A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.     

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.     

Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2

A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin- induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies.     

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive G alpha i-3 protein.

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.     

Possible involvement of a GTP-binding protein in a late event during endogenous ganglioside-modulated cellular proliferation

The B subunit of cholera toxin, a protein which binds specifically to ganglioside GM1 on the cell surface, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts as measured by an increase in [3H]thymidine incorporation. Pertussis toxin pretreatment markedly inhibits B subunit-induced DNA synthesis. The inhibitory effects of pertussis toxin were observed even in the presence of insulin which greatly potentiates the mitogenic response to the B subunit. Treatment with either pertussis toxin or insulin did not alter the binding of the B subunit to the cells. The dose-response for pertussis toxin-induced inhibition of DNA synthesis correlated closely with the dose-response for ADP-ribosylation of a 41-kDa membrane protein, suggesting the involvement of a GTP-binding protein that is a substrate for pertussis toxin (Gi) in mitogenesis induced via cross-linking of endogenous gangliosides. Pertussis toxin, in a similar concentration-dependent manner, also inhibited the mitogenic response to unfractionated fetal calf serum and to bombesin in the absence or presence of insulin. The inhibitory effect of pertussis toxin was clearly unrelated to any effects on known G proteins coupled to adenylate cyclase or phospholipase C. In addition, pertussis toxin did not impair the early increase in cytosolic free Ca2+ induced by the B subunit or bombesin. Pertussis toxin-induced inhibition of DNA synthesis could still be observed even when the toxin was added as late as 6 h after addition of the growth-promoting agents. This suggests the involvement of a GTP-binding protein in a late step of the B subunit- and bombesin-mediated pathways of mitogenesis. The possibility that other growth factors bypass this pathway is shown by their lack of sensitivity to pertussis toxin.     

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.     

Expression and potential functions of G-protein alpha subunits in embryos of Xenopus laevis.

During early embryonic development, many inductive interactions between tissues depend on signal transduction processes. We began to test the possibility that G-proteins participate in the signal transduction pathways that mediate neural induction. The expression during Xenopus development of three G alpha subunits, G alpha 0, G alpha i-1 and G alpha s-1, was characterized. The three maternally expressed genes showed different expression patterns during early development. Whole-mount in situ hybridization revealed that all three genes were expressed almost exclusively in the gastrula ectoderm and predominantly in the neuroectoderm in the neurula embryo. In order to investigate the involvement of these proteins in neural induction, we overexpressed the G-protein alpha subunits by injecting the G alpha mRNAs into fertilized eggs. Overexpression of G alpha s-1 increased the ability of gastrula ectoderm to become induced to neural tissue approximately four-fold. Overexpression of G alpha 0 and G alpha i-1 had less pronounced effects on neural competence, and inhibition of the G alpha 0 and G alpha i-1 proteins by pertussis toxin did not change the neural competence of the exposed gastrula ectoderm. Overexpression of the G alpha 0 and G alpha i-1 genes did, however, inhibit the normal disappearance of the blastocoel during gastrulation, suggesting a role for these G-proteins in regulating this process. The data also suggest a specific role for the G alpha s subunit in mediating the initial phases of neural induction.     

Independent activation of endogenous p21-activated protein kinase-3 (PAK3) and JNK by thrombin in CCL39 fibroblasts

Thrombin, a potent mitogen for CCL39 hamster lung fibroblasts, activates the seven membrane-spanning receptor PAR1. To better understand the signaling pathways controlled by this receptor we analyzed a potential downstream effector, p21-activated protein kinase (PAK). Thrombin and PAR1 agonist peptide, as well as serum and lysophosphatidic acid, were found to stimulate HA-mPAK3 activity in CCL39 cells transfected with a plasmid encoding the epitope-tagged kinase. Similar results were obtained using antibodies developed against the endogenous kinase. PAK3 activation is sensitive to pertussis toxin, but insensitive to LY 294002, an inhibitor of phosphatidylinositol 3'-kinase. Thrombin and serum also activate c-jun amino terminal kinase (JNK). Similar to PAK3 activation, thrombin-stimulated JNK activity is inhibited by pertussis toxin, but not by LY 294002. In a CCL39-derived cell line expressing constitutively active mPAK3 in a tetracyline-dependent manner, induction of PAK activity does not lead to corresponding increases in JNK activity. Our findings indicate that PAK3 is responsive to thrombin and other G protein-coupled receptor systems. Furthermore, our data suggest that in CCL39 cells, JNK activation by thrombin occurs independently of PAK3.     

