Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T alpha subunit. There was no significant incorporation on the T beta gamma subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp(NH)p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of T alpha and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp(NH)p complex was about 3-fold slower than that of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the T alpha subunit. These results indicate that FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of T alpha, which ultimately leads to the activation of cGMP phosphodiesterase.     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Characterization of transducin from bovine retinal rod outer segments. Use of monoclonal antibodies to probe the structure and function of the subunit

A panel of monoclonal antibodies has been developed against the T alpha, T beta and T gamma subunits of bovine transducin. Two anti-T alpha antibodies from this panel (TF15 and TF16) and a third one (4A) against frog T alpha (Witt, P. L., Hamm, H. E., and Bownds, M. D. (1984) J. Gen. Physiol. 84, 251-263) were characterized. Each of these monoclonal antibodies recognizes a different region of T alpha and has a specific effect on the function of transducin. The binding of TF15 is reversibly enhanced by treating T alpha with either 1 M guanidinium chloride or, to a smaller extent, by the removal of bound guanine nucleotide. Its epitope is located in a 12-kDa tryptic fragment containing the binding site for the guanine moiety of GTP. Taken together, these results support previous observations that the conformation of T alpha is modulated by the occupancy of the guanine nucleotide binding site. In contrast to TF15, TF16 recognizes only the native form of T alpha. Its epitope resides within the central portion of the T alpha molecule. While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin. These effects are unlikely to be caused by the occupation of the guanine nucleotide binding site by TF16 because this antibody quantitatively precipitates T alpha-GTP gamma S. We propose that bound TF16 locks T alpha in a conformation that prevents the entrance of guanine nucleotide and favors T beta gamma association. In contrast to TF16, the epitope of 4A was mapped to the amino-terminal region of T alpha. This monoclonal antibody blocks pertussis toxin-catalyzed ADP-ribosylation, GTP gamma S uptake, and T alpha-T beta gamma association. Moreover, the binding site for 4A becomes inaccessible when transducin binds to photolyzed rhodopsin. These results suggest that the inhibitory effects of 4A are due to a simultaneous steric blockage of both the interaction of T alpha with T beta gamma and their binding to photolyzed rhodopsin. The results obtained from these studies are correlated with the structure and function of T alpha.     

Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups

The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

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.     

Regulation of retinal transducin by C-terminal peptides of rhodopsin.

Transducin is a multi-subunit guanine-nucleotide-binding protein that mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase in retinal rod outer segments. Whereas the T alpha subunit of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunit may function to link physically T alpha with photolysed rhodopsin. In order to determine the binding sites of rhodopsin to transducin, we have synthesized eight peptides (Rhod-1 etc.) that correspond to the C-terminal regions of rhodopsin and to several external and one internal loop region. These peptides were tested for their inhibition of restored GTPase activity of purified transducin reconstituted into depleted rod-outer-segment disc membranes. A marked inhibition of GTPase activity was observed when transducin was pre-incubated with peptides Rhod-1, Rhod-2 and Rhod-3. These peptides correspond to opsin amino acid residues 332-339, 324-331 and 317-321 respectively. Peptides corresponding to the three external loop regions or to the C-terminal residues 341-348 did not inhibit reconsituted GTPase activity. Likewise, Rhod-8, a peptide corresponding to an internal loop region of rhodopsin, did not inhibit GTPase activity. These findings support the concept that these specific regions of the C-terminus of rhodopsin serve as recognition sites for transducin.     

A structural model for the alpha-subunit of transducin. Implications of its role as a molecular switch in the visual signal transduction mechanism

Transducin is a GTP-binding protein which mediates the light activation signal from photolyzed rhodopsin to cGMP phosphodiesterase and is pivotal in the visual excitation process. Biochemical studies suggest that the T alpha subunit of transducin is composed of three functional domains, one for rhodopsin/T beta gamma interaction, another for guanine nucleotide binding, and a third for the activation of phosphodiesterase. The integration of the primary sequence of T alpha along with secondary structure, hydropathy and folding topology predictions, and a comparison with homologous proteins have led to the construction of a three-dimensional model of the T alpha subunit. A molecular mechanism which underlies the coupling action of T alpha is suggested on the basis of this model.     

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.     

Characterization of transducin from bovine retinal rod outer segments. Participation of the amino-terminal region of T alpha in subunit interaction

The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.     

