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PubMed2003

Light-dependent translocation of arrestin in the absence of rhodopsin phosphorylation and transducin signaling.

Visual arrestin plays a crucial role in the termination of the light response in vertebrate photoreceptors by binding selectively to light-activated, phosphorylated rhodopsin. Arrestin localizes predominantly to the inner segments and perinuclear region of dark-adapted rod photoreceptors, whereas light induces redistribution of arrestin to the rod outer segments. The mechanism by which arrestin redistributes in response to light is not known, but it is thought to be associated with the ability of arrestin to bind photolyzed, phosphorylated rhodopsin in the outer segment. In this study, we show that light-driven translocation of arrestin is unaffected in two different mouse models in which rhodopsin phosphorylation is lacking. We further show that arrestin movement is initiated by rhodopsin but does not require transducin signaling. These results exclude passive diffusion and point toward active transport as the mechanism for light-dependent arrestin movement in rod photoreceptor cells.

Animals↗
PubMed1992

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.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

The rod transducin alpha subunit amino terminus is heterogeneously fatty acylated.

Rod transducin (Tr), a heterotrimeric GTP-binding protein composed of alpha, beta, and gamma subunits, couples photolysis of rhodopsin to the activation of cyclic GMP phosphodiesterase in the vertebrate visual signal transduction cascade. To determine if T alpha r is covalently modified, we analyzed tryptic fragments of bovine retinal T alpha r using electrospray mass spectrometry, liquid chromatography/mass spectrometry, tandem mass spectrometry, and gas chromatography. A novel heterogeneous fatty acylation was detected at the NH2 terminus. Four types of NH2-terminal tryptic fragments of T alpha r were isolated, and each contained either a lauroyl (C12:0), myristoyl (C14:0), (cis-delta 5)-tetradecaenoyl (C14:1) or (cis,cis-delta 5, delta 8)-tetradecadienoyl (C14:2) fatty acyl residue amide-linked to the NH2-terminal glycine residue. NH2-terminal fatty acylation does not anchor T alpha r permanently in the membrane, since T alpha r used in these experiments was eluted without detergent from rod outer segment membranes.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Enhanced GTPase activity of transducin when bound to cGMP phosphodiesterase in bovine retinal rods.

The generation of the physiological response of a retinal rod cell to an incident photon involves activation of a cGMP phosphodiesterase (PDE) by a GTP-binding protein, transducin (T). This activation has been shown to occur by formation of a membrane-bound T alpha GTP-PDE complex (Clerc, A., and Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627; Catty, P., Pfister, C., Bruckert, F., and Deterre, P. (1992) J. Biol. Chem 267, 19489-19493). The recovery of the response involves turning-off of T by its intrinsic GTPase activity. We show here that the formation of the membrane-bound T alpha GTP-PDE complex correlates with an enhanced rate of GTP hydrolysis. In vivo, this would provide an appropriate mechanism for fast turn-off of cGMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

N-myristoylation of the rod outer segment G protein, transducin, in cultured retinas.

Bovine retinas incubated with [3H]myristic acid incorporated detectable radiolabel into only a few proteins. The most heavily labeled was the alpha subunit of the rod outer segment G protein transducin (Gt alpha). The radiolabeled protein was specifically eluted from illuminated membranes in the presence of GTP, displaying the unique solubility properties of Gt alpha. It comigrated with Gt alpha in electrophoresis and chromatography and was immunoprecipitated by Gt alpha-specific antibodies. The radiolabel was confirmed by hydrolysis, chemical derivatization, and chromatography to be amide-linked myristic acid. The solubility of the myristoylated Gt alpha indicates that myristoylation is not sufficient to cause tight membrane association of this normally membrane-bound subunit. Incorporation of [3H]myristate was blocked by the protein synthesis inhibitor cycloheximide, suggesting that that fatty acid group is introduced during or soon after translation in the rod inner segment.

Animals↗
PubMed1992

Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy.

