The regulation of the cyclic GMP phosphodiesterase by the GDP-bound form of the alpha subunit of transducin

The functional interactions of the retinal G protein, transducin, with the cyclic GMP phosphodiesterase (PDE) have been examined using the different purified subunit components of transducin and the native and trypsin-treated forms of the effector enzyme. The limited trypsin treatment of the PDE removes the low molecular weight gamma subunit (Mr approximately 14,000) of the enzyme, yielding a catalytic moiety comprised of the two larger molecular subunits (alpha, Mr approximately 85,000-90,000; beta, Mr approximately 85,000-90,000), which is insensitive to the addition of either the pure alpha T.GTP gamma S species or the pure beta gamma T subunit complex. However, the addition of the pure alpha T.GDP species to the trypsin-treated PDE (tPDE) results in a significant (90-100%) inhibition of the enzyme activity. This inhibition can be reversed by excess beta gamma T, suggesting that the holotransducin molecule does not (functionally) interact with the tPDE. However, the inhibition by alpha T.GDP is not reversed by the alpha T.GTP gamma S complex, over a range of [alpha T.GTP gamma S] which elicits a marked stimulation of the native enzyme activity, suggesting that the activated alpha T species does not effectively bind to the tPDE. The alpha T.GDP complex also is capable of inhibiting the alpha T.GTP gamma S-stimulated cyclic GMP hydrolysis by the native PDE. This inhibition can be reversed by excess alpha T.GTP gamma S, as well as by beta gamma T, indicating that the binding site for the activated alpha T species is in close proximity and/or overlaps the binding site for the alpha T.GDP complex on the enzyme. Overall, these results are consistent with a scheme where (a) both the small and larger molecular weight subunits of PDE participate in alpha T-PDE interactions, (b) the activation of PDE by the alpha T.GTP gamma S (or alpha T.GTP) species does not result in the complete dissociation of the gamma subunit from the enzyme, and (c) the deactivation of this signal transduction system results from a direct interaction between the alpha T.GDP species and the catalytic moiety of the effector enzyme.     

Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.     

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)     

Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].     

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes.

To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.     

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.     

G protein-effector coupling: binding of rod phosphodiesterase inhibitory subunit to transducin

The cyclic GMP phosphodiesterase of retinal rods is composed of three distinct polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). In this multimeric form, the enzyme is inhibited. Its activity is stimulated by the interaction with the GTP-bound form of the T alpha subunit of transducin and reversed upon the recombination of the inhibitory gamma subunit with the catalytic alpha beta subunit. We show here by a novel coimmunoprecipitation technique that the gamma subunit, but not the alpha beta subunit, forms a 1:1 complex with T alpha. The binding of gamma to T alpha is nucleotide-dependent and is facilitated by GTP gamma S or Gpp(NH)p. This study provides convincing evidence that the T alpha-GTP subunit of transducin stimulates phosphodiesterase activity by binding to gamma and physically carrying it away from alpha beta.     

Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated alpha-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade.

Resonance energy-transfer approaches have been used to directly monitor the interactions of the GTP gamma S-bound alpha subunit of transducin (alpha T GTP gamma S) with the retinal cyclic GMP phosphodiesterase (PDE). The PDE was labeled with 5-(iodoacetamido) fluorescein (IAF-PDE) and served as the fluorescence donor in these experiments while the alpha T GTP gamma S was labeled with eosin-5-isothiocyanate (EITC-alpha T GTP gamma S) and served as the energy acceptor. The EITC-alpha T GTP gamma S species was able to quench a significant percentage of the IAF-PDE fluorescence (typically greater than or equal to 30%) due to resonance energy transfer between the IAF and EITC moieties. The quenching by the EITC-alpha T GTP gamma S species was dose-dependent, saturable (Kd = 21 nM), and specific for the GTP gamma S-bound form of the alpha T subunit. Limited trypsin treatment of the IAF-PDE, which selectively removes a fluorescein-labeled gamma subunit (gamma PDE), completely eliminates the quenching of the IAF fluorescence by the EITC-alpha T GTP gamma S complex. Although the EITC-alpha T GTP gamma S complex competes with the unlabeled alpha T GTP gamma S for a binding site on the IAF-PDE, as well as for a site on the native PDE, it is not able to stimulate PDE activity. Thus, the modification of a single EITC-reactive residue on the alpha T GTP gamma S complex prevents this subunit from eliciting a key activation event within the retinal effector enzyme.     

