Toxin-catalyzed ADP-ribosylation Research Articles

Stimulation of mono-ADP ribosylation in rat liver plasma membranes after long-term alcohol intake.

ADP ribosylation is considered one of the important covalent modifications of cellular proteins catalyzed by ADP ribosyltransferase, which transfers ADP ribose moiety of NAD to an acceptor protein. Because a growing body of evidence has suggested significant biological roles for mono-ADP ribosylations in transmembrane signal transduction and other cell metabolism, how alcohol intake alters them is of interest. Cholera toxin and pertussis toxin have been widely used as probes to investigate the roles of GTP-binding proteins (G-proteins) in the transduction of hormonal and sensory signals. We first tested effects of long-term alcohol intake on these toxin-catalyzed ADP ribosylations of G-proteins in rat liver plasma membranes. Treatment of rat liver plasma membrane with [32P]NAD and thiol-preactivated cholera toxin resulted in the labeling of a 44-kD band, most likely an alpha-subunit of the stimulatory GTP-binding protein, the extent of which was much greater in alcohol-fed rats than in pair-fed controls. Analogous experiments with pertussis toxin also demonstrated enhancement of toxin-catalyzed ADP ribosylation of the inhibitory GTP-binding protein after long-term alcohol intake. More interesting was that long-term alcohol intake remarkably stimulated endogenous mono-ADP ribosylation of a 58-kD protein in a GTP-dependent manner. In vitro, ethanol (50 mmol/L) or a single load of ethanol (3 gm/kg) did not stimulate the reaction. Thus long-term alcohol intake stimulated both toxin-catalyzed and endogenous mono-ADP ribosylations of proteins in rat liver plasma membranes. Pursuit of alcohol interaction with mono-ADP ribosylation may provide an interesting approach to the study of alcohol's effects on the liver.

Insulin Stimulates Association of a 41-kDa G-Protein (G IR41) with the Insulin Receptor

Insulin has a paradoxical effect on a 41 kDa Gi-like protein: Although insulin-treatment of rat adipocytes inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41 kDa G-protein in membranes in a dose-dependent manner, it simultaneously increased ADP-ribosytation of a 41 kDa G-protein that co-immunoprecipitates with the insulin receptor (G IR41). The latter effect was insulin concentration- and time-dependent. The dose-dependent stimulatory effect of insulin on the autophosphorylation of the insulin receptor and on the ADP-ribosylation of the G IR41 in the insulin receptor immunoprecipitates closely paralleled each other. The time course of insulin-stimulated increase in the ADP-ribosylated G IR41, although rapid, was slower than the autophosphorylation of the receptor. The G IR41 is associated with and regulated by the insulin receptor further supporting an important role for this G-protein in modulating insulin action at the receptor level.

Identification of GTP Binding Proteins in Brain Microvessels and Their Role in Phosphoinositide Turnover

The identity of GTP binding proteins in cerebral microvessels was investigated by immunoblotting. Data indicate the presence of a characteristic pattern of Gi, Gs and Go. The most remarkable finding is the presence of Go protein in cerebral microvessels that consist predominantly of capillary segments free of neuronal contaminants. The pattern of pertussis toxin-catalyzed ADP-ribosylation of a 40 kDa polypeptide is characteristic of the Gi/Go type α-subunits. In addition, we have observed that GTPτS, a non-hydrolizable guanine nucleotide, exerts a dual regulatory effect on phosphoinositide metabolism depending on the concentration, thus 10 −7 M concentration inhibits the 32P incorporation into PIP 2 and PA by a pertussis toxin-sensitive mechanism; on the contrary, the stimulatory effect of higher concentration than 10 −6 M of GTPτS is pertussis toxin-insensitive.

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits.

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.

The GTP-binding regulatory proteins, Gs and G(i), are altered in erythrocyte membranes of patients with ischemic heart disease resulting from coronary atherosclerosis.

