Pertussis Toxin Substrate Research Articles

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.

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).

Transfection of NG108-15 cells with antisense opioid-binding cell adhesion molecule cDNA alters opioid receptor-G-protein interaction.

We previously reported that transfection of antisense OBCAM (opioid-binding cell adhesion molecule) cDNA into NG108-15 neuroblastoma x glioma hybrid cells, which contain delta-opioid receptors, results in greatly reduced opioid binding (Ann, D. K., Hasegawa, J., Ko, J. L., Chen, S. T., Lee, N. M., and Loh, H. H. (1992) J. Biol. Chem. 267, 7921-7926. Here we report that these cells show altered coupling between opioid receptors and G-proteins. G-proteins were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for Gi2 and Go alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into a 39-41-kDa protein with the same electrophoretic mobility as Gi2 and Go alpha subunits. This band, which was also a pertussis toxin (PTX) substrate, exhibited decreased CTX-induced ADP-ribosylation in membranes of cells treated chronically with D-Ala2-D-Leu5-enkephalin (DADLE). In cells transfected with antisense cDNA for OBCAM, labeling of this band was also decreased, compared with either sense-transfected or untransfected cells. DADLE inhibition of adenylyl cyclase and DADLE stimulation of GTPase were also greatly impaired in antisense cells, as well as GTP and GppNHp inhibition of basal and forskolin-stimulated adenylyl cyclase. These results provide further evidence for a role of OBCAM in opioid receptor function.

Tissue- and subunit-specific regulation of G-protein expression by hypo- and hyperthyroidism

Thyroid hormone status has profound effects on signal transduction in various tissues throughout the body. Therefore, we quantified the signal transducing G-proteins in the rat heart, cerebral cortex, vas deferens and liver by immunoblotting and pertussis toxin labeling in response to chemically induced hypothyroidism (treatment with propylthiouracil) and hyperthyroidism (treatment with triiodothyronine). Levels of the pertussis toxin (PTX) substrates G(Iniα) and G oα in the heart and vas deferens were inversely correlated with thyroid hormone levels, i.e. G iα and G oα were decreased or unchanged in hyperthyroid rats and increased in hypothyroid rats compared to control animals. The cerebral cortex and liver expression of PTX substrates G iα and G oα was not affected by changes in thyroid hormone. Regulation of G sα protein was more complex in that G sα was elevated in the hearts from both hypothyroid and hyperthyroid rats compared to control rats, while G sα was unaffected in the other tissues tested. Expression of G-protein β-subunits was not affected by thyroid status in the heart, liver, or cerebral cortex. Our results suggest that tissue- and G-protein-specific factors are involved in the regulation of G-protein subunits by thyroid hormone. Moreover, cardiac expression of G sα is upregulated by increases or decreases in the normal level of thyroid hormone.

Coupling of the expressed alpha 1B-adrenergic receptor to the phospholipase C pathway in Xenopus oocytes. The role of Go.

alpha 1B-Adrenergic receptor mRNA was injected into Xenopus oocytes, resulting in a norepinephrine-evoked Cl- current. The response was proportional to norepinephrine concentration, blocked by prazosin, and dependent on intracellular Ca2+ derived from inositol trisphosphate-sensitive stores. Oocytes treated with 2 micrograms/ml pertussis toxin showed a time-dependent decrease of the norepinephrine response, taking up to 72 h to show an 80% decrease. Overnight treatment with 10 micrograms/ml pertussis toxin also resulted in 80% reduction. Responses to two other cloned receptors (M1-muscarinic and serotonin-1c) expressed in oocytes were also reduced 50% or more by 72 h of pertussis toxin treatment. Pertussis toxin labeling of the cloned Xenopus alpha o-subunit translated in vitro showed that it was a significantly poorer substrate for pertussis toxin than the two mammalian alpha o-subunits expressed and assayed under identical conditions. This unexpected biochemical behavior of the Xenopus alpha o-subunit is in agreement with the rather unusual treatment conditions required to observe the effects of pertussis toxin on the receptor-evoked Cl- current in the oocyte. Injection of mammalian heterotrimeric G(o) but not Gi3 significantly enhanced the norepinephrine-evoked Cl- current in oocytes. Injection of mixtures of anti-sense oligonucleotides to the Xenopus alpha o-subunit reduced the norepinephrine-evoked Cl- current by 60% within 24 h, compared with oocytes injected with the oligonucleotides encoding sense sequences. These studies indicate that the expressed alpha 1B-adrenergic receptor, like the native muscarinic receptor, utilizes G(o) to couple to the phospholipase C-mediated Cl- current in Xenopus oocytes.

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.