In this work we have used fluorescence spectroscopic approaches to examine the binding of the beta gamma T subunit complex of transducin to the photoreceptor, rhodopsin. To do this, we have covalently labeled the beta gamma T subunit complex with the environmentally sensitive fluorescent cysteine reagent 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). By using the MIANS moiety as a fluorescent reporter group, we were able to monitor directly the binding of the MIANS-beta gamma T complex to light-activated rhodopsin, which was reconstituted into phosphatidylcholine vesicles, through an enhancement (30-50%) in the MIANS fluorescence. Phosphatidylcholine vesicles, alone, elicited only minor changes in the MIANS-beta gamma T fluorescence (i.e. less than 10% enhancement), whereas the addition of rhodopsin in the absence of lipid vesicles and in minimal detergent fully mimicked the effects of reconstituted rhodopsin and caused a significant enhancement of the MIANS fluorescence. The interactions between the MIANS-beta gamma T complex and rhodopsin also resulted in a quenching of the rhodopsin tryptophan fluorescence (approximately 30%), which most likely reflected resonance energy transfer between the tryptophan residues and the MIANS moieties. The binding of the MIANS-beta gamma T species to the alpha T subunit was accompanied by an enhancement of the MIANS fluorescence (30-50%) and a slight blue shift of the emission maximum, as described previously (Phillips, W. J., and Cerione, R. A. (1991) J. Biol. Chem. 266, 11017-11024). However, the alpha T-induced enhancement of the MIANS-beta gamma T fluorescence was not additive with the enhancement elicited by rhodopsin. Conditions which resulted in the activation of the alpha T subunit reversed the alpha T-induced enhancement of the MIANS emission, whereas the rhodopsin-induced enhancement persisted, thereby suggesting that the rhodopsin-beta gamma T complex can remain intact throughout the G protein activation event. Studies with synthetic peptides representing different regions of the cytoplasmic domain of rhodopsin demonstrated that a portion of the putative carboxyl-terminal tail (amino acid residues 310-324) was capable of eliciting changes in the MIANS-beta gamma T fluorescence as well as inhibiting the MIANS-beta gamma T-induced quenching of the rhodopsin tryptophan fluorescence. These results suggest that this region of the rhodopsin molecule may constitute a portion of the binding domain for the beta gamma T complex.

Anilino Naphthalenesulfonates↗
PubMed1992

Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.

Aluminofluoride and beryllofluoride complexes can activate the heterotrimeric G-proteins by binding next to GDP in the nucleotide site of their G alpha subunit and acting as analogs of the gamma-phosphate of a GTP. However, the exact structures of the activatory complexes in solution as well as those of the bound complexes in the nucleotide site are still disputed. We have studied, by monitoring the activation-dependent tryptophan fluorescence of transducin T alpha subunit, the pF (-log[F-]) and pH dependencies of the kinetics of activation and deactivation of T alpha GDP in the presence of NaF and aluminum or beryllium salts. Comparisons were made with the calculated pF and pH dependencies of the distribution of the metallofluoride complexes, in order to identify the activating species. We observed that the contribution of a magnesium-dependent mechanism of activation by fluoride (Antonny, B., Bigay, J., and Chabre, M. (1990) FEBS Lett. 268, 277-280) and effects due to slow equilibration kinetics between various aluminofluoride complexes could give rise to puzzling kinetics that had caused misinterpretations of previous results. Once corrected for these effects, our results suggest that with aluminum AlF3(OH)- is, rather than AlF4-, the main activating species and that the bound form of the complex is tetracoordinated GDP-AlF3. Deactivation kinetics depend on the free fluoride concentration in the medium, suggesting that the simple bimolecular scheme: T alpha GDP-AlF3 in equilibrium with T alpha GDP+AlF3(OH) does not fully describe the interaction. Fluorides in the bound complexes can also exchange with free F- ions in solution. With beryllium, two complexes are activatory: BeF3-.H2O and BeF2(OH)-.H2O. In the nucleotide site these give two tetracoordinated complexes, GDP.BeF3 and GDP.BeF2(OH), as shown by their different dissociation rates.

Aluminum↗
PubMed1991

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.

Animals↗
PubMed1990

Pineal transduction. Adrenergic----cyclic AMP-dependent phosphorylation of cytoplasmic 33-kDa protein (MEKA) which binds beta gamma-complex of transducin.

