G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules.

In muscle, it has been established that guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.     

G-protein dependent potentiation of calcium release from sarcoplasmic reticulum of skeletal muscle

Skinned fibre experiments were conducted to determine if guanine nucleotide-binding proteins play a role in excitation-contraction coupling of skeletal muscle. By itself, the GTP-gamma S, a non hydrolysable GTP analogue was unable to induce calcium release from the sarcoplasmic reticulum, even at concentrations as high as 500 microM. However, calcium- or caffeine-induced calcium releases were enhanced by GTP-gamma S in micromolar concentrations. This response was blocked by GDP-beta S or Pertussis toxin. 32P-ADP-ribosylation catalysed by Pertussis toxin, radiolabelled G-protein alpha subunits in the range of 40 kDa on membrane subcellular fractions of rat skeletal muscle. Using Western blot analysis with antibodies raised against the bovine transducin, G-proteins were identified in frog and rat skeletal muscle subcellular fractions. In most of the muscle fractions (plasma membrane, T-tubules, triads, sarcoplasmic reticulum), the anti-beta subunit antibodies recognized a 36 kDa protein which comigrated with transducin beta subunit. It appears therefore that some of the G-proteins identified by ADP-ribosylation or immunostaining in several subcellular fractions from skeletal muscle, are implicated in the modulation of calcium release from sarcoplasmic reticulum. These results suggest that a Pertussis toxin sensitive G-protein is present at the loci of E-C coupling, and that it serves to regulate the calcium release.     

Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.     

Identification of proteins resembling G-protein alpha subunits in locust muscle

In locust skeletal muscle, FMRFamide-like peptides decrease a K+ conductance. Functional data suggest the involvement of G-proteins. For identification of G-protein alpha-subunits, membranes of locust skeletal muscle were probed with ADP-ribosylating bacterial toxins, the photoreactive GTP analog, [alpha-32P]GTP azidoanilide, and with antibodies against mammalian alpha-subunits. Multiple guanine nucleotide-binding proteins of approximately 24-95 kDa were detected. Pertussis toxin catalyzed the ADP-ribosylation of two proteins comigrating with the ADP-ribosylated alpha-subunits of the mammalian G-proteins Go and Gi. Cholera toxin promoted ADP-ribosylation of a protein comigrating with mammalian cholera toxin substrates (i.e., Gs alpha-subunits). An antibody against mammalian Go alpha-subunits detected a 54-kDa protein. Thus proteins with properties of mammalian G-protein subunits are present in insect muscle.     

G-proteins in Torpedo marmorata electric organ. Differential distribution in pre- and post-synaptic membranes and synaptic vesicles

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP-ribosylation of the alpha-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go alpha-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedo synaptic vesicles either by ADP-ribosylation or by immunoblotting.     

Demonstration of the presence of G-proteins in hepatic microsomal fraction

The presence of G-proteins in isolated hepatic microsomal vesicles is demonstrated. The G-proteins were identified by their capacity to be ADP-ribosylated by cholera and pertussis toxins. Cholera toxin identified 42 and 45 kDa proteins, corresponding to alpha s-1 and alpha s-2, respectively. Pertussis toxin identified a 40 kDa protein corresponding to alpha i. The microsomal G-proteins are identical to the corresponding G proteins of the plasma membrane, but are present in different proportions; the microsomes have considerably less alpha s proteins than the plasma membrane.     

Presence of three pertussis toxin substrates and Go alpha immunoreactivity in both plasma and granule membranes of chromaffin cells

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Gi alpha. Significant immunoreactivity with anti-Go alpha antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.     

Identification and purification of GTP-binding regulatory proteins from plasma membranes of swine heart