Light-regulated biochemical events in invertebrate photoreceptors. 1. Light-activated guanosinetriphosphatase, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes

The occurrence of a guanine nucleotide binding protein activated by squid rhodopsin was established by examination of GTPase activity, guanine nucleotide binding, and cholera toxin catalyzed labeling of squid photoreceptor membranes. Purified squid (Loligo opalescens) photoreceptors exhibited GTPase activity that increased 3-4-fold by illumination. Half-maximal GTPase activity was observed when 2% of the rhodopsin was photoconverted to metarhodopsin. The Km of the light-regulated activity was 1 microM GTP. Binding of the hydrolysis-resistant GTP analogue guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] was enhanced greater than 10 times by illumination. A protein, Mr 44 000, was identified as a component of the light-activated guanine nucleotide binding protein/GTPase through its specific labeling with [32P]NAD catalyzed by cholera toxin: light increased the extent of 32P incorporation 7-fold. The addition of ATP to the membrane suspension enhanced labeling, while guanine nucleotides inhibited labeling with the relative potency GTP gamma S much greater than GDP greater than GTP greater than Gpp(NH)p. The 44 000-dalton protein was membrane bound irrespective of variations in ionic strength and divalent ion concentration over a wide range. These results suggest that a G protein, which incorporates both GTP binding and hydrolysis functions, is intimately involved in the visual process of invertebrate photoreceptors.     

Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.     

An antibody directed against the carboxyl-terminal decapeptide of the alpha subunit of the retinal GTP-binding protein, transducin. Effects on transducin function

An antibody (AS/7) prepared against the carboxyl-terminal decapeptide of the alpha subunit of transducin (alpha T) has been used in various reconstitution studies aimed at characterizing the role of the carboxyl-terminal domain in the different functional activities of transducin. The peptide-specific antibody is a potent inhibitor of the rhodopsin-stimulated GTPase activity in phospholipid vesicle systems containing pure rhodopsin and pure holo-transducin, or rhodopsin and the purified alpha T and beta/gamma (beta gamma T) subunit components, with the highest levels of inhibition (80-95%) occurring under conditions where the molar ratio of holo-transducin (or alpha T) to AS/7 approximately equal to 1. The inhibition of the receptor-stimulated GTPase does not represent an interference in the interactions between the alpha T subunit and the beta gamma T complex, since essentially identical levels of inhibition are observed when AS/7 is preincubated with either free alpha T, holo-transducin, or alpha T in the presence of excess beta gamma T, prior to assay. The AS/7-induced inhibition also does not appear to reflect an alteration in the ability of alpha T to bind or hydrolyze GTP and, in fact, the incubation of alpha T with AS/7 results in a stimulation of the intrinsic GTPase activity for alpha T alone (i.e. in the absence of rhodopsin). Thus, we conclude that the inhibition of the rhodopsin-stimulated GTPase activity by AS/7 is due to the direct blocking (by the antibody) of rhodopsin-alpha T interactions. While AS/7 is capable of uncoupling rhodopsin-transducin interactions, it appears to promote the stimulation of the cyclic GMP phosphodiesterase (PDE) by an activated alpha T subunit. Specifically, when the pure alpha T-guanosine 5-O-(3-thiotriphosphate) (alpha TGTP gamma S) species is preincubated with AS/7 prior to its addition to an assay solution containing PDE, there is at least a 4-fold increase in the resultant cyclic GMP hydrolysis relative to the activities measured with alpha TGTP gamma S, alone, or with alpha TGTP gamma S preincubated with nonimmune (control) rabbit IgG. The AS/7-induced promotion is specific for the active form of alpha T; the inactive alpha TGDP species does not stimulate PDE activity either in the presence or absence of the antibody. The different effects by AS/7 on the various activities of the alpha T subunit highlight the existence of distinct functional domains on alpha T.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase

Work in several laboratories has shown that Gi, the inhibitory guanyl nucleotide-binding protein of the adenylate cyclase system, is similar in many ways to transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system. Separated subunits of purified transducin, T alpha (approximately 39 kDa) and T beta gamma (approximately 35 and approximately 10 kDa), do not exhibit GTPase activity; GTPase activity is observed when the subunits are combined in the presence of rhodopsin ( Fung , B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502). Subunits of Gi, Gi alpha (approximately 41 kDa), and Gi beta gamma (approximately 35 and approximately 10 kDa) were prepared from rabbit liver membranes. It was found that Gi beta gamma could replace T beta gamma in reconstituting the rhodopsin-stimulated GTPase activity of T alpha. Gi alpha exhibited rhodopsin-stimulated GTPase activity when reconstituted with Gi beta gamma or T beta gamma. GTPase activity was a function of Gi alpha concentration when Gi beta gamma or T beta gamma was constant, and the GTPase activity of a given amount of Gi alpha was dependent on Gi beta gamma concentration. These studies demonstrate that the GTPase activity of Gi resides in Gi alpha and further establish that Gi alpha and Gi beta gamma are functionally analogous to T alpha and T beta gamma, respectively.     

Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.

In these studies we have investigated the role of the beta gamma T subunit complex in promoting the rhodopsin-stimulated guanine nucleotide exchange reaction (i.e. the activation event) of the alpha T subunit. The results of these studies demonstrate that although the beta gamma T subunit complex increases the association of the alpha T subunit with lipid vesicles that lack the photoreceptor, the beta gamma T complex is not necessary for the binding of alpha T to lipid vesicles containing rhodopsin, provided sufficient amounts of rhodopsin are present. The rhodopsin-promoted GDP/guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) exchange reaction, within the rhodopsin-alpha T complex, then results in the dissociation of the alpha TGTP gamma S species from the rhodopsin-containing phospholipid vesicles. A second line of evidence for the occurrence of rhodopsin/alpha T interactions, in the absence of beta gamma T, comes from phosphorylation studies using the beta 1 isoform of protein kinase C. The phosphorylation of the alpha T subunit by protein kinase C is inhibited by beta gamma T, both in the absence and in the presence of rhodopsin, but is enhanced by rhodopsin in the absence of beta gamma T. These rhodopsin-alpha T complexes also appear to be capable of undergoing a rhodopsin-stimulated guanine nucleotide exchange event. When the guanine nucleotide exchange is allowed to occur prior to the addition of protein kinase C, the phosphorylation of the alpha T subunit is inhibited. Although beta gamma T is not absolutely required for the rhodopsin/alpha T interaction, it appears to increase the apparent affinity of the alpha T subunit for rhodopsin, both when rhodopsin was inserted into phosphatidylcholine vesicles and when soluble lipid-free preparations of rhodopsin were used. This results in a significant kinetic advantage for the rhodopsin-stimulated guanine nucleotide exchange event, such that the addition of beta gamma T causes a 10-fold promotion of the rhodopsin-stimulation [35S]GTP gamma S binding to alpha T after 1 min but provides less than a 20% promotion of the rhodopsin-stimulated binding after 1 h. The ability of beta gamma T to increase the association of alpha T with the lipid vesicle surface does not appear to contribute significantly to the ability of rhodopsin to couple functionally to alpha T subunits, and there appears to be no requirement for beta gamma T in the alpha T activation event, once the rhodopsin-alpha T complex has formed.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.     

Beta gamma-subunit of bovine transducin composed of two components with distinctive gamma-subunits

During the process of transduction of a photon signal in vertebrate rod outer segments, transducin, a guanine nucleotide binding protein, mediates between a photobleaching intermediate of rhodopsin and a cGMP-phosphodiesterase. We report here that the beta gamma-subunit of bovine transducin (T beta gamma) characterized so far consists of two components (T beta gamma-1 and T beta gamma-2), which can be separated by anion exchange chromatography under nondenaturing conditions. Both components consisted of two polypeptides of Mr 36,000 (T beta) and about 8,000 (T gamma) in sodium dodecyl sulfate polyacrylamide (13%) gel electrophoresis. On a further analysis by 8 M urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, T gamma subunits of T beta gamma-1 and T beta gamma-2 showed Mr values of 8,000 (T gamma-1) and 6,000 (T gamma-2), respectively. Amino acid compositions of both T gamma-1 and T gamma-2 roughly corresponded with that of T gamma previously reported and were quite different from that of gamma-subunit of cGMP-phosphodiesterase. Western blot analysis of freshly isolated rod outer segments by an antiserum raised against a mixture of T beta gamma-1 and T beta gamma-2 revealed the presence of both components in the membranes of a starting material. This observation excludes the possibility that one of the components might be produced artificially in the course of the purification. In the presence of a photobleaching intermediate of either unphosphorylated or phosphorylated rhodopsin, the binding of guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp) to the alpha-subunit of transducin (T alpha) was remarkably enhanced with increasing concentrations of purified T beta gamma-2. On the contrary, T beta gamma-1 retained little ability, if any, to enhance the GppNHp binding to T alpha; the ability of T beta gamma-1 was at least 30 times lower than that of T beta gamma-2. Such a low activity of T beta gamma-1 was attributed to inability for coupling of T alpha with a photobleaching intermediate of rhodopsin. These results indicate that T gamma-2 is essential for the GTP binding of transducin. The role of T gamma-1 in vertebrate photoreceptor cells was discussed.