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Activated cGMP phosphodiesterase of retinal rods. A complex with transducin alpha subunit.

Purified G-protein (transducin) activated with the nonhydrolyzable analog guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and cGMP phosphodiesterase (PDE) from retinal rods are added to protein-stripped disc membranes. Specific binding of the mainly soluble alpha subunit of G-protein with GTP gamma S bound (G alpha GTP gamma S, activator of the PDE) to the disc membrane in the presence of PDE is measured from gel scans or experiments with labeled G-protein alpha subunit (G alpha). Its variation as a function of G concentration matches the theoretical variation of G alpha involved in the activation of PDE calculated with previously estimated dissociation constants (Bennett, N., and Clerc, A. (1989) Biochemistry 28, 7418-7424), and the G alpha bound/PDE ratio at saturation is close to 2. No increase of G alpha binding to the membrane is observed when purified inhibitory subunit of PDE (PDE gamma) is added together with or instead of total PDE, and excess PDE gamma remains soluble. These results suggest that activated PDE is a complex with the activator G alpha GTP rather than PDE from which the inhibitory subunits have been removed. A method for purifying PDE gamma with a high yield of recovery and activity is described.     

The role of nucleoside-diphosphate kinase reactions in G protein activation of NADPH oxidase by guanine and adenine nucleotides

NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.     

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.     

ADP-ribosylation of transducin by pertussis toxin

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.     

Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Role of the C-terminal domain of the beta-subunit.

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.     

An antibody-induced enhancement of the transducin-stimulated cyclic GMP phosphodiesterase activity

In this work we have characterized the ability of a carboxyl peptide-specific antibody (AS/7), raised against the alpha subunit of transducin (alpha T), to potentiate the stimulation of the cyclic GMP phosphodiesterase (PDE) by transducin. The complexation of the purified guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-bound form of alpha T (alpha T.GTP gamma S) with AS/7 results in a 2-5-fold enhancement in the total levels of cyclic GMP hydrolysis measured after 1 min. This potentiation by AS/7 cannot be attributed simply to an increase in the apparent affinity of alpha T.GTP gamma S for the effector enzyme, nor to an increased affinity of the enzyme for the substrate cyclic GMP. The AS/7-induced potentiation is specific for alpha T.GTP gamma S-PDE interactions; this antibody has no effect on the activity of the trypsin-activated PDE nor on the ability of the GDP-bound form of alpha T to inhibit the trypsin-activated enzyme (Kroll, S., Phillips, W. J., and Cerione, R. A. (1989) J. Biol. Chem. 264, 4490-4497). Phosphatidylcholine vesicles also will enhance the alpha T.GTP gamma S-stimulated PDE activity (1.5-2-fold) relative to that measured in the absence of a lipid milieu. However, the potentiations of alpha T-stimulated cyclic GMP hydrolysis elicited by AS/7 and lipids represent separate events. Titration profiles describing the AS/7-induced potentiation, as a function of the amount of antibody added to the assay mixtures, indicate that maximal activity occurs when there is one molecule of AS/7 per two molecules of alpha T.GTP gamma S; the AS/7-induced potentiation is lost when AS/7 much greater than alpha T. GTP gamma S, i.e. conditions which favor the formation of monovalent AS/7-alpha T.GTP gamma S complexes. When the AS/7 is papain-treated to yield monovalent antibody molecules, complexation between these monovalent antibodies and alpha T still occurs (as reflected by the ability of these antibodies to block rhodopsin-alpha T coupling); however, the potentiation of the alpha T.GTP gamma S-stimulated PDE activity is lost. Taken together, these results suggest that the AS/7-induced potentiation of alpha T-stimulated activity is dependent on the bivalent nature of the antibody, and maximal stimulation of PDE activity is achieved by the interactions of two activated-alpha T molecules with a single molecule of PDE.     

