mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells.

Opioids are regarded to act via receptors interacting with heterotrimeric pertussis toxin (PTX)-sensitive G proteins. In membranes of SH-SY5Y cells, the mu-selective agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) and the delta-selective agonist [D-Pen2,Pen5]-enkephalin (DPDPE) stimulated incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into proteins comigrating with the alpha subunits of G(i1), G(i2), G(i3), G(o1), and another form of G(o), presumably G(o2). In membranes of PTX-treated cells, both agonists were ineffective. Subtype-specific immunoprecipitation of G protein alpha subunits photolabeled in the absence or presence of agonists revealed profound differences between mu and delta opioid receptors in coupling to PTX-sensitive G proteins. Whereas activated delta opioid receptors preferentially coupled to G(i1), activated mu opioid receptors more effectively coupled to G(i3). Additionally, we provide evidence that G(o) subtypes are also differentially activated by the two receptors. Thus, mu and delta opioid receptors appear to discriminate between PTX-sensitive G proteins and lead to activation of distinct G protein subtypes.

Differential activation of Gi and Gs proteins by E- and I-type prostaglandins in membranes from the human erythroleukaemia cell line, HEL.

The group of prostaglandin (PG) E2- and prostacyclin receptors consists of different subtypes, which exhibit different affinities for prostaglandins and synthetic analogues. PGE2 activities the E-type PG receptor subtypes EP1, EP2 and EP3, whereas the PGE2 analogue, sulprostone, binds only to the EP1 and EP3 receptor subtypes. The stable PGI2 analogues, iloprost and cicaprost, both activate the PGI2 receptor (IP) and iloprost, additionally, bind to the EP1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with Gs and Gi-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE2, iloprost and cicaprost were only partially inhibited by PTX. PGE2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified Gi protein alpha subunits. Furthermore, PGE2, iloprost and cicaprost enhanced GTP azidoanilide-labelling of Gs protein alpha subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP1 and EP3 receptor subtypes activate G1 proteins, that (2) the EP2 receptor subtype activates Gs proteins and that (3) the IP receptor activates both Gi and Gs proteins.

Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells.

In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.

Detection of G-protein heterotrimers on large dense core and small synaptic vesicles of neuroendocrine and neuronal cells.

Heterotrimeric G proteins, initially believed to be exclusively present in the plasma membrane, have also been found to be associated with intracellular membrane compartments. There they are involved in various membrane trafficking processes including regulated secretion (reviewed in Bomsel, M., K. Mostov, Mol. Biol. Cell 3, 1317-1328 (1992)). Vesicles of two distinct types enter the regulated secretory pathway, i.e. large dense core vesicles and small synaptic vesicles, which differ in their membrane composition and content. Little is known about an association of heterotrimeric G proteins with regulated secretory vesicles, that would explain some aspects of the role heterotrimeric G proteins have during secretion. By immunofluorescence microscopy and immunoreplica analysis, we provide the first demonstration of the presence of complete sets of heterotrimeric G proteins, consisting of alpha-, beta-, and gamma-subunits, on large dense core vesicles from bovine adrenal medulla (chromaffin granules) and small synaptic vesicles from rodent and bovine brain. Each of the two types of secretory vesicles contains beta-subunits (at least beta 1 and beta 2), as well as gamma-subunits (at least gamma 2 or gamma 3). Interestingly, they differ in their composition of alpha-subunits. On small synaptic vesicles, we found two G(o) alpha-subunits (alpha o1 and alpha o2) and two Gi alpha-subunits (alpha i1 and alpha i2). In contrast, on chromaffin granules so far only one alpha o-subunit but no alpha i-subunits could be detected. Functional properties such as transmitter storage and/or exocytotic membrane fusion may be modulated by the various G-protein subunits associated with chromaffin granules and small synaptic vesicles.

Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells.