Pertussis toxin expression in Drosophila alters the visual response and blocks eating behaviour

Pertussis toxin inactivates certain G-proteins by introducing an ADP-ribose group near the carboxyl-terminus of the α-subunit. The major pertussis toxin substrate in Drosphilla tissues is G oα. We introduced a pertussis toxin gene under control of the hsp70 heat-shock promoter into the Drosphila genome. When heat-shocked, transformed flies produced active pertussis toxin which ADP-ribosylates endogenous G oα. Pertussis toxin is expressed in photoreceptors, in the lamina of the eye and in epithelial cells lining the gut. As expected from the absence of G oα in photoreceptors, pertussis toxin does not affect the photoreceptor component of the Drosophila visual response. However, it abolishes light on- and off-transients in the electroretinogram. These normally arise from the lamina, a tissue where G oα transcripts have been detected. Pertussis toxin expression also blocks embryonic development and shortens the lifetime of adult Drosophila. Following heat-shock, transformed adult are active, but they fail to take up nutrients because they stop eating. High energy metabolites are significantly depleted shortly after pertussis toxin expression is induced and the flies die within 48 h.

Guanine nucleotide binding proteins mediate the chemotactic signal of transforming growth factor-beta 1 in rat IL-2 activated natural killer cells.

Transforming growth factor (TGF)-beta 1 induced rat IL-2-activated natural killer (IANK) cell chemotaxis. Various doses of cholera toxin (CT) or pertussis toxin (PT) inhibited the activity of TGF-beta 1 suggesting a role for guanine nucleotide binding (G) proteins. ADP-ribosylation assay showed that rat IANK cell membranes possess a 39 kDa PT substrate and two, 41 and 42 kDa, CT substrates. ADP-ribosylation also showed that incubating IANK cell membranes with TGF-beta 1 in the presence of guanosine 5'-O-(3-thiotriphosphate) resulted in the disappearance of the PT substrate. Immunoblot analysis showed that rat IANK cell membranes possess one Gi (39 kDa), one G0 (39 kDa) and three Gs (40, 41, and 42 kDa) proteins. Pretreatment of IANK cell membranes with TGF-beta 1 in the presence of guanosine-5'-O-(3-thiotriphosphate) reduced the intensity of the 39 kDa G(0) and the 40 kDa Gs but not the 39 kDa Gi or the 41 kDa or 42 kDa Gs. Furthermore, TGF-beta 1 stimulated GTP binding and increased GTPase activity in IANK cell membranes. Both activities were inhibited by PT or CT. This inhibition was associated with the modification of G proteins by the toxins suggesting that bacterial toxin substrates are linked to TGF-beta 1 receptors. Our results suggest that G0 and Gs are involved in mediating the chemotactic signal of TGF-beta 1 in rat IANK cells.

Identification of heterotrimeric and low molecular weight GTP-binding proteins in rabbit skeletal muscle longitudinal sarcoplasmic reticulum

Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [ 32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [ 32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of G sα, showed an immunoreactive band at about 45 kDa. [ 32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)- ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [ 32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a G sα, the absence of G i and G o, and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.

Opposing influences of dexamethasone and retinoic acid on adenylate cyclase activity in ROS 17/2.8 cells.

Exposure of ROS 17/2.8 cells to dexamethasone (DEX) or retinoic acid (RA) increases and decreases, respectively, adenylate cyclase activity (ACA) in response to isoproterenol, forskolin, guanylylimidodiphosphate, or NaFl. Despite dramatic changes in ACA, there were no significant changes in levels of cholera toxin- or pertussis toxin (PT)-dependent ADP-ribosylation of membranes prepared from cells after DEX or RA exposure as compared to controls. Similarly, immunochemical detection of alpha S, alpha i1-3, and alpha O, as well as Northern blot analysis of messenger RNA for each of the respective GTP binding proteins, also failed to demonstrate an influence of DEX or RA when contrasted with controls. In a novel use of the cyc- reconstitution assay, wherein the influence of inhibitory guanine nucleotide binding proteins in the extracts of control, DEX-, and RA-treated membranes is removed by a previous 24-h incubation with PT in the intact cell, we demonstrate that this PT treatment markedly enhances ACA in the cyc- reconstitution assay for all three preparations, but that the fold-increase due to PT-treatment is greatest in RA-treated cells. The greater magnitude of the effect of PT on RA-treated ROS 17/2.8 cells, in the absence of any obvious quantitative changes in the levels of the PT substrates, suggests that the effect of RA on ROS 17/2.8 cells appears to be an augmentation of the influence of inhibitory guanine nucleotide binding proteins, ultimately leading to reduced ACA.

Cellular signalling by lipoproteins in cultured smooth muscle cells from spontaneously hypertensive rats.

