A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an alpha beta gamma-heterotrimeric structure. The alpha-subunit of the purified protein (alpha 40 beta gamma) had a molecular mass of 40 kDa and differed from that of Gi (alpha 41 beta gamma) or Go (alpha 39 beta gamma) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three alpha were different from one another. However, the beta gamma-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their beta-subunits were immunologically indistinguishable from one another. Thus, the alpha 40 beta gamma is a new IAP substrate protein different from Gi or Go, in the alpha-subunit only.     

Purification of the major GTP-binding proteins from human placental membranes

Using minor modifications of procedures developed to purify GTP-binding proteins (G-proteins) from rabbit liver, we have purified the major G-proteins present in human placental membranes. One, referred to as Gi, is the major substrate for pertussis toxin-catalyzed ADP-ribosylation and has an alpha-subunit of 41,000 daltons, and beta-subunit of 36,000 and 35,000 daltons, and a gamma-subunit of 10,000 daltons. The other protein, referred to as Gp, was identified by its ability to bind guanine nucleotides specifically with high affinity. This activity was resolved from Gs and Gi by the second step of purification (AcA-34 chromatography) and was further purified through heptylamine-Sepharose and hydroxylapatite. The guanine nucleotide-binding site, which can be resolved by high performance liquid chromatography procedures and identified by a photolyzable GTP analogue, is associated with a 21,000-dalton protein (Gp alpha) that copurifies with beta gamma-subunits indistinguishable from the beta gamma-subunits associated with Gs and Gi. This protein represents a potentially novel member of the structurally and functionally homologous family of G-proteins that are transducing elements in the receptor-mediated regulation of a variety of cellular processes.     

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.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.     

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.     

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.     

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.     

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified beta gamma-subunits of alpha beta gamma-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the alpha- and beta-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.     

Interaction of GTP-binding proteins with calmodulin

Two GTP-binding proteins (Gi and Go), which were the substrates for islet-activating protein, pertussis toxin, were purified from bovine cerebral cortical membranes. Both Gi and Go completely inhibited calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. The same concentrations of these proteins, however, had no appreciable effect on the basal phosphodiesterase activity. The isolated Gi alpha and beta gamma subunits of GTP-binding proteins were potent inhibitors of the calmodulin-stimulated phosphodiesterase activity, but Go alpha was very weak. Therefore, the beta gamma subunits were likely to be the major active molecules in the brain membranes. GTP-binding proteins were shown to bind directly to calmodulin in a Ca2+-dependent manner by a gel permeation binding experiment.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities

Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.     

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)     

Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles

The involvement of GTP-binding proteins (G proteins) in insulin action has been investigated in an in vitro system. Insulin receptors that have been purified by wheat germ lectin chromatography and either tyrosine-agarose chromatography, sucrose density centrifugation, or insulin-Sepharose chromatography have been co-inserted into phospholipid vesicles with different purified G proteins. The results of these studies indicate that a specific insulin-promoted phosphorylation of two G proteins, Go and Gi, can occur in these phospholipid vesicles. Bovine retinal transducin is a poor substitute for Go and Gi, being only weakly phosphorylated by the insulin receptor, and bovine brain Gs is not a substrate. The phosphorylation of Gi and Go occurs primarily on the alpha-subunits. Under optimal conditions, about one alpha o- or alpha i-subunit is phosphorylated on a tyrosine residue for every two beta-subunits of the insulin receptor, suggesting a 1:1 interaction between these G proteins and the heterotetrameric (alpha 2 beta 2) insulin receptor molecular. The inactive (GDP-bound) form of the alpha-subunits appears to be the preferred substrate, with the phosphorylation being significantly reduced in alpha o and alpha i upon the binding of guanosine 5'-O-thiotriphosphate (GTP gamma S) and completely eliminated in the pure alpha-GTP gamma S complex of transducin. The Gi and Go proteins also cause an enhancement of the insulin-stimulated receptor autophosphorylation. This enhancement is a reflection of an increased incorporation of the insulin receptor into lipid vesicles which is induced by these G proteins. Taken together these results provide evidence for the interactions of G proteins with the insulin receptor in a lipid milieu.     

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.     

Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.     

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.     

Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.     

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.     

Subunit interactions of the Go protein.

The monoclonal antibody, MONO, recognizes an epitope on the G protein alpha o-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTP gamma S, all beta gamma-subunits are released from the alpha o-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTP gamma S or GTP gamma S-induced subunit dissociation. However, we have been unable to induce a similar dissociation of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent Kd of alpha o-GDP for binding beta gamma of 340-390 nM has been reported [(1989) J. Biol. Chem. 264, 20688-20696] our results would suggest that binding of MONO to the alpha o-subunit induces an increased affinity of alpha o-GDP for beta gamma. Alternatively, these results could be explained if, under the conditions used, the Kd of alpha o-GDP for beta gamma were at least two orders of magnitude lower than estimated previously.