Negative regulation of the platelet Na+/H+ exchanger by trimeric G-proteins.

Human platelets contain a Na+/H+ exchanger (NHE) that regulates the cytosolic pH. The role of trimeric G-proteins in NHE control was investigated in plasma membrane vesicles by measuring exchange of intravesicular protons for extravesicular Na+. Exchange was saturable, independent of membrane potential and inhibited by ethylisopropyl amiloride (Ki 0.05 micromol.L-1), demonstrating the involvement of NHE-1. The G-protein activators AlF4- and GMP-P(NH)P reduced exchange by increasing the Km for Na+ from 11.3 +/- 2.1 mM to 21.6 +/- 1.4 mM (AlF4-) and 19.8 +/- 1.1 mM (GMP-P(NH)P), leaving Vmax and the Hill coefficient unchanged. This effect was abolished by inhibitors of Gi-proteins (N-ethylmaleimide, holoenzyme- and A-protomer of pertussis toxin) and by an anti-Galpha Ig and GDP(beta)S. Activation of Gi-proteins by mastoparan and its synthetic analogue Mas7 also strongly reduced NHE activity. These data show that in platelets NHE-1 is under negative control of the Gi-family of trimeric G-proteins.     

Altered expression of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in experimental hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Giα proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Giα1, Giα1/2, and Giα3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Giα substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Giα activator) increased [³H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [³H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Giα-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro. J. Cell. Physiol. 175:295–304, 1998. © 1998 Wiley-Liss, Inc.     

Comparative study of molecular mechanisms of natural and synthetic polycationic peptides action on the activity of the adenylyl cyclase signaling system

The molecular mechanisms of action of natural and synthetic polycationic peptides, forming amphiphilic helices, on the heterotrimeric G-proteins and enzyme adenylyl cyclase (AC), components of hormone-sensitive AC system, were studied. It is shown that synthetic peptides C-epsilonAhx-WKK(C10)-KKK(C10)-KKKK(C10)-YKK(C10)-KK (peptide I) and (GRGDSGRKKRRQRRRPPQ)2-K-epsilonAhx-C(Acm)(peptide II) in dose-dependent manner stimulate the basal AC activity, inhibit forskolin-stimulated AC activity and decrease both stimulating and inhibiting AC effects of the hormones in the tissues (brain striatum, heart muscle) of rat and in smooth muscles of the mollusc Anodonta cygnea. AC effects of these peptides are decreased after membrane treatment by cholera and pertussis toxins and are inhibited in the presence of the peptides, corresponding to C-terminal regions 385-394 alphas- and 346-355 alphai2-subunits of G-proteins. These data give evidence that the peptides I and II act on the signaling pathways which are realized through Gs- and Gi-proteins. At the same time, natural polycationic peptide mastoparan acts on AC system through Gi-proteins and blocks hormonal signals mediated via Gi-proteins only. Consequently, the action of mastoparan on G-proteins is selective and differs from the action of the synthetic peptides. It is also shown that peptide II, with branched structure, directly interacts not only with G-proteins (less effective in comparison with peptide I with hydrophobic radicals and mastoparan), but also with enzyme AC, the catalytic component of AC system. On the basis of data obtained the following conclusions were made: 1) the formation of amphiphilic helices is not enough for selective activation of G-protein by polycationic peptides, and 2) the primary structure of the peptides, the distribution of positive charged amino acids and hydrophobic radicals in them are very important for selective interaction between polycationic peptides and G-proteins.     

Nod factors activate both heterotrimeric and monomeric G-proteins in <Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Interaction of activator of G-protein signaling 3 (AGS3) with LKB1, a serine/threonine kinase involved in cell polarity and cell cycle progression: phosphorylation of the G-protein regulatory (GPR) motif as a regulatory mechanism for the interaction of GPR motifs with Gi alpha

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the alpha subunit of Gi/Go (Gialpha > Goalpha) stabilizing the GDP-bound conformation of Galpha and apparently competing with Gbetagamma for GalphaGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.     

G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain.

Attachment of heterotrimeric G-proteins to the inner face of the plasma membrane is fundamental to their role as signal transducers by allowing interaction with both receptors and effectors. Certain G-protein alpha subunits are anchored to the membrane by covalent myristoylation. The beta gamma complex is required for G-protein interaction with receptors and is independently membrane associated through an unknown mechanism. A series of carboxyl-terminal modifications including isoprenylation which may contribute to membrane attachment has been identified recently in G-protein gamma subunits. Expression and membrane targeting of beta and gamma subunits were examined in COS cells. The expression of either subunit was found to require cotransfection with both beta and gamma cDNAs. Mutation of the carboxyl-terminal cysteine residue of gamma shown to undergo isoprenylation and carboxymethyl-esterification preserved beta gamma expression but blocked isoprenylation and membrane attachment. These results implicate the carboxyl-terminal processing of G-protein gamma subunits and beta coexpression as necessary and sufficient for membrane targeting of the beta gamma complex.     

