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)     

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.     

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.     

The amino terminus of G protein alpha subunits is required for interaction with beta gamma

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.     

Expression and purification of functional G protein alpha subunits using a baculovirus expression system.

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S.     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Mechanism of inhibition of transducin GTPase activity by fluoride and aluminum

Transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system, is structurally and functionally similar to the inhibitory and stimulatory guanyl nucleotide-binding proteins, Gi and Gs, of the adenylate cyclase complex. All are heterotrimers composed of alpha, beta, and gamma subunits. Gs and Gi can be activated by NaF with AlCl3 as well as by agonists acting through specific receptors. The effects of NaF and AlCl3 on transducin were investigated in a reconstituted system consisting of the purified subunits of transducin (T alpha, T beta, gamma) and rhodopsin. NaF noncompetitively inhibited the GTPase activity of T alpha in a concentration- and time-dependent manner. Inhibition by NaF was enhanced synergistically by AlCl3 which alone only slightly inhibited GTPase activity. None of the other anions tested reproduced the effect of fluoride. Fluoride inhibited [3H]guanosine 5'-(beta, gamma-imido)triphosphate binding to T alpha and release of bound GDP. The ADP-ribosylation of T alpha by pertussis toxin and binding of T alpha to rhodopsin, both of which are enhanced in the presence of T beta gamma, were inhibited by NaF and AlCl3. These findings are consistent with the hypothesis that fluoride enhances the dissociation of T alpha from T beta gamma, resulting in the inhibition of GTP-GDP exchange, and therefore, GTP hydrolysis.     

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.     

Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions.

Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.     

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.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

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.     

Studies on nucleotide and receptor regulation of Gi proteins: effects of pertussis toxin

In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of Gi proteins are affected by PT. Experiments with the Gs-deficient Gi-regulated adenylyl cyclase of cyc- S49 cell membranes indicated that PT blocks Gi activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-Gi to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of Gi. Experiments in which Gi was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of Gi, but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Chromatographic resolution and immunologic identification of the alpha 40 and alpha 41 subunits of guanine nucleotide-binding regulatory proteins from bovine brain

A guanine nucleotide-binding regulatory protein (G protein), with subunits designated as alpha 40 beta gamma, was identified and partially resolved from two other purified G proteins, Go (alpha 39 beta gamma) and Gi (alpha 41 beta gamma), found in bovine brain. The alpha 40 G protein subunit served as a substrate for ADP-ribosylation catalyzed by Bordetella pertussis toxin, as did alpha 39 and alpha 41. alpha 40 was shown to be closely related to, but distinct from, alpha 41 by reaction with various peptide antisera. An antiserum generated against a peptide derived from the sequence of a Gi alpha clone isolated from a rat C6 glioma cDNA library (Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K., and Kaziro, Y. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3776-3780) reacted with alpha 40 to the exclusion of all other alpha subunits tested. Another antiserum generated against a peptide derived from an analogous region of a different Gi alpha clone from a bovine brain cDNA library (Nukuda, T., Tanabe, T., Takahashi, H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa, K., Hiranaga, M., Matsuo, H., and Numa, S. (1986) FEBS Lett. 197, 305-310) reacted exclusively with alpha 41. Evidence is given for the existence of another form of alpha 41 that did not react with either of these two peptide antisera. The antisera were used to survey various rat tissues for the expression of alpha 40 and alpha 41.     

Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.     

The GTP-binding protein of rod outer segments. I. Role of each subunit in the GTP hydrolytic cycle

The GTP-binding protein of Bufo marinus rod outer segments (ROS) is composed of 3 subunits: G alpha, 39,000; G beta, 36,000; and G gamma, approximately 6,500. A stepwise analysis of the GTP hydrolytic cycle (GTP binding, GTP hydrolysis, and GDP release) was facilitated by using purified subunits of the GTP-binding protein. When G alpha and G beta, gamma concentrations were held constant, the initial rate of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha was dependent upon the amount of bleached rhodopsin present (as illuminated, urea-washed ROS disc membranes). When G alpha and the quantity of these membranes was held constant, the initial rate of GTP gamma-s binding to G alpha was markedly enhanced by increasing the amount of G beta, gamma. G beta preparations (free of G gamma) also stimulated the binding of GTP gamma-s to G alpha to the same extent as G beta, gamma preparations, suggesting that G gamma is not an essential component of the G beta, gamma-dependent stimulation of the rate of GTP gamma-s binding to G alpha. Nonlinear regression analysis revealed a single class of binding sites with an apparent stoichiometry of 1 mol of site/mol of G alpha under optimal binding conditions. Following GTP binding to G alpha, the GTP X G alpha complex dissociates from G beta, gamma which remains primarily bound to the ROS disc membranes. Moreover, while GTP remains in excess, the rates of GTP hydrolysis exhibited saturation in the presence of increasing amounts of G beta, gamma. Nonlinear regression analysis of these data argues against a direct role for G beta, gamma in the hydrolysis of GTP. Thus, both topologic and kinetic data support the concept that GTP hydrolysis is carried out by G alpha alone. After hydrolysis of GTP, the GDP X G alpha complex returned to the ROS disc membrane when G beta, gamma was present on the membrane surface, in the presence and absence of light. Without guanine nucleotides GDP release occurred in the presence of illuminated ROS disc membranes and G beta, gamma. Guanine nucleotides (GTP gamma-s approximately equal to GTP approximately equal to guanosine 5'-(beta, gamma-imido)triphosphate greater than GDP) could effectively displace GDP from G alpha under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)