We investigated pivotal signalling responses of cultured aortic smooth muscle cells (VSMC) from the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto rat (WKY) to lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL3) stimulated a time- and dose-dependent accumulation of inositol phosphates in VSMC. SHR and WKY VSMC exhibited comparable half-maximal dose requirements (approximately 13 micrograms/ml for LDL and approximately 14 micrograms/ml for HDL3), although, at any given dose, the response of SHR VSMC was significantly greater than WKY VSMC. Simultaneous addition of LDL and HDL3 to VSMC resulted in additive stimulatory effects on phosphoinositide catabolism. Pertussis toxin pretreatment of VSMC completely negated the stimulatory effects of LDL and HDL3 on IP accumulation. [32P]-ADP ribosylation and immunoblotting studies revealed the guanine nucleotide-binding (G protein) substrate(s) for pertussis toxin to be a Gi protein(s). SHR and WKY VSMC did not differ with respect to levels of Gi alpha or G beta, and thus, the amplified responsiveness in SHR VSMC cannot be attributed to alterations in levels of pertussis toxin-sensitive G protein. The spectrum of signalling responses elicited by LDL and HDL3 are similar to those elicited by vasoactive hormones, and thus lipoproteins may, via stimulation of phosphoinositide catabolism, 45Ca2+ uptake and Na+/H(+)-exchange, directly regulate smooth muscle cell growth and contraction.

Impaired G-proteins and cyclic nucleotide phosphodiesterase activity in T-lymphocytes from patients with sarcoidosis.

Among the various immune abnormalities which characterize active sarcoidosis, a low proliferative response of peripheral blood lymphocytes to mitogenic lectins has long been observed. Since membrane-associated G-proteins are very likely to be crucial elements in lectin signal transduction, we investigated the binding of 5'-guanylylimidodiphosphate (GppNHp), a non hydrolyzable GTP analogue, to blood total lymphocyte membranes and to blood T-lymphocyte membranes from patients with active sarcoidosis, and from healthy control subjects. GppNHp binding was markedly decreased in peripheral cells from patients with sarcoidosis as compared to controls, suggesting the occurrence of a defect at the level of G-protein(s). A further characterization of G-proteins in these cells by means of ADP-ribose-labelling in the presence of bacterial toxins brought forward a significant decrease in the labelling of a 40 kDa protein, the major pertussis toxin substrate, in membranes from sarcoid patients, while the labelling of the major 44 kDa cholera toxin substrate proved to be unchanged with respect to control membranes. It is hypothesized that, in sarcoid lymphocytes, a defect in the negative control of adenylate cyclase mediated by the inhibitory G-protein Gi, prevents the lowering of cAMP necessary to normal mitogenic response of blood lymphocytes to stimulation. cAMP degradation by the specialized enzyme phosphodiesterase constitutes another critical step in the control of cAMP levels. Both cAMP and cGMP phosphodiesterase activities were found decreased in blood total lymphocyte preparations from sarcoid patients. With purified T-cells, although the mean cAMP and cGMP phosphodiesterase activities from sarcoid patients were found more markedly decreased with respect to healthy donors, only the decrease in cGMP phosphodiesterase was found statistically significant. The role these defects in cyclic nucleotide degradation potentially play in the disturbance of blood lymphocytes response associated with sarcoidosis is discussed.

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.

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.

Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats.

The question of this study was whether alterations in the inhibitory guanine-nucleotide binding protein alpha-subunits (G(i)alpha) contribute to alterations in adenylate cyclase regulation in hypertensive cardiomyopathy of spontaneously hypertensive rats (SHR). G(i)alpha was measured by pertussis toxin-catalysed 32P-adenosine 5'-pyrophosphate (ADP)-ribosylation and radioimmunochemically by competition of rat myocardial membrane extracts to DS 4 antiserum binding to the 125I-radiolabelled C terminus of retinal transducin alpha (125I-KENLKDCGLF). Cardiac beta-adrenoceptors, m-cholinoceptors as well as isoprenaline-, guanine-nucleotide [Gpp(NH)p]- and forskolin-stimulated adenylate cyclase activity and inotropic responses to isoprenaline and carbachol were studied in SHR and age-matched Wistar-Kyoto (WKY, control) rats. In native membranes of SHR there was an increase in pertussis toxin substrates, but a larger increase in the presence of non-ionic detergent Lubrol PX. The radioimmunological quantification of G(i)alpha revealed an increase in membrane extracts of SHR. In addition, myocardial beta-adrenoceptors and myocardial m-cholinoceptors were reduced in SHR compared with in WKY rats. Basal adenylate cyclase, isoprenaline-, Gpp(NH)p- and forskolin-stimulated adenylate cyclase activities were also reduced. However, in the presence of 5 mmol/l MnCl2 no differences in adenylate cyclase activities between SHR and WKY rats were detected under either condition. The present study shows that the amount of G(i)alpha-proteins and not only pertussis toxin substrates are increased in membranes of hypertrophic hearts from SHR without heart failure. The results obtained with pertussis-labelling depended strongly on the substrate quality of G(i)alpha. Increased G(i)alpha expression and reduced beta-adrenoceptor number might have functional relevance in the regulation of adenylate cyclase activity and force of contraction in SHR. An increase in G(i)alpha expression might play a pathophysiological role, not only in terminal heart failure, but also in hypertrophic cardiomyopathy.