Acute ischemic heart disease is associated with alterations in the cardiac adenylate cyclase system response, although the specificity and mechanism of these events are unknown. We studied the characteristics of inhibitory (G(i)) and stimulatory (Gs) GTP-binding regulatory proteins (G proteins) of adenylate cyclase in erythrocyte membranes of patients (n = 16) with nonacute ischemic heart disease resulting from coronary atherosclerosis. Gs was measured by reconstitution with the resolved catalytic unit of adenylate cyclase and by cholera toxin-catalyzed ADP-ribosylation of a 42-kD protein; G(i) was tested as a 41-kD substrate of pertussis toxin-catalyzed ADP-ribosylation. Gs activity was decreased by 27 +/- 2% in the cholate extract and by 25 +/- 3% in the supernatant of guanosine 5'-(gamma-thio)triphosphate-treated membranes. The amount of cholera toxin substrate was decreased by 33 +/- 3%, and the pertussis toxin substrate was increased by 27 +/- 5% compared with healthy subjects (n = 10). All changes in G-protein characteristics appear to be specific relative to other erythrocyte membrane proteins and hemoglobin. Those patients who have a decreased Gs possess approximately normal Gi, and those with increased G(i) showed no change in Gs. Patients with increased G(i) (normal Gs) exhibited more severe deterioration of their coronary arteries than did patients with decreased Gs (normal G(i)) (P < .05), but these two groups did not differ significantly in serum lipids, hormones, drug therapy, historical data, or baseline assessment (P < 0.05).

GTP-Binding Protein Couples with Metabotropic Glutamate Receptor in Bovine Retinal On-Bipolar Cell

GTP-binding protein (G protein) linking to metabotropic glutamate receptor of bovine retinal on-bipolar cell was studied by use of pharmacologically selective ligands, 2-amino-4-phosphonobutyric acid (APB) on bacterial toxin-catalyzed ADP-ribosylation and GTPγS-binding. In contrast to the electrophysiological findings reported, G protein coupling to APB-sensitive glutamate receptor served as a substrate for pertussis toxin but did not for cholera toxin. Several glutamate analogues effective on on-bipolar cell, as well as APB, increased GTPγS binding to retinal membranes devoid of rod outer segments. The enhancement of GTPγS binding by APB was completely abolished when the membranes were pretreated with pertussis toxin and NAD. These results suggest that, in retinal on-bipolar cell, the G protein which couples metabotropic glutamate receptor to hyperpolarizing response of the cell is sensitive to pertussis toxin.

An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins

Cholera toxin-catalysed [ 32P]ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the α2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M r polypeptides which represent forms of G sα, there was weak labelling of a 40,000 M r polypeptide(s). Addition of the α2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M r polypeptide(s) but did not alter labelling of the forms of G s. We have previously demonstrated that the 40,000 M r polypeptide(s) labelled in such a manner represents a combination of the α subunits of G i2 and G i3 [Milligan et al. (1991) J. biol. Chem. 266 6447–6455]. Mercuric acetate treatment of membranes prelabelled with [ 32P]ADP-ribose by exposure to pertussis toxin and [ 32P]NAD removed totally the radiolabel from both G i2 and G i3. However, cholera toxin-catalysed [ 32P]ADP-ribosylation of either the α subunits of the G i-subtypes or of forms of G s was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the G i-subtypes and from G s but did not remove [ 32P]ADP-ribose incorporated by pertussis toxin from the G i-subtypes. It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue. It is suggested that this residue may be Arg 179 in G i2α and Arg 178 in G i3α.

Different functional forms of G-protein βγ-subunits, βγ-I and βγ-II, in bovine brain

Heterotrimeric GTP binding regulatory proteins (G proteins) are involved in the signal transduction process in cells. We have previously demonstrated that G protein (Gi/o) in bovine brain contains two subspecies of the βγ-subunit, βγ-I and βγ-II, with distinct γ subunits, i.e., γ-I and γ-II, but identical β-subunit. We found that γ-I agreed with the γ-subunit reported elsewhere, while γ-II was a novel γ-subunit. In the present study we separated the fractions containing G-protein isoforms, Go∗, Gi1, Go and Gi2, with a Mono Q column and they were subjected to 15% polyacrylamide gel electrophoresis. All isoforms were shown to possess both γ subunits, γ-I and γ-II. The molar ratio of the two γ-subunit isoforms was one to one, based on the relative intensity of Coomassie blue-stained bands. Differences in biological activity between G proteins composed of βγ-I and βγ-II were investigated. The amount of GTPγS bound to the α-subunit was larger in αβγ-I than in αβγ-II. The pertussis toxin-catalyzed ADP-ribosylation on α-subunit was enhanced by either βγ-subunit subspecies, but the effect was larger with βγ-I than with βγ-II. From gel filtration with a Sephacryl S 300, it appeared that all α- and either βγ-subunit, i.e., βγ-I or βγ-II, formed a trimer complex. These findings suggest the possible existence of two different functional forms in each G-protein isoform depending on the βγ-subunit subspecies.