The alpha-subunits of the widely expressed G-proteins Gq and G11 indistinguishably activate beta-isoforms of phospholipase C. In this report we have tested whether differences exist in the activation of both G-proteins via phospholipase C-linked receptors. We found that bombesin and vasopressin, with very similar potencies and time dependencies, induce the activation of both Gq and G11 in Swiss 3T3 cells, suggesting that these G-proteins, at least in part, serve interchangable functions.

Heterogeneity of three electrophoretically distinct Go alpha-subunits in mammalian brain.

So far three splice variants of the alpha o-gene coding for two alpha o proteins have been identified by molecular cloning, and the corresponding proteins purified. In the present study subtype-specific peptide antibodies revealed the existence of an electrophoretically distinct third form of alpha o in mammalian brain membranes which migrates more slowly on SDS-PAGE and shows a more acidic pI value than the other alpha o-subunits. Each of the three alpha o-subunits is detected as two isoforms when enriched from brain membranes. Rodent alpha o-subunits differ from non-rodent species in their electrophoretic mobilities. The results suggest that (i) there may exist a novel alpha o-subunit which reacts with an alpha o1-subunit-specific antibody, (ii) each alpha o-subunit may exist in more than one co- or posttranslationally modified isoform in brain membranes, and (iii) differences between alpha o-subunits from different species exist which are detectable by gel electrophoretic methods.

Expression, characterization and purification of soluble G-protein beta gamma dimers composed of defined subunits in baculovirus-infected insect cells.

Recombinant beta 1 gamma 2 dimers of signal-transducing guanine nucleotide-binding proteins (G-proteins) carrying a mutation known to block isoprenylation of the gamma 2 subunit were expressed as a soluble protein in baculovirus-infected insect cells. The soluble beta gamma dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an S20,w value of 4.1 +/- 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus-infected insect cells can be employed for high level production of pure G-protein beta gamma dimers suitable for functional and structural characterization.

The alpha-subunits of G-proteins G12 and G13 are palmitoylated, but not amidically myristoylated.

The alpha-subunits of the G-proteins G12 and G13 were expressed with a baculovirus system in insect cells and analysed for acylation. Both proteins incorporated tritiated palmitic and to a lesser extent also tritiated myristic acid. Radiolabel from both fatty acids was sensitive to treatment with neutral hydroxylamine. This result supports a thioester-type fatty acid bond and argues against amidical N-myristoylation. Fatty acid analysis after labeling with [3H]palmitic acid showed that palmitate represents the predominant fatty acid linked to G alpha 12 and G alpha 13. Separation of cells into cytosolic and membranous fractions revealed that palmitoylated alpha-subunits of G12 were exclusively membrane-bound, whereas [35S]methionine-labeled proteins were detected in soluble and particulate fractions. Inhibition of protein synthesis with cycloheximide did not block palmitoylation of the alpha-subunits, which indicates that palmitoylation occurs independently of protein synthesis.

Agonist-sensitive binding of a photoreactive GTP analog to a G-protein alpha-subunit in membranes of HL-60 cells.

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

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

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

Receptor-mediated activation of G-proteins by kappa opioid agonists in frog (Rana esculenta) brain membranes.

This study delineates the heterotrimeric guanine nucleotide binding regulatory protein (G-protein) types in frog (Rana esculenta) brain membranes and their activation by kappa opioid agonists. Ethylketocyclazocine (EKC), trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)b enzeneacetamide (U-50,488) and bremazocine displayed dose-dependent, norbinaltorphimine-reversible stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding in crude membrane preparations. G-proteins were identified by Western-blotting using previously characterized specific antisera that were generated against mammalian G-protein alpha-subunits and beta-subunits. A photoreactive guanosine 5'-triphosphate (GTP) analog, [alpha-32P]GTP azidoanilide ([alpha-32P]AA-GTP) irreversibly labeled four proteins in the molecular weight range of 39-43 kDa. Ethylketocyclazocine and U-50,488 stimulated photolabelling of these proteins among which the 39 kDa band comigrated with the protein specifically labelled with the alpha(i2) antibody and the 40 kDa band was identified as alpha(o1). The other two bands were also stained with the alpha(common) antibody, but were not further identified. These results suggest that the endogenously expressed kappa opioid receptors that are present in frog brain interact with multiple G-proteins in situ. Furthermore, the structure of most G-proteins seems to be well preserved during phylogenesis.