Adrenergic regulation of phosphorylation of pineal proteins was studied. Norepinephrine treatment of intact pinealocytes incubated with 32Pi enhanced phosphorylation of a 33-kDa phosphoprotein (33PP). The effect of NE was rapid, sustained, and appeared to be mediated by a beta-adrenergic----cyclic AMP mechanism. Studies using broken cell preparations revealed that 33PP was phosphorylated by cyclic AMP-dependent protein kinase (PKA). It was also possible to demonstrate PKA-dependent phosphorylation of the 33-kDa protein in cytosol from rat retina and in cow and sheep pineal glands. Two-dimensional polyacrylamide gel electrophoresis revealed that 33PP is acidic (pI congruent to 4.5), appears to exist as two isoforms with slightly different charge, and has the same mobility as the retinal 33-kDa PKA substrate. Immunological analysis indicated 33PP in both tissues is a previously reported 33-kDa protein (MEKA); this protein is a PKA substrate which has been reported to form a cytoplasmic complex with the beta gamma complex of transducin. Consistent with this, it was possible to identify the beta-subunit in pineal cytoplasm and in the same congruent to 70-kDa gel permeation fraction which contained the 33-kDa protein identified as MEKA. Thus, it appears possible that MEKA is present in pineal cytoplasm in a 70-kDa complex with G beta gamma, as is the case in retina. The finding of MEKA in the pineal makes it the latest addition to a family of retinal/pineal proteins which are thought to have evolved from a common ancestral photochemical transduction system.

Animals↗
PubMed1990

Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound.

An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.

Animals↗
PubMed1990

Displacement of rhodopsin by GDP from three-loop interaction with transducin depends critically on the diphosphate beta-position.

We have studied the effect of GDP and its analog guanyl-5'-yl thiophosphate (GDP beta S) on the interaction between rhodopsin and transducin (Gt). Stabilization of the light-induced active intermediate, metarhodopsin II (MII), by bound Gt (extra-MII effect) monitored the catalytic interaction between the proteins. Extra-MII can be completely abolished by GDP, with a half-suppression at 10 microM under the conditions (4 degrees C, pH 8, 7.5 nM photoactivated rhodopsin). The effect of GDP did not depend on divalent cations, in contrast to GTP-induced dissociation of the complex. The GDP analog GDP beta S did not affect extra-MII although it binds to the MII-Gt complex with only three times lower affinity (reversal of the GDP effect by GDP beta S). However, GDP beta S enhanced considerably the efficiency of synthetic rhodopsin peptide competition against the formation of extra-MII. GDP and GDP beta S slow the Gt activation rate (monitored by kinetic light scattering), with the same relative efficiencies. We therefore assume that GDP, GDP beta S, and GTP bind at the same site. We discuss a generalized induced fit mechanism, where MII induces opening of the Gt nucleotide site and release of GDP which in turn is obligatory to establish the MII-stabilizing rhodopsin-Gt three-loop interaction (König, B., Arendt, A., McDowell, J.H., Kahlert, M., Hargrave, P.A., and Hofmann, K.P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6878-6882). The GDP beta S/GDP difference is discussed in terms of bound GDP disturbing the interaction with two and GDP beta S with only one of the rhodopsin binding sites. Mechanistically, our results indicate a critical role of the beta-phosphate interaction with the nucleotide binding site in the GDP-induced transformation of Gt.

Animals↗
PubMed1987

Mechanism of action of monoclonal antibodies that block the light activation of the guanyl nucleotide-binding protein, transducin.