Molecular characterization of a conserved, guanine nucleotide-dependent ADP-ribosylation factor in Drosophila melanogaster

ADP-Ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin-catalyzed ADP-ribosylation in vitro. Because the functional role(s) of ARF in mammalian systems is (are) elusive, we looked for ARF in Drosophila melanogaster, and report the partial purification and molecular cloning of an ARF from Drosophila. We cloned the Drosophila ARF 1 gene without library screening by a combination of 5 polymerase chain reactions (PCRs), yielding a 546-base open reading frame encoding 182 amino acids, which are > 93% identical to those of mammalian class I ARFs. This ARF gene maps to 79F3-6 in the proximal region of the left arm of Drosophila chromosome 3. The Drosophila ARF1 gene structure, including placement of introns, is highly conserved relative to mammalian class 1 ARF genes. A single ARF mRNA species of 1.8 kb was abundant in all Drosophila body segments. Recombinant Drosophila ARF 1 synthesized in Escherichia coli had biochemical and immunochemical activities similar to those of mammalian ARF. The similarities of sequence and biochemical properties between Drosophila and mammalian ARFs contrast with their differences from Drosophila arl (ARF-like protein), which does not stimulate cholera toxin-catalyzed ADP-ribosylation, and is only approximately 52-56% identical in amino acid sequence to mammalian ARFs.

Estradiol up-regulates the stimulatory GTP-binding protein expression in the MCF-7 human mammary carcinoma cell line

The effect of estradiol treatment of the human mammary carcinoma cell MCF-7 on the adenylyl cyclase system was examined. Treatment with 10 nM estradiol for 72 h increased the basal level ofcAMP, and isoproterenol-, PGE 2- or calcitonin-stimulated cAMP production. Estradiol also increased the response to cholera toxin but did not alter the response to forskolin. No significant change in growth rate was observed during the 72 h of estradiol treatment. In MCF-7 cell membranes the responsiveness to isoproterenol, PGE 2, or cholera toxin was also enhanced by estradiol treatment. The cholera toxin-catalyzed ADP-ribosylation of gsoe in MCF-7 cell membranes was significantly increased by 72 h of treatment with estradiol. Consistent with this observation, the level of Gsα immunoreactivity was increased in the estradiol-treated cell membranes. On the other hand, pertussis toxin did not change the responsiveness to isoproterenol, PGE 2 or calcitonin in either control or estradiol-treated cells. In addition, ADP-ribosylation with pertussis toxin also did not reveal any change in Gi. These results clearly indicate that Gs expression is under the control of estradiol, and that this effect may contribute to the increased sensitivity of hormone-stimulated adenylyl cyclase activities in MCF-7 cells.

Protein intake affects levels of G-protein subunits Gαi2, Gαi3, and Gβ in rat glomerular membranes

Using toxin-catalyzed ADP-ribosylation and specific immunoblots we examined whether the mass of G-protein subunits, G alpha s, G alpha i (includes G alpha i2, G alpha i3, and G alpha 0) and G alpha beta, in glomerular membranes was altered by dietary protein intake. ADP-ribosylation catalyzed by cholera toxin (CT) or pertussis toxin (PT) detected significant amounts of G alpha s or G alpha i in glomerular membranes from rats fed a low (6% casein) or a high (40% casein) protein diet. There was no significant difference in G alpha s content between glomerular membranes from low or high protein-fed rats. However, the amounts of G alpha i were significantly lower in glomerular membranes from rats fed a high protein diet when compared to glomerular membranes from rats fed a low protein diet. Two isoforms of immunoreactive G alpha s, 45 and 52 kDa proteins, were detected in glomerular membranes. The predominant isoform of G alpha s was a 52 kDa protein. As with ADP-ribosylation, immunoblots showed no significant difference in G alpha s content between glomerular membranes obtained from the two diet groups of rats. Also, immunoreactive G alpha i2, G alpha i3 and G beta were present in glomerular membranes. The mass of G alpha i2 and G alpha i3 was significantly lower in glomerular membranes of rats fed a high protein diet than in those of rats fed a low-protein diet. The decreased mass of total G alpha i, that is G alpha i2 and G alpha i3, was comparable to that seen with PT-catalyzed ADP-ribosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin Induces an Unmasking of the Carboxyl Terminus of Gi Proteins in Rat Adipocytes