G-proteins mediate inhibition and activation of Ca(2+)-induced exocytosis from SLO-permeabilized peptidergic nerve endings.

In SLO-permeabilized isolated nerve endings from the rat neurohypophysis, GTP, guanosine 5'[y-thio]triphosphate (GTPyS) and guanosine 5'(beta y-imido]triphosphate (GMPPNP) inhibit the Ca(2+)-evoked vasopressin release. Pretreatment with pertussis toxin enhances the inhibitory effects of both GTP-analogues. Omission of Mg2+ overcomes the effect of GMPPNP and reverses the inhibitory effect of GTP and GTPyS. In the absence of Mg2+, GTP and GTPyS now potentiate Ca(2+)-evoked secretion.

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens.

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method.

Changes in G protein pattern and in G protein-dependent signaling during erythropoietin- and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells.

We have studied the expression of G protein subtypes and the role of G protein-dependent signaling in two subclones of RED-1 cells, an erythropoetin(Epo)-sensitive, murine erythroleukemia cell line. Clone 6C8 showed terminal erythroid differentiation in response to a combined treatment with Epo and dimethylsulfoxide. Clone G3 was resistant to these inducers, but responded to Epo with enhanced proliferation. We measured G protein alpha subunit levels by toxin-catalyzed adenosine diphosphate (ADP)-ribosylation with [32P]-nicotinamide adenine dinucleotide (NAD) and by semiquantitative immunoblotting with specific antisera. Native RED-1 cells expressed G alpha i2, alpha i3, alpha s, and alpha q/11, but not alpha i1 and alpha o. Terminal differentiation was associated with a selective loss (approximately 80%) of G alpha i3 and an increase in a truncated cytosolic form of G alpha i2, while the membrane levels of alpha i2, alpha q/11, and alpha s did not change significantly. Treatment of G3 cells with the inducers was without effect on G protein abundance. However, except for alpha s, G3 cells contained significantly higher levels of the different G protein alpha subunits tested. Stimulation of G protein-coupled receptors by thrombin and ADP caused a pertussis toxin (PTX)-inhibitable transient increase in intracellular Ca2+ that was markedly reduced in differentiated cells. In G3 cells, but not in 6C8 cells, thrombin also caused a PTX-sensitive inhibition of isoprenaline-stimulated cyclic 3',5'-adenosine monophosphate (cAMP) formation. Our results show that specific alterations in G protein expression and function are associated with erythroid differentiation of erythroleukemia cells but do not prove a causal relationship. The loss of G alpha i3 may affect cellular responses that are mediated via P2T purine or thrombin receptors.

Adenylyl cyclase inhibition and altered G protein subunit expression and ADP-ribosylation patterns in tissues and cells from Gi2 alpha-/-mice.

The inhibition of alpha i2-/- mouse cardiac isoproterenol-stimulated adenylyl cyclase (AC; EC 4.6.1.1) activity by carbachol and that of alpha i2-/- adipocyte AC by phenylisopropyladenosine (PIA), prostaglandin E2, and nicotinic acid were partially, but not completely, inhibited. While the inhibition of cardiac AC was affected in all alpha i2-/- animals tested, only 50% of the alpha i2-/- animals showed an impaired inhibition of adipocyte AC, indicative of a partial penetrance of this phenotype. In agreement with previous results, the data show that Gi2 mediates hormonal inhibition of AC and that Gi3 and/or Gi1 is capable of doing the same but with a lower efficacy. Disruption of the alpha i2 gene affected about equally the actions of all the receptors studied, indicating that none of them exhibits a striking specificity for one type of Gi over another and that receptors are likely to he selective rather than specific in their interaction with functionally homologous G proteins (e.g., Gi1, Gi2, Gi3). Western analysis of G protein subunit levels in simian virus 40-transformed primary embryonic fibroblasts from alpha i2+/+ and alpha i2-/- animals showed that alpha i2 accounts for about 50% of the immunopositive G protein alpha subunits and that loss of the alpha i2 is accompanied by a parallel reduction in G beta 35 and G beta 36 subunits and by a 30-50% increase in alpha i3. This suggests that G beta-gamma levels may be regulated passively through differential rates of turnover in their free vs. trimeric states. The existence of compensatory increase(s) in alpha i subunit expression raises the possibility that the lack of effect of a missing alpha i2 on AC inhibition in adipocytes of some alpha i2-/- animals may be the reflection of a more pronounced compensatory expression of alpha i3 and/or alpha i1.