Retinal cyclic-GMP phosphodiesterase gamma-subunit: identification of functional residues in the inhibitory region.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides. The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues # 24-45 and an inhibitory region for PDE alpha/beta within residues # 80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, peptides of PDE gamma # 63-87 and mutant peptides were synthesized and utilized in PDE inhibition assays. The following peptides exhibited a decreased ability to inhibit PDE alpha/beta: All were from PDE gamma # 63-87; PDE gamma Tyr 84----Gly, PDE gamma Phe 73----Gly and PDE gamma Gln 83----Gly.     

Rhodopsin-stimulated activation-deactivation cycle of transducin: kinetics of the intrinsic fluorescence response of the alpha subunit

The intrinsic tryptophan fluorescence of the alpha subunit of transducin (alpha T) has been shown to be sensitive to the binding of guanine nucleotides, with the fluorescence being enhanced by as much as 2-fold upon the binding of GTP or nonhydrolyzable GTP analogues [cf. Phillips and Cerione (1988) J. Biol. Chem. 263, 15498-15505]. In this work, we have used these fluorescence changes to analyze the kinetics for the activation (GTP binding)-deactivation (GTPase) cycle of transducin in a well-defined reconstituted phospholipid vesicle system containing purified rhodopsin and the alpha T and beta gamma T subunits of the retinal GTP-binding protein. Both the rate and the extent of the GTP-induced fluorescence enhancement are dependent on [rhodopsin], while only the rate (and not the extent) of the GTP gamma S-induced enhancement is dependent on the levels of rhodopsin. Comparisons of the fluorescence enhancements elicited by GTP gamma S and GTP indicate that the GTP gamma S-induced enhancements directly reflect the GTP gamma S-binding event while the GTP-induced enhancements represent a composite of the GTP-binding and GTP hydrolysis events. At high [rhodopsin], the rates for GTP binding and GTPase are sufficiently different such that the GTP-induced enhancement essentially reflects GTP binding. A fluorescence decay, which always follows the GTP-induced enhancement, directly reflects the GTP hydrolytic event. The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP. The GTP-induced fluorescence changes (i.e., the enhancement and ensuing decay) could be fit to a simple model describing the activation-deactivation cycle of transducin. The results of this modeling suggest the following points: (1) the dependency of the activation-deactivation cycle on [rhodopsin] can be described by a simple dose response profile; (2) the rate of the rhodopsin-stimulated activation of multiple alpha T(GDP) molecules is dependent on [rhodopsin] and when [alpha T] greater than [rhodopsin], the activation of the total alpha T pool may be limited by the rate of dissociation of rhodopsin from the activated alpha T(GTP) species; and (3) under conditions of optimal rhodopsin-alpha T coupling (i.e., high [rhodopsin]), the cycle is limited by GTP hydrolysis with the rate of Pi release, or any ensuing conformational change, being at least as fast as the hydrolytic event.     

Retinal cyclic-GMP phosphodiesterase gamma-subunit: use of mutant synthetic peptides to define function.

Previously, we have domain-mapped the 87 amino acid PDE gamma inhibitory subunit of the retinal phosphodiesterase (PDE) alpha beta gamma 2 complex using synthetic peptides (1). The PDE gamma subunit has a binding domain for transducin-alpha (T alpha) and for PDE alpha/beta within residues #24-45. An inhibitory region for PDE alpha/beta is within residues #80-87. In order to establish the role of individual amino acids in the function of the PDE gamma inhibitory subunit, mutants were synthesized and utilized in PDE inhibition assays. The following mutants exhibited a decreased ability to inhibit PDE alpha/beta: Tyr84----Gly; Arg24----Gly; and Arg33----Pro. Sequence comparisons with cone PDE gamma indicate that there is identity within these functional regions.