A 23 kDa GTP-binding protein was purified from pig heart sarcolemma. This protein was not ADP-ribosylated by cholera, pertussis and botulinum C3 toxins. In pig heart sarcolemma pertussis toxin ADP-ribosylated 40 kDa subunit of Gi-protein, cholera toxin--45 kDa subunit of Gs-protein, botulinum C3 toxin ADP-ribosylated a group of proteins with Mr 22, 26 and 29 kDa. Antiserum generated against the peptide common for all alpha-subunits of G-proteins did not react with purified 23 kDa protein. Trypsin cleaved the 23 kDa protein in the presence of guanyl nucleotides into a 22 kDa fragment. Proteolysis of the 39 kDa alpha 0-subunit from bovine brain plasma membranes and ADP-ribosylated 40 kDa alpha i-subunit from pig heart sarcolemma in the presence of GTP gamma S yielded the 37 and 38 kDa fragments, respectively. In the presence of GTP and GDP the proteolysis of alpha 0 yielded the 24 and 15 kDa fragments, while the proteolysis of ADP-ribosylated alpha i-subunit yielded a labelled 16 kDa peptide. Irrespective of nucleotides trypsin cleaved the ADP-ribosylated 26 kDa substrate of botulinum C3 toxin into two labelled peptides with Mr 24 and 17 kDa. The data obtained indicate the existence in pig heart sarcolemma of a new 23 kDa GTP-binding protein with partial homology to the alpha-subunits of "classical" G-proteins.     

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.     

Multiple isoforms of ADP-ribosylated G-like proteins from mammalian thyroid membranes

Bovine, canine, and porcine thyroid membrane proteins which were [32P] ADP-ribosylated by cholera and pertussis toxin in vitro were analyzed by one and two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These three mammalian species have similar cholera toxin substrates (Mr 42,000 and 48,000) and pertussis toxin substrates (Mr 40,000). Resolution by two dimensional gel electrophoresis of these ribosylated proteins revealed that they each consist of at least 6 distinct polypeptides with similar isoelectric points ranging from approximately 5.5-7.0.     

Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs).

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent 'down-regulation' of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.     

A high-affinity bradykinin receptor in membranes from rat myometrium is coupled to pertussis toxin-sensitive G-proteins of the Gi family

In rat myometrial membranes, two 3H-Bradykinin binding sites with KD values of 16 pM and 1.0 nM were identified. Employed at pM concentrations, bradykinin stimulated high affinity GTPases. This effect was abolished by the bradykinin antagonist, [D-Arg(Hyp3-Thi5,8, D-Phe7)]bradykinin (10 microM), and by treatment of membranes with pertussis toxin. Myometrial membranes contained two pertussis toxin substrates of 40 and 41 kDa, which corresponded immunologically to alpha-subunits of Gi-type G-proteins. The faster migrating substrate was tentatively identified as Gi2 alpha-subunit. The electrophoretic mobility of the slower migrating Gi alpha-subunit was very similar to that of the Gi3 alpha-subunit. Go alpha-subunits were not detected. Thus, in uterine smooth muscle, G-proteins of the Gi-family (Gi2, Gi3) couple high-affinity bradykinin receptors to their effector enzymes.     

Involvement of pertussis toxin-sensitive G-proteins in the hormonal inhibition of dihydropyridine-sensitive Ca2+ currents in an insulin-secreting cell line (RINm5F).

Adrenaline inhibits insulin secretion via pertussis toxin-sensitive mechanisms. Since voltage-dependent Ca2+ currents play a key role in insulin secretion, we examined whether adrenaline modulates voltage-dependent Ca2+ currents of the rat insulinoma cell line, RINm5F. In the whole-cell configuration of the patch-clamp technique, dihydropyridine- but not omega-conotoxin-sensitive Ca2+ currents were identified. Adrenaline via alpha 2-adrenoceptors inhibited the Ca2+ currents by about 50%. Somatostatin which also inhibits insulin secretion was less efficient (inhibition by 20%). The hormonal inhibition of Ca2+ currents was not affected by intracellularly applied cAMP but blocked by the intracellularly applied GDP analog guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. In contrast to adrenaline and somatostatin, galanin, another inhibitor of insulin secretion, reduced Ca2+ currents by about 40% in a pertussis toxin-insensitive manner. Immunoblot experiments performed with antibodies generated against synthetic peptides revealed that membranes of RINm5F cells possess four pertussis toxin-sensitive G-proteins including Gi1, Gi2, Go2, and another Go subtype, most likely representing Go1. In membranes of control but not of pertussis toxin-treated cells, adrenaline via alpha 2-adrenoceptors stimulated incorporation of the photo-reactive GTP analog [alpha-32P]GTP azidoanilide into pertussis toxin substrates comigrating with the alpha-subunits of Gi2, Go2, and the not further identified Go subtype. The present findings indicate that activated alpha 2-adrenoceptors of RINm5F cells interact with multiple G-proteins, i.e. two forms of Go and with Gi2. These G-proteins are likely to be involved in the adrenaline-induced inhibition of dihydropyridine-sensitive Ca2+ currents and in other signal transduction pathways contributing to the adrenaline-induced inhibition of insulin secretion.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.     