Purification of the G-protein G13 from rat brain membranes.

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.

Rapid isolation of G alpha 13 from bovine brain membranes: supportive effect of ethylene glycol.

G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated G alpha 13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of G alpha 13 markedly. Only in the presence of ethylene glycol (30% v/v) a clear separation of G alpha 13 from other G-proteins was achieved during the initial anion exchange chromatography. This allowed isolation of G alpha 13 by subunit exchange chromatography on beta gamma-agarose. G alpha 13 was only released from immobilized beta gamma-dimers via activation by AMF but not by GTP gamma S, pointing to a poor basal nucleotide exchange of this protein. In contrast, N-terminally truncated G alpha 13 did not bind to immobilized beta gamma-dimers.

G proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets.

Using subtype-specific antisera, we were able to identify the recently described alpha subunits of G12 and G13 in platelet membranes as 43-kDa proteins. Activation of the thromboxane A2 and the thrombin receptors in platelet membranes led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha 12 and alpha 13, indicating that both receptors couple to G12 and G13. In addition, both activated receptors were demonstrated to couple to one or more members of the Gq family. In the absence of receptor agonists, incorporation of [alpha-32P]GTP azidoanilide into alpha 12 and alpha 13 was low over a long time period (up to 45 min) due to an obviously low basal nucleotide exchange rate, whereas an agonist-stimulated photolabeling of alpha 12 and alpha 13 could be observed after 4-8 min and reached a maximum after 30-45 min. Effective activation of G12 and G13 via the thromboxane A2 and the thrombin receptors was not dependent on the presence of GDP. Our results provide evidence that G12 and G13 play a functional role in transmembrane signal transduction and suggest that both proteins are involved in pathways leading to platelet activation.

Immunocytochemical localization of the G-protein sub-unit, G(o) alpha, in rat heart. Implications for a role of G(o) alpha in secretion of cardiac hormones.

The cellular and sub-cellular localization of the G-protein subunit, G(o) alpha, in rat heart was determined by immunofluorescence and immunoelectron microscopy. Using antibodies directed against purified G(o) alpha and an antiserum raised against the C-terminal decapeptide of G(o) alpha, strong immunoreactivity was found in the conducting system of the heart, neurons, and atrial cardiomyocytes. Labeling of ventricles was weak compared to that of atria. In neurons, immunoelectron microscopy revealed G(o) alpha was localized along the inner surface of axolemma and on axoplasmal vesicles. G(o) alpha immunoreactivity in atrial and ventricular myocytes was not restricted to sarcolemma, but was also found on sub-sarcolemmal vesicles with characteristic caveolar morphology. At the level of intercalated discs, labeling was spread over the periphery of intercalated discs avoiding its membrane structures. Additionally, in atrial endocrine cardiomyocytes, approximately 60% of secretory granules revealed G(o) alpha-labeling on the cytoplasmic surface. A small number of these granules stood out due to particularly intense labeling. The observation that these granules were found most-frequently in sub-sarcolemmal areas suggests that they may be mature granules undergoing exocytosis. Therefore, G(o) alpha found on secretory granules of endocrine cardiomyocytes may have a function in regulated exocytosis of cardiac hormones. Sarcolemmal localization of G(o) alpha in atrial and ventricular cardiomyocytes supports the role of G(o) alpha in transmembrane signal transduction. Furthermore, caveolar localization of G(o) alpha may provide a compartmental basis for integrating G(o)