G protein subunit, alpha i-3, activates a pertussis toxin-sensitive Na+ channel from the epithelial cell line, A6

In nonpolar excitable cells, guanine nucleotide regulatory (G) proteins have been shown to modulate ion channel activity in response to hormone receptor activation. In polarized epithelia, hormone receptor-G protein coupling involved in the generation of cAMP occurs on the basolateral membrane, while the physiological response to this messenger is a stimulation of ion channel activity at the apical membrane. In the present study we have utilized the patch-clamp technique to assess if the polarized renal epithelia, A6, have topologically distinct G proteins at their apical membrane capable of modulating Na+ channel activity. In excised inside-out patches of apical membranes, spontaneous Na+ channel activity (conductance 8-9 picosiemens) was inhibited by the addition of 0.1 mM guanosine 5'-O-(2-thio)diphosphate to the cytosolic membrane surface without an effect on single channel conductance. In contrast, the percent open time of spontaneous Na+ channels increased from 6 to 50% following the addition of 0.1 mM GTP. The addition of preactivated pertussis toxin (100 ng/ml) to the cytosolic bathing solution of the excised patch inhibited spontaneous Na+ channel activity within a minute by 85% from approximately 47 to 7% open time and reduced the percent open time for Na+ channel activity to zero after approximately 3 min. The addition of 0.1 mM guanosine 5'-(3-O-thio)triphosphate or the addition of 20 pM purified human alpha i-3 subunit to pertussis toxin-treated membrane patches restored Na+ channel activity from zero to 35% open time. As little as 0.2 pM alpha i-3 subunit was capable of restoring Na+ channel activity. These data provide evidence for a role of pertussis toxin-sensitive G proteins in the apical plasma membrane of renal epithelia distal to signal transduction pathways in the basolateral membrane of these cells. This raises the possibility of a topologically distinct signal transducing pathway co-localized with the Na+ channel.     

Subtype-specific increase in G-protein alpha-subunit mRNA by interleukin 1 beta

The guanine nucleotide regulatory proteins (G-proteins) which are substrates for ADP-ribosylation by pertussis toxin (alpha i-1, alpha i-2, alpha i-3 and alpha o) transduce a variety of hormonal signals. Endothelial cells express mRNA for three alpha i subtypes although the level of alpha i-1 mRNA is very low. Interleukin 1 beta (IL 1 beta), a pleiotropic inflammatory mediator which stimulates a complex series of responses in human endothelial cells leading to increased coagulation and platelet adhesion, increases expression of one subtype of alpha i (alpha i-2) mRNA in human endothelial cells as determined by Northern blot analysis without affecting the level of mRNA for other alpha-subunits. These studies show that mRNA levels for alpha i subtypes are independently regulated, suggesting that there may be subtype specificity in the cell's requirements for the Gi class of signal-transducing proteins.     

Coupling of the insulin-like growth factor-I receptor tyrosine kinase to Gi2 in human intestinal smooth muscle: Gbetagamma -dependent mitogen-activated protein kinase activation and growth

Endogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, G(i), which mediates G(beta)gamma-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of G(i2). G(beta)gamma subunits selectively mediated IGF-I-dependent MAP kinase activation; G(alpha)i2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase activity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of G(i)/G(o) activation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of G(i2) activation. These data suggest that IGF-I specifically activates G(i2), resulting in concurrent G(beta)gamma-dependent stimulation of MAP kinase activity and growth, and G(alpha)i2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression.     

Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

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 (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L.A. (1989) Dev. Biol. 133, 605-608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.     

Pertussis toxin inhibits EGF-, phorbol ester- and insulin-stimulated DNA synthesis in BALB/c3T3 cells: evidence for post-receptor activation of Gi alpha

The contribution of the GTP-binding protein, Gi, to EGF, phorbol dibutyrate (PdBu)-, and insulin-stimulated DNA synthesis was examined in BALB/c3T3 cells. Pertussis toxin inhibited DNA synthesis by each agonist, particularly at suboptimal agonist concentrations, but the inhibition could be partially overcome with higher agonist concentrations and combinations of these agonists. This suggested that (1) some, but not all, of the mitogenic signals for all three agonists were transduced by Gi (2) Gi may be activated by post-receptor mechanisms involving protein kinase C. Gi alpha-specific antibodies and ADP-ribosylation by pertussis toxin using 32P-NAD each labelled a single protein band, representing one or more species of Gi alpha. Pertussis toxin treatment increased the synthesis of Gi alpha. These results are discussed in relation to possible direct effects of Gi alpha on nuclear control during division.     