GTP-binding protein-independent potentiation by mastoparan of IL-1β-induced nitric oxide release from insulin-secreting HIT-T15 cells

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.     

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells.

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.     

Regulation of adenylate cyclase by hormones and G-proteins

Over the past few years, it has become apparent that a large number of transmembrane signaling systems operate through heterotrimeric G-proteins [( 1] Gilman, A.G. (1984) Cell 36, 577-579; [2] Baker, P.F. (1986) Nature 320, 395). Adenylate cyclase is regulated by stimulatory hormones through Gs(alpha s beta gamma) and inhibitory hormones through Gi(alpha i beta gamma) [( 2]; Katada, T. et al. (1984) J. Biol. Chem. 259, 3586-3595), whereas the breakdown of phosphatidylinositol bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DG) by phospholipase C is probably also mediated by a heterotrimeric G-protein (Go or Gi) [1,2]. Similarly, the activation of cGMP phosphodiesterase by light-activated rhodopsin is mediated through the heterotrimeric G-protein transducin (Stryer, L. (1986) Rev. Neurosci. 9, 89-119). Other transmembrane signaling systems may also be found to involve G-proteins similar to those already recognized. Because of the emerging universality of G-proteins as transducers of receptor-triggered signals, it may be useful to evaluate the current models prevailing in the adenylate cyclase field, as these models seem to guide our way in evaluating the role of G-proteins in transmembrane signaling, in general.     

Endogenous RGS proteins attenuate Galpha(i)-mediated lysophosphatidic acid signaling pathways in ovarian cancer cells

Lysophosphatidic acid is a bioactive phospholipid that is produced by and stimulates ovarian cancer cells, promoting proliferation, migration, invasion, and survival. Effects of LPA are mediated by cell surface G-protein coupled receptors (GPCRs) that activate multiple heterotrimeric G-proteins. G-proteins are deactivated by Regulator of G-protein Signaling (RGS) proteins. This led us to hypothesize that RGS proteins may regulate G-protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared LPA activity in SKOV-3 ovarian cancer cells expressing G(i) subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS wild-type (RGSwt) counterparts. Both forms of the G-protein contained a point mutation rendering them insensitive to inhibition with pertussis toxin, and cells were treated with pertussis toxin prior to experiments to eliminate endogenous G(i/o) signaling. The potency and efficacy of LPA-mediated inhibition of forskolin-stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi G(i) proteins as compared to RGSwt G(i). We further showed that LPA signaling that is subject to RGS regulation terminates much faster than signaling thru RGS insensitive G-proteins. Finally, LPA-stimulated SKOV-3 cell migration, as measured in a wound-induced migration assay, was enhanced in cells expressing Galpha(i2) RGSi as compared to cells expressing Galpha(i2) RGSwt, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate this LPA effect. These data establish RGS proteins as novel regulators of LPA signaling in ovarian cancer cells.     

A 43 kDa form of the GTP-binding protein Gi3 in human erythrocytes

Purified preparations of human erythrocyte G-proteins contain a 43 kDa pertussis toxin substrate which appears to be the alpha-subunit of a heterotrimeric GTP-binding protein. The 43 kDa protein is recognized by antisera that are sequence-specific for peptides encoding a sequence common to all 39-53 kDa G-protein alpha-subunits. G alpha o-specific antiserum did not recognize 43 or 40-41 kDa alpha-subunits. AS/6, which recognizes the alpha i proteins, recognized 43 kDa as well as 40-41 kDa proteins. Of the three antisera specific for individual members of the alpha i family, only the Gi3-specific antiserum recognized the 43 kDa erythrocyte G-protein. However, 40-41 kDa forms of all three alpha is are present. These observations indicate that human erythrocytes contain a novel 43 kDa form of Gi3.     

Heterotrimeric G protein Gi is involved in a signal transduction pathway for ATP release from erythrocytes

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein G(s) resulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the G(i) subtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins G(alphai1), G(alphai2), and G(alphai3) but not G(alphao) were identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples G(i/o) from their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 microM) or Mas-7 (1 microM), which are compounds that directly activate G(i) proteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 microM), which is an inactive Mas analog. In separate experiments, Mas (10 microM) but not Mas-17 (10 microM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein G(i) is a component of a signal transduction pathway for ATP release from erythrocytes.     