Genes coding for proteins in central nervous system myelin.

There has been considerable interest recently in a genetic component as a causative factor in multiple sclerosis, but the identity of putative susceptibility genes is unknown. In the past decade, the primary amino acid sequences of the four proteins making up 90% of the protein content of central nervous system myelin (proteolipid protein, myelin basic protein, 2',3'-cyclic nucleotide-3'-phosphohydrolase, and myelin-associated glycoprotein) have been determined in several species. Additionally, the structural genes coding for these proteins have been analysed and their human chromosomal localization determined. We have been analysing these genes for possible variants conferring susceptibility to multiple sclerosis. Recent results have shown that cholera and pertussis toxin substrates and low molecular-weight GTP-binding proteins are also present in central nervous system myelin. This implies the presence of signal transducing systems whose purpose is currently obscure. The emerging picture of central nervous system myelin is of a complex dynamic structure composed of many more proteins than was previously thought.

Differential isoprenylation of carboxy-terminal mutants of an inhibitory G-protein alpha-subunit: neither farnesylation nor geranylgeranylation is sufficient for membrane attachment.

To determine the effect of protein isoprenylation with farnesyl vs geranylgeranyl groups on membrane association in vivo, COS cells were transfected with cDNAs encoding the wild-type G-protein alpha i1 (WT) subunit, the soluble nonmyristoylated G-protein alpha i1 glycine to alanine mutant (GA), a double mutant in which the carboxy-terminal residues CGLF of GA were mutated to CVLS (GA-CVLS), and a double mutant in which the carboxy terminus of GA was mutated to CALL (GA-CALL). As opposed to the WT and GA proteins, the GA-CVLS and GA-CALL proteins were not pertussis toxin substrates nor were they recognized by antibodies that recognize the nonmutated alpha i1 carboxy terminus. Only the GA-CVLS and GA-CALL proteins incorporated [3H]mevalonate in the form of a farnesyl and a geranylgeranyl moiety, respectively. Subcellular localization, as assessed by immunoblotting and immunoprecipitation, revealed that the WT protein localizes almost exclusively to the membrane fraction, whereas the GA, GA-CVLS, and GA-CALL proteins localize predominantly to the soluble fraction. The soluble GA-CVLS and GA-CALL proteins were not carboxyl methylated, but the small amount localized to the membrane was partially carboxyl methylated. These results indicate that neither farnesylation nor geranylgeranylation is sufficient alone to lead to membrane association.

Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus.

The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues. Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor. To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates. As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate. Other inactive mutants were G352D and L353 delta/Y354 delta. Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta. Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G. ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin. It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha).

IL-3 stimulated haemopoietic stem cell proliferation: evidence for G protein independent mitogenic signalling events.

Interleukin-3 stimulates the survival and proliferation of the FDCP-Mix 1 multipotent stem cell line. We have investigated the possible involvement of a guanyl nucleotide regulatory (G) protein(s) in the IL-3 stimulated proliferative response. We report here that pertussis toxin (PT) can partially inhibit IL-3 stimulated DNA synthesis and that this inhibition is bypassed by TPA. The ADP-ribosylation of the PT substrate G protein in vivo is complete in 2 hours without concomitant inhibition of IL-3 stimulated hexose transport or Na+/H+ exchange. When loaded into FDCP-Mix 1 cells fluoroaluminate and GTP-gamma-S, which can directly activate G proteins, are not capable of mimicking the effects of IL-3. Evidence is also presented that IL-3 does not stimulate a membrane-bound high affinity GTPase activity in the FDCP-Mix 1 cell line. These data suggest that a PT substrate G protein(s) can influence the IL-3 signalling cascade in an indirect or permissive manner, but that the IL-3 receptor does not directly couple to a PT substrate G protein.

Properties of 1-methyladenine receptors in starfish oocyte membranes: Involvement of pertussis toxin-sensitive GTP-binding protein in the receptor-mediated signal transduction

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a guanine nucleotide-binding protein (G protein) serving as the substrate of pertussis toxin is involved in the 1-MA receptor-mediated signal. We thus investigated properties of 1-MA receptors by means of binding of the radiolabeled ligand to the oocyte membranes. There were apparently two forms of 1-MA receptors with high and low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. A 39-kDa protein, which had been identified as the alpha-subunit of the major substrate G protein for pertussis toxin, was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the 39-kDa pertussis toxin-substrate G protein in starfish oocyte membranes.