Several groups have shown a relationship between the insulin receptor and inhibitory G proteins, Gi. An antisera, 8729, to a peptide sequence (KNNLKDCGLF) corresponding to the carboxyl termini of Giα subunits was used to investigate this relationship by immunoelectron microscopy. Rat adipocytes were incubated in the absence or presence of 100 ng/ml insulin for 1 h and fixed for immunoelectron microscopy. Insulin-treated adipocytes stained with 8729 were labeled at the cell surface at a much higher density than control adipocytes. Subcellular fractionation of insulin-treated and control cells was followed by PAGE and Western blots of the plasma membrane and low-density microsomes with 8729. The density of the bands did not change in response to insulin treatment. Antibodies to noncarboxyl terminus sequences of the α subunit were used for immunoelectron microscopy and no difference was noted between insulin-treated and control adipocytes. These results indicated that 8729 was detecting a conformational change in the structure of Giα subunit in the plasma membrane in response to insulin. This unmasking of the carboxyl terminus was also seen in response to treatment with phenylisopropyladenosine and prostaglandin E2. Pertussis toxin-catalyzed ADP ribosylation also unmasked the carboxyl terminus. In contrast, isoproterenol, an agonist of stimulatory G proteins (Gs), did not induce an unmasking of the carboxyl terminus. These results support the hypothesis that some of insulin's effects are mediated through Gi proteins in adipocytes.

Effects of brefeldin A and accessory proteins on association of ADP-ribosylation factors 1, 3, and 5 with Golgi

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance in vitro cholera toxin-catalyzed ADP-ribosylation and subsequently shown to participate in vesicular transport in the Golgi and other cellular compartments. By cDNA and genomic cloning, at least six mammalian ARFs were identified. Brefeldin A (BFA) disrupts Golgi membranes and inhibits binding of soluble high molecular weight proteins to Golgi fractions. We examined the effects of BFA on binding of ARF1, -3, and -5 to a Golgi fraction in the presence of an ATP-regenerating system and a fraction of soluble, high molecular weight, accessory proteins (SAP), presumably containing complexes identified by others as coatomers that are involved in vesicular transport. ARF binding in all instances was dependent on guanosine 5'-O-(3-thiotriphosphate) and increased by the ATP-regenerating system. Binding of ARF1 and -3, but not ARF5, was enhanced by SAP. BFA inhibited the SAP-dependent, but not the SAP-independent, binding of ARF1 and -3. It had no effect on the increment in binding produced by an ATP-regenerating system. B36, an inactive derivative of BFA, did not inhibit SAP-dependent binding of ARF1 and -3. Binding of ARF5, which was SAP-independent, was not affected by BFA. These observations are consistent with the conclusion that mammalian ARFs differ in their dependence on accessory proteins for interaction with Golgi and, perhaps, other cellular membranes and that BFA specifically inhibits SAP-dependent ARF binding.

Somatostatin induces release of the α subunits of pertussis toxin-sensitive G proteins in native membranes and in intact GH 4 C 1 rat pituitary cells

Incubation of GH 4 C 1 rat pituitary cell membranes with the poorly hydrolyzable GTP analogue, GTP-γS, produces a decrease in the pertussis toxin-catalyzed ADP-ribosylation of 40-kDa protein in the membrane pellet and the release of an α-like substrate from the membrane into the supernatant fraction; these effects do not occur with the inactive GDP analogue, GDPβS. The resolved supernatant fraction from GTPγS-stimulated membranes is significantly activated to pertussis toxin-catalyzed [ 32P]ADP-ribosylation by the addition of purified βγ complex. Immunoblot analysis identifies the released pertussis toxin substrate as α subunits of G i2, G i3, and G o in the resolved supernatant. The physiological agonist, somatostatin, also stimulates the release of G 2 and G o α subunits but not G i3 from GH 4C 1 cell membranes in the presence of a low concentration of GTPγS (20 nM). The effects of somatostatin are inhibited by pretreatment of GH 4C 1 cells with pertussis toxin. Furthermore, the addition of somatostatin to intact GH 4C 1 cells decreases the level of G 2 α subunits in the crude membrane whereas immunoblot analysis of the 274,000 × g supernatant (cytosolic fraction) clearly shows the presence of G i2 α subunits. These data indicate that pertussis toxin-sensitive G proteins in GH 4C 1 cells dissociate into α subunits and αγ complex with the release of the α subunits from the membranes upon somatostatin activation.