Transforming growth factor beta enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast-like cells

The effects of transforming growth factor beta (TGF beta) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Purified TGF beta incubated with UMR-106 cells for 48 hr produced a concentration-dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGF beta. TGF beta also enhanced receptor-mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptor-mediated enzyme activation by cholera toxin and forskolin. In cells in which PTH-stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGF beta was reduced by 33%. However, TGF beta neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP-ribosylation of a 41,000-Da protein, presumably the alpha subunit of the inhibitory guanine nucleotide-binding regulatory component (Gi) of adenylate cyclase, in cholate-extracted UMR-106 cell membranes. TGF beta also had no effect on the levels of alpha or beta subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (less than or equal to 30 min) exposure of cells to TGF beta during early culture was sufficient to increase PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. TGF beta enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGF beta enhances PTH response in osteoblast-like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGF beta may involve Gi, although in a manner unrelated to either pertussis toxin-catalyzed ADP-ribosylation of the alpha subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGF beta on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGF beta may regulate osteoblast responses to systemic hormones.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

A novel strategy to engineer functional fluorescent inhibitory G-protein alpha subunits

Signaling studies in living cells would be greatly facilitated by the development of functional fluorescently tagged G-protein alpha subunits. We have designed G(i/o)alpha subunits fused to the cyan fluorescent protein and assayed their function by studying the following two signal transduction pathways: the regulation of G-protein-gated inwardly rectifying K(+) channels (Kir3.0 family) and adenylate cyclase. Palmitoylation and myristoylation consensus sites were removed from G(i/o) alpha subunits (G(i1)alpha, G(i2)alpha, G(i3)alpha, and G(oA)alpha) and a mutation introduced at Cys(-4) rendering the subunit resistant to pertussis toxin. This construct was fused in-frame with cyan fluorescent protein containing a short peptide motif from GAP43 that directs palmitoylation and thus membrane targeting. Western blotting confirmed G(i/o)alpha protein expression. Confocal microscopy and biochemical fractionation studies revealed membrane localization. Each mutant G(i/o) alpha subunit significantly reduced basal current density when transiently expressed in a stable cell line expressing Kir3.1 and Kir3.2A, consistent with the sequestration of the Gbetagamma dimer by the mutant Galpha subunit. Moreover, each subunit was able to support A1-mediated and D2S-mediated channel activation when transiently expressed in pertussis toxin-treated cells. Overexpression of tagged G(i3)alpha and G(oA)alpha alpha subunits reduced receptor-mediated and forskolin-induced cAMP mobilization.     

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.     

Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.     

Opposing effects of fibroblast growth factor and pertussis toxin on alkaline phosphatase, osteopontin, osteocalcin, and type I collagen mRNA levels in ROS 17/2.8 cells

In rat osteosarcoma (ROS 17/2.8) cells, which express osteoblastic features in culture, basic fibroblast growth factor (bFGF) reduces the level of alkaline phosphatase, type I collagen, and osteocalcin mRNA and increases osteopontin mRNA, independent of growth stimulation. The fibroblast growth factor (FGF) effects are dose dependent (EC50 about 6 pM) and are detected 24 h after addition of the growth factor. bFGF also reduces parathyroid hormone-stimulatable adenylate cyclase and alkaline phosphatase activity in these cells. Concomitant treatment with pertussis toxin (20 ng/ml) opposes the FGF effects. Although cyclic AMP elevating agents mimic pertussis toxin action on some parameters, they produce opposite effects on others, indicating that antagonism between pertussis toxin and bFGF is not mediated by cyclic AMP. bFGF caused a small reduction in steady state NAD-dependent ADP-ribosylation and had no detectable effects on the steady-state levels of the Gi alpha (alpha subunit of the inhibitory G protein) 1, 2, and 3, visualized with specific antibodies in these cells. Although the site of interaction of pertussis toxin and FGF remains to be determined, the findings presented here suggest separate control of growth and differentiation by bFGF and show that pertussis toxin treatment can modulate differentiation in these cells, presumably via Gi proteins.     

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.