Isoprenylation of C-terminal cysteine in a G-protein gamma subunit

The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue and X is any amino acid). This domain represents the site of Cys thioether modification by isoprenoids in p21ras, nuclear lamins, and fungal mating factors. We now show that G gamma 6, translated in reticulocyte lysate, is efficiently labeled with the isoprenoid precursor, [3H]mevalonate. Alteration of the sequence of G gamma 6 so that a Gly was substituted for Cys in the C-terminal Cys-A-A-X element rendered the protein incapable of undergoing isoprenoid modification. In contrast to G gamma 6, the Gi alpha 1 subunit did not appear to undergo isoprenylation when translated in reticulocyte lysate. Transient expression of the protein in COS cells, which were able to isoprenylate the p21 product of transfected H-ras, also failed to demonstrate isoprenylation of Gi alpha 1. The modification of the gamma subunit by a hydrophobic moiety may have important implications for the assembly of the brain G-protein beta gamma complexes into the cell membrane.     

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.     

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.     

Mechanisms of mastoparan-stimulated surfactant secretion from isolated pulmonary alveolar type 2 cells.

Mastoparan, a tetradecapeptide component of wasp venom, is a potent activator of secretion in a variety of cell types, and has been shown to activate purified G-proteins reconstituted into phospholipid vesicles with a preferential activation of Gi over Gs (Higashijima, T., Uzu, S., Nakajima, T., and Ross, E. R. (1988) J. Biol. Chem. 263, 6491-6494). To identify the biochemical activities of mastoparan in a cellular system, we characterized the effects of mastoparan on signal transduction pathways in rat pulmonary alveolar type 2 epithelial cells, which synthesize and secrete pulmonary surfactant. Mastoparan inhibited adenylylcyclase activity in a manner that was dose-dependent (IC50 = 30 microM), but sensitive to neither guanine nucleotide nor pertussis toxin (PT). Mastoparan induced a PT-sensitive increase in cellular inositol trisphosphate and a rapid rise in cytosolic calcium released from intracellular stores; the time to onset of the calcium rise, but neither the rate nor the amplitude of the rise, were PT-sensitive. Mastoparan also caused a dose- (EC50 = 16 microM) and time-dependent activation of arachidonic acid release that was completely insensitive to pretreatment with PT. Secretion of pulmonary surfactant was increased by mastoparan approximately 8-fold over constitutive levels at 1 h with an EC50 = 20 microM, and mastoparan-stimulated secretion was partially sensitive to PT at late time points and to inhibitors of arachidonic acid metabolism, but not to the protein kinase C inhibitor H7. These findings are consistent with the activation of Gi proteins in type 2 cells by mastoparan, although the lack of predicted triphosphoguanine nucleotide and PT sensitivity for some activities indicates that mastoparan does not act in a manner strictly analogous to liganded receptors or that some activities are not mediated by activation of Gi. While mastoparan is a potent secretagogue in several cell types, its secretory activity appears to have only a limited dependence on the activation of Gi proteins in type 2 cells.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Relationships within the family of GTP-binding proteins isolated from bovine central nervous system

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.     

Regulatory contribution of heterotrimeric G-proteins to oocyte maturation in the sea urchin

Regulation of animal oocyte maturation is hypothesized to involve heterotrimeric G-proteins. It is difficult to test this hypothesis though without knowing what G-proteins are present in these cells and where are they localized. We set out to test the hypothesis that G-proteins regulate maturation in the sea urchin oocyte by identifying resident G-proteins in oocytes and eggs, and then investigating their function. We find four families of G-protein alpha-subunits (Galphai, Galphaq, Galphas, and Galpha12) present in both oocytes and eggs of the sea urchin. Three of them, Galphai, Galphaq, and Galphas are present on the plasma membrane of the oocyte, while the fourth is located on cytoplasmic vesicles. Upon oocyte maturation, these proteins remain in eggs, and continue to be expressed in embryonic tissues. To test the functional contribution of the G-proteins to the regulation of oocyte maturation, we employ specific intervening reagents, including antibodies and competitor peptides to each Galpha subunit, and specific Galpha toxins. We find that Gi is a main candidate for a positive regulator of sea urchin oocyte maturation. These studies provide a foundation to further test specific hypotheses of the G-protein mediated regulation of oocyte maturation, fertilization, and early development in the sea urchin.