Bilateral ureteral obstruction alters levels of the G-protein subunits Gαs and Gαq/11

To evaluate the effects of bilateral ureteral obstruction (BUO) on the levels of G-protein subunits in glomeruli, we examined the types and amounts of G-protein subunits in glomerular membranes from sham-operated control (SOC) rats and rats with BUO of 24 hours duration utilizing bacterial toxin-catalyzed ADP-ribosylation and specific antibodies. ADP-ribosylation catalyzed by cholera or pertussis toxin demonstrated the presence of Gs and Gi proteins in glomerular membranes. Immunoblots further revealed the existence of two types of G alpha s (45 and 52 kDa), as well as G alpha i2 (40 kDa), G alpha i3 (41 kDa), G alpha q/11 (42 kDa) and G beta (35 to 36 kDa) in glomerular membranes. The predominant subspecies of G alpha s was the 52 kDa protein. Detectable amounts of G alpha o were not found in glomerular membranes. Moreover, G-protein subunits were not detected in cytosolic extracts of glomeruli. Both forms of G alpha s and G alpha q/11 were significantly reduced in glomerular membranes from rats with BUO when compared to SOC rats. No significant difference in total G alpha i, G alpha i2 and G alpha i3 and G beta content was observed between the two groups of rats. In vivo pretreatment of rats with simultaneous administration of the angiotensin-converting enzyme inhibitor, enalaprilat, and the thromboxane synthase inhibitor, OKY-046, maintained the amount of G alpha s and G alpha q/11 in rats with BUO at the levels seen in SOC rats. The two drugs did not affect the amounts of G-protein subunits in glomerular membranes of SOC rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfected Go1 alpha inhibits the calcium dependence of beta-adrenergic stimulated cAMP accumulation in C6 glioma cells.

Increasing evidence indicates that heterotrimeric G proteins, and in particular Go, regulate ionic channel activities. In order to investigate the role of Go proteins in the modulation of the Ca2+ influx, C6 glioma cells were stably transfected with alpha o1 cDNA. Expression of the Go1 alpha protein was checked by Bordetella pertussis toxin-catalyzed ADP-ribosylation and Western blots using one- and two-dimensional gel analyses. Three clones were selected based on their degree of Go1 alpha expression. In alpha o1-transfected cells, cAMP accumulations, in response to isoproterenol or forskolin, were lower than in control cells. This inhibitory effect was a function of the amount of expressed Go1 alpha. In contrast, Go1 alpha expression was not followed by a significant inhibition of isoproterenol- or forskolin-stimulated adenylyl cyclase activities in particulate fractions. In C6 parental cells, 50-60% of the isoproterenol-induced cAMP accumulation was dependent on external Ca2+ concentration. This Ca(2+)-dependent cAMP accumulation was related to an induced transient Ca2+ influx. In transfected cells, expression of Go1 alpha inhibited the Ca2+ influx and the Ca(2+)-dependent component of isoproterenol-induced cAMP accumulation. In conclusion, beta-adrenergic agonists stimulate an entry of Ca2+ which exerts a positive feedback on cAMP production, and Go1 alpha blocks this positive feedback by inhibiting the Ca2+ influx.

Tryptophan207 is involved in the GTP-dependent conformational switch in the alpha subunit of the G protein transducin: chymotryptic digestion patterns of the GTP gamma S and GDP-bound forms.

The limited proteolytic pattern of transducin, Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the alpha t subunit were identified. The alpha t subunit in the GTP gamma S bound form was cleaved into a major 38 kD fragment, whereas alpha t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The beta gamma t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The gamma t subunit was not cleaved and only a small portion of beta t was digested into several fragments. In order to determine which proteolytic fragment of alpha t still contained the carboxyl terminal region, chymotrypsinization was carried out using Gt previously 32P-labeled at Cys347 by pertussis toxin-catalyzed ADP-ribosylation. The 32P-label was mainly associated with the alpha t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not 32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of alpha t, at Leu15 and Leu19. Chymotrypsin removed 15-19 amino acid residues from the amino terminus of alpha t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of alpha t-GTP gamma S was resistant to proteolysis but alpha t-GDP and the 38 kD fragments of alpha t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac β-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy

The role of the β-adrenoceptor-G-protein-adenylate cyclase system in the pathogenesis of cardiac hypertrophy was studied. We have used a minipig model of pressure-overload cardiac hypertrophy secondary to aortic banding. Four groups of five animals were used: minipigs made hypertrophic were evaluated 2 months (CH2 group) and 9 months (CH9 group) later and compared to controls (C2 and C9 groups, respectively). A decrease in β-adrenergic receptor density and an increase in antagonist affinity were shown in left ventricular membranes of hypertrophied animals compared with controls. In both groups, CH2 and CH9, an increase in EC 50 for isopreterenol-stimulated adenylate cyclase activity, an increase in forskolin-stimulated adenylate cyclase activity and a diminished inhibition by carbachol of isoproterenol-stimulated adenylate cyclase were observed. In contrast, fluoride-stimulated adenylate cyclase activity was markedly increased only in the end stage of hypertrophy. α s-cholera toxin-catalysed ADP-ribosylation is increased in early hypertrophy and then decreases with late hypertrophy and a similar pattern is observed with α O pertussis toxin-catalysed ADP-ribosylation, whereas α i-ADP-ribosylation remains unchanged. Tissue content of G s-, G i- and G 0-proteins, as assessed by specific antibodies, was found unchanged in CH9 and CH2 groups when compared with that in C9 and C2 control groups, respectively. Modifications in G s functional activity in later hypertrophic stages, expressed as alterations in cholera toxin ADP-ribosylation and adenylate cyclase fluoride responsiveness, may be important in the pathogenesis of decompensation from compensated hypertrophy to cardiac failure.

Baculovirus Expression and Purification of a Soluble, Mutant G-Protein α Subunit

The cDNA for the α i1 protein that had undergone site-directed mutagenesis to change glycine-2 to alanine was ligated into a baculovirus transfer vector. A recombinant virus was obtained by transfecting Sf9 cells with both the wild-type baculovirus DNA and the transfer vector and screening for recombinant plaques. Infection with the recombinant virus led to a high level of expression of the mutated α i1 protein in the soluble fraction of the cell. The protein was purified by ammonium sulfate precipitation, gel filtration, and immobilized dye chromatography. The typical yield was 7.5 mg from two 800-ml cultures. The protein showed immunoreactivity to three different α i-specific antibodies. It bound guanosine 5′-(γ-thio)triphosphate (GTPγS) with a stoichiometry of 0.63 to 0.91 mol/mol and with a rate constant ( k GTPγS) of binding of 0.126 min −1. When GTPγS bound, the protein was protected from complete tryptic cleavage. The recombinant protein was able to undergo pertussis toxin-catalyzed ADP ribosylation and bind βγ subunits but with a reduced affinity compared to that of a transducin. Thus using a recombinant baculovirus, a nonmyristylated G protein α subunit was abundantly expressed in Sf9 cells and milligram quantities of a functional protein were easily purified.

Prenyl modification of guanine nucleotide regulatory protein gamma 2 subunits is not required for interaction with the transducin alpha subunit or rhodopsin.

Guanine nucleotide-binding regulatory protein (G protein) beta gamma dimers that were active in reconstitution assays were produced in insect cells using the baculovirus/Sf9 insect cell expression system. Sf9 cells were infected either singly or in combination with recombinant baculoviruses containing a human G-protein beta 1 gene or a bovine G-protein gamma 2 gene. It was possible to express the beta 1 and gamma 2 gene products independently of each other in this system, as determined by using immunological and metabolic labeling techniques. Further, the ability of recombinant beta and/or gamma chains to function in defined biochemical assays of beta gamma activity was assessed for membrane extracts and supernatant fractions from infected Sf9 cells. Extracts of cells expressing beta or gamma chain alone were inactive in these assays, whereas those from cells coinfected with beta 1 and gamma 2 did display activity. These assays were used to identify recombinant beta gamma dimer migration during chromatographic purification, and the recombinant dimers were purified to near homogeneity. Both the membrane-associated and soluble beta gamma dimers facilitated rhodopsin-catalyzed guanosine 5'-[gamma-thio]triphosphate binding to Gt alpha, the GTP-binding subunit of the retinal G protein transducin (K0.5 of 13 +/- 2 and 36 +/- 5 nM, respectively). Both recombinant beta gamma dimers also facilitated the pertussis toxin-catalyzed ADP-ribosylation of Gt alpha with equal potency (K0.5 of 9 +/- 1 and 10 +/- 3 nM for membrane and soluble dimers, respectively). [3H]Mevalonolactone labeling showed that the gamma 2 subunits of membrane-associated beta gamma dimers incorporated radiolabel, whereas in the soluble form they did not. Thus, prenyl modification of gamma 2 directs the membrane association of the beta 1 gamma 2 dimer and increases its apparent affinity for receptor, but it is not required for the functional interaction(s) of the dimer.