T alpha GDP by fluoride complexes and the activation of the PDE by T alpha GTP. ADP-ribosylation of transducin by CTx requires the GTP-dependent activation of ADP-ribosylation factors (ARF), takes place only on the high-affinity, nucleotide-free complex, Rh*-T alpha empty-T beta gamma and does not activate T alpha. Subsequent to CTx-catalyzed ADP-ribosylation the following occurs: (a) addition of GDP induces the release from Rh* of inactive CTxT alpha GDP (CTxT alpha, ADP-ribosylated alpha subunit of transducin) which remains associated to T beta gamma; (b) CTxT alpha GDP-T beta gamma exhibits the usual slow kinetics of spontaneous exchange of GDP for GTP[gamma S] in the absence of Rh*, but the association and dissociation of fluoride complexes, which act as gamma-phosphate analogs, are kinetically modified, suggesting that the ADP-ribose on Arg174 specifically perturbs binding of the gamma-phosphate in the nucleotide site; (c) CTxT alpha GDP-T beta gamma can still couple to Rh* and undergo fast nucleotide exchange; (d) CTxT alpha GTP[gamma S] and CTxT alpha GDP-AlFx (AlFx, Aluminofluoride complex) activate retinal cGMP-phosphodiesterase (PDE) with the same efficiency as their unmodified counterparts, but the kinetics and affinities of fluoride activation are changed; (e) CTxT alpha GTP hydrolyses GTP more slowly than unmodified T alpha GTP, which entirely accounts for the prolonged action of CTxT alpha GTP on the PDE; (f) after GTP hydrolysis, CTxT alpha GDP reassociates to T beta gamma and becomes inactive. Thus, CTx catalyzed ADP-ribosylation only perturbs in T alpha the GTP-binding domain, but not the conformational switch nor the domains of contact with the T beta gamma subunit, with Rh* and with the PDE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATION*

Agonist stimulation of G protein-coupled receptors (GPCRs) can transactivate epidermal growth factor receptors (EGFRs), but the precise mechanisms for this transactivation have not been defined. Key to this process is the protease-mediated “shedding” of membrane-tethered ligands, which then activate EGFRs. The specific proteases and the events involved in GPCR-EGFR transactivation are not fully understood. We have tested the hypothesis that transactivation can occur by a membrane-delimited process: direct increase in the activity of membrane type-1 matrix metalloprotease (MMP14, MT1-MMP) by heterotrimeric G proteins, and in turn, the generation of heparin-binding epidermal growth factor (HB-EGF) and activation of EGFR. Using membranes prepared from adult rat cardiac myocytes and fibroblasts, we found that MMP14 activity is increased by angiotensin II, phenylephrine, GTP, and guanosine 5′-O-[γ-thio]triphosphate (GTPγS). MMP14 activation by GTPγS occurs in a concentration- and time-dependent manner, does not occur in response to GMP or adenosine 5′-[γ-thio]triphosphate (ATPγS), and is not blunted by inhibitors of Src, PKC, phospholipase C (PLC), PI3K, or soluble MMPs. This activation is specific to MMP14 as it is inhibited by a specific MMP14 peptide inhibitor and siRNA knockdown. MMP14 activation by GTPγS is pertussis toxin-sensitive. A role for heterotrimeric G protein βγ subunits was shown by using the Gβγ inhibitor gallein and the direct activation of recombinant MMP14 by purified βγ subunits. GTPγS-stimulated activation of MMP14 also results in membrane release of HB-EGF and the activation of EGFR. These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation via direct G protein activation of MMP14 and identify MMP14 as a heterotrimeric G protein-regulated effector.     

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.     

Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The delta subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments

The delta subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (). To determine the physiological effect of the delta subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of delta subunit GST fusion protein (delta-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nm delta subunit. Several experiments suggest that this effect was not due to the effects of delta-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous delta-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of delta-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of delta-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the delta-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of delta-GST. These results demonstrate that delta-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the delta subunit may play a similar role in the intact outer segment.     

Regulation of Actin Polymerization in Cell-free Systems by GTPγS and Cdc42

We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPγS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPγS appeared to act via a small G-protein, since it was active in lysates ofD. discoideum mutants missing either the α₂- or β-subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPγS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42.

In a high speed supernatant, GTPγS alone was ineffective, but GTPγS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPγS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP₂ synthesis.

     

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.     

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.     

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.     

A dominant-negative Galpha mutant that traps a stable rhodopsin-Galpha-GTP-betagamma complex

Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex.     

Domain Organization and Conformational Plasticity of the G Protein Effector,PDE6

The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration.     