Guanosine 5'-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries.

To determine whether direct stimulation of endothelial G-proteins causes relaxations of the underlying vascular smooth muscle, the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and sodium fluoride were studied in porcine coronary arteries and endothelial cells. Isometric tension was measured in coronary rings contracted with prostaglandin F2 alpha. GTP gamma S (in the presence of saponin) and sodium fluoride (in the presence of AlCl3) relaxed rings with, but not those without endothelium. The responses were inhibited by nitro-L-arginine and pertussis toxin. In membrane fractions of coronary endothelial cells, GTP gamma S and sodium fluoride inhibited the ADP-ribosylation of G-proteins catalyzed with [32P]-NAD and pertussis toxin. These data suggest that direct stimulation of G-proteins in endothelial cells by GTP gamma S and sodium fluoride causes a pertussis toxin-sensitive relaxation which may be attributed to the release of nitric oxide.     

K-ras transformation greatly increases the toxin-dependent ADP-ribosylation of GTP binding proteins in thyroid cells. Involvement of an inhibitor of the ADP-ribosylation reaction.

The GTP binding (G) proteins of normal (FRTL5) and ras-transformed thyroid cells (KiKi) were characterized by cholera and pertussis toxin-induced ADP-ribosylation and immunoblot analysis. Two pertussis toxin substrates with molecular masses of 40 and 41 kDa were identified in normal cells as the alpha i2 and alpha i3 subunits. The molecular masses of the cholera toxin substrates were 42 and 45 kDa. The same cholera and pertussis toxin substrates were present in the K-ras-transformed cell line. However, the toxin-dependent ADP-ribosylation was markedly higher in KiKi than in normal cell membranes (more than 50-fold). The reason for this difference was investigated; it could not be explained by the relative amounts of G proteins in the two cell systems, since the levels of alpha i2 subunit as measured by quantitative immunoblot in K-ras-transformed cells were only slightly (65%) higher than in normal cells. The difference in ADP-ribosylation was not due to poly-ADP-ribosylation nor to a different degree of subunit dissociation of G proteins in the two cell lines. Rather, the enhanced ADP-ribosylation in K-ras-transformed cells appears to be due to the loss of an inhibitory factor present in the normal cells. Partial characterization indicates that such a factor is a peripheral membrane protein of less than 25 kDa capable of directly interfering with the ADP-ribosylation reaction.     

Widespread distribution of Gq alpha/G11 alpha detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide

Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS-PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gs alpha.     

Two distinct Gi-proteins mediate formyl peptide receptor signal transduction in human leukemia (HL-60) cells

In membranes of myeloid differentiated HL-60 cells, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine stimulates phospholipase C via a pertussis toxin-sensitive G-protein but does not inhibit adenylyl cyclase. In these membranes, the chemotactic peptide markedly stimulates the cholera toxin-dependent [32P]ADP-ribosylation of two proteins with approximate molecular masses of 40 and 41 kDa, respectively. The radiolabeled proteins comigrate on sodium dodecyl sulfate-polyacrylamide gels with the two pertussis toxin substrates present in HL-60 membranes, alpha i2 and alpha i3. The effect of the chemotactic peptide is blocked by treatment of intact HL-60 cells with pertussis toxin. Peptide mapping studies using Staphylococcus aureus protease V8 reveal that the two radiolabeled proteins are structurally distinct. Thus, the agonist-activated formyl peptide receptor functionally interacts with two distinct pertussis toxin substrates, most likely with Gi2 and Gi3. As the third Gi protein, Gi1, appears to be absent from both HL-60 cells and from systems that clearly reveal hormonal inhibition of adenylyl cyclase, the results strongly suggest that primary structure alone does not suffice to determine which effector mechanism is regulated by a given Gi-protein.     

Purification and immunochemical characterization of the major pertussis-toxin-sensitive guanine-nucleotide-binding protein of bovine-neutrophil membranes

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as alpha n, was purified to near homogeneity from this fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified alpha n was shown to interact with beta gamma subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified alpha n on SDS/polyacrylamide gels was intermediate between those of the alpha subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the alpha subunit of transducin (Gt). Several polyclonal antisera against the alpha subunits of bovine Gt and Go did not react with alpha n on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine alpha i, reacted only minimally with alpha n. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated Gn, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.