Seven monoclonal antibodies to the alpha subunit (G alpha) of the frog photoreceptor guanyl nucleotide-binding protein (transducin or G-protein) have been characterized as to their effect on G-protein function, and this has been correlated in the accompanying paper (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) with the antibody-binding sites on G alpha tryptic fragments. Antibodies 4A, 7A, 7B, 7C, and 7D are members of a class of antibodies that block G-protein activation by light and therefore also block activation of the cGMP phosphodiesterase. All these blocking antibodies also block the interaction of G-protein with rhodopsin as measured by the light-scattering "binding signal," and as measured by the stabilization of meta-rhodopsin II by bound G-protein (extra-meta-rhodopsin II). The antibodies (or Fab fragments) also solubilize G alpha beta gamma from the membrane in the dark under isosmotic conditions and thus interfere with G alpha interaction with the membrane. Antibody 4A also blocks the extra-meta-rhodopsin II generated by G-protein-rhodopsin interaction in detergent solubilized membranes. Thus, even in the absence of phospholipids, antibody 4A blocks G-protein-rhodopsin interaction. Therefore, we suggest that the antibodies recognize a region of G alpha involved with binding to rhodopsin. An alternative hypothesis is that this antigenic site is a region of interaction between the alpha and beta gamma subunits, disruption of this interaction leading to removal of both the alpha and beta gamma subunits from the membrane and blocking interaction with rhodopsin. This does not seem to be the case because the antibodies immunoprecipitate the alpha beta gamma complex, and not just the alpha subunit. Other antibodies, 4C and 4H, do not block phosphodiesterase activation, the light-scattering signal, extra-meta-rhodopsin II formation, or interaction with the membrane in the dark and therefore recognize other sites on G alpha.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Topographic analysis of antigenic determinants recognized by monoclonal antibodies to the photoreceptor guanyl nucleotide-binding protein, transducin.

Antigenic sites for six monoclonal antibodies that bind to the alpha subunit (G alpha) of the photoreceptor guanyl nucleotide-binding protein (G-protein or transducin) have been determined. Five of these antibodies (4A, 7A, 7B, 7C, and 7D) were shown in the preceding paper (Hamm, H. E., Deretic, D., Hofmann, K. P., Schleicher, A., and Kohl, B. (1987) J. Biol. Chem. 262, 10831-10838) to block G-protein-rhodopsin interaction. We have blotted tryptic and chymotryptic peptides of G-protein to nitrocellulose paper and found that these antibodies bind to peptides that contain the COOH-terminal end of the protein assessed by 32P-ADP-ribosylation of the COOH-terminus by pertussis toxin. The antigenic site is not exactly at the COOH-terminus since the antibodies also bind two peptides which lack a 2-kDa piece from the COOH-terminus. Antigenic sites are therefore on the 7-kDa chymotryptic peptide and 5-kDa tryptic peptide more than 2 kDa away from the COOH-terminus. Further evidence for this antigenic site comes from the ability of these antibodies to block pertussis toxin-mediated ADP-ribosylation while still binding to the previously ADP-ribosylated protein both on nitrocellulose blots and in immunoprecipitations. Antibody 4H, which was shown not to interrupt any of the functions studied, binds to the 11-kDa major tryptic fragment. To aid in the mapping of these sites onto the surface of G alpha, a model of the three-dimensional structure of G alpha has been generated using the G alpha primary sequence, predicted secondary structure, hydropathy plot, and the constraints of the GDP-binding site of the GTP-binding protein elongation factor Tu solved by Jurnak (Jurnak, F. (1985) Science 230, 32-36).

Adenosine Diphosphate Ribose↗
PubMed1985

[Effect of bacterial toxins on the GTPase activity of transducin from bovine rod outer segments].

The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD-GTP complex.

Animals↗
PubMed1986

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system.

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1988

A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin.

In structure-function studies on bovine rhodopsin by in vitro site-specific mutagenesis, we have prepared three mutants in the cytoplasmic loop between the putative transmembrane helices E and F. In each mutant, charged amino acid residues were replaced by neutral residues: mutant 1, Glu239----Gln; mutant 2, Lys248----Leu; and mutant 3, Glu247----Gln, Lys248----Leu, and Glu249----Gln. The mutant rhodopsin genes were expressed in monkey kidney (COS-1) cells. After the addition of 11-cis-retinal to the cells, the rhodopsin mutants were purified by immunoaffinity adsorption. Each mutant gave a wild-type rhodopsin visible absorption spectrum. The mutants were assayed for their ability to stimulate the GTPase activity of transducin in a light-dependent manner. While mutants 1 and 3 showed wild-type activity, mutant 2 (Lys248----Leu) was inactive.

Amino Acid Sequence↗