Stabilization of an intermediate activation state for transducin by a fluorescent GTP analogue

The GTP-binding protein (G protein), transducin, serves as a key molecular switch in vertebrate vision through the tight regulation of its GTP-binding (activation)/GTP hydrolytic (deactivation) cycle by the photoreceptor rhodopsin. To better understand the structure-function characteristics of transducin activation, we have set out to identify spectroscopic probes that bind to the guanine nucleotide-binding site of this G protein and maintain its ability to interact with its specific cellular target/effector, the cyclic GMP phosphodiesterase (PDE). In this study, we describe the characterization of a fluorescently labeled GTP analogue, BODIPY-FL GTPgammaS (BOD-GTPgammaS), that binds to the alpha subunit of transducin (alpha(T)) in a rhodopsin- and Gbetagamma-dependent manner, similar to the binding of GTP or GTPgammaS, with an apparent dissociation constant of 100 nM. The rhodopsin-dependent binding of BOD-GTPgammaS to alpha(T) is slow, relative to the rate of binding of GTPgammaS, particularly under conditions where rhodopsin must act catalytically to stimulate the exchange of BOD-GTPgammaS for GDP on multiple alpha(T) subunits. This reflects a slower rate of dissociation of rhodopsin and Gbetagamma from alpha(T)-BOD-GTPgammaS complexes, relative to their rates of dissociation from alpha(T)-GTPgammaS. The binding of BOD-GTPgammaS occurs without a change in the intrinsic tryptophan fluorescence of alpha(T), indicating that only a subtle movement of the Switch 2 domain on alpha(T) accompanies the binding of this GTPgammaS analogue. Nevertheless, the BOD-GTPgammaS-bound alpha(T) subunit is able to bind with high affinity to the recombinant, purified gamma subunit of PDE (gamma(PDE)) labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS (K(d) approximately 13 nM)), as well as bind to and stimulate the activity of PDE, albeit less efficiently compared to alpha(T)-GTPgammaS. Taken together, these findings suggest that the binding of BOD-GTPgammaS to transducin causes it to adopt a distinct conformation that appears to be intermediate between the inactive and fully active states of alpha(T), and this fluorescent nucleotide analogue can be used as a reporter group to characterize the interactions of alpha(T) in this conformational state with its biological target/effector.     

Rhodopsin kinase and recoverin modulate phosphodiesterase during mouse photoreceptor light adaptation

Light stimulates rhodopsin in a retinal rod to activate the G protein transducin, which binds to phosphodiesterase (PDE), relieving PDE inhibition and decreasing guanosine 3′,5′-cyclic monophosphate (cGMP) concentration. The decrease in cGMP closes outer segment channels, producing the rod electrical response. Prolonged exposure to light decreases sensitivity and accelerates response kinetics in a process known as light adaptation, mediated at least in part by a decrease in outer segment Ca²⁺. Recent evidence indicates that one of the mechanisms of adaptation in mammalian rods is down-regulation of PDE. To investigate the effect of light and a possible role of rhodopsin kinase (G protein–coupled receptor kinase 1 [GRK1]) and the GRK1-regulating protein recoverin on PDE modulation, we used transgenic mice with decreased expression of GTPase-accelerating proteins (GAPs) and, consequently, a less rapid decay of the light response. This slowed decay made the effects of genetic manipulation of GRK1 and recoverin easier to observe and interpret. We monitored the decay of the light response and of light-activated PDE by measuring the exponential response decay time (τ_REC) and the limiting time constant (τ_D), the latter of which directly reflects light-activated PDE decay under the conditions of our experiments. We found that, in GAP-underexpressing rods, steady background light decreased both τ_REC and τ_D, and the decrease in τ_D was nearly linear with the decrease in amplitude of the outer segment current. Background light had little effect on τ_REC or τ_D if the gene for recoverin was deleted. Moreover, in GAP-underexpressing rods, increased GRK1 expression or deletion of recoverin produced large and highly significant accelerations of τ_REC and τ_D. The simplest explanation of our results is that Ca²⁺-dependent regulation of GRK1 by recoverin modulates the decay of light-activated PDE, and that this modulation is responsible for acceleration of response decay and the increase in temporal resolution of rods in background light.     

Kinetics of turn-offs of frog rod phototransduction cascade

The time course of the light-induced activity of phototrandsuction effector enzyme cGMP-phosphodiesterase (PDE) is shaped by kinetics of rhodopsin and transducin shut-offs. The two processes are among the key factors that set the speed and sensitivity of the photoresponse and whose regulation contributes to light adaptation. The aim of this study was to determine time courses of flash-induced PDE activity in frog rods that were dark adapted or subjected to nonsaturating steady background illumination. PDE activity was computed from the responses recorded from solitary rods with the suction pipette technique in Ca²⁺-clamping solution.     

Phosducin regulates the expression of transducin betagamma subunits in rod photoreceptors and does not contribute to phototransduction adaptation

For over a decade, phosducin's interaction with the βγ subunits of the G protein, transducin, has been thought to contribute to light adaptation by dynamically controlling the amount of transducin heterotrimer available for activation by photoexcited rhodopsin. In this study we directly tested this hypothesis by characterizing the dark- and light-adapted response properties of phosducin knockout (Pd−/−) rods. Pd−/− rods were notably less sensitive to light than wild-type (WT) rods. The gain of transduction, as measured by the amplification constant using the Lamb-Pugh model of activation, was 32% lower in Pd−/− rods than in WT rods. This reduced amplification correlated with a 36% reduction in the level of transducin βγ-subunit expression, and thus available heterotrimer in Pd−/− rods. However, commonly studied forms of light adaptation were normal in the absence of phosducin. Thus, phosducin does not appear to contribute to adaptation mechanisms of the outer segment by dynamically controlling heterotrimer availability, but rather is necessary for maintaining normal transducin expression and therefore normal flash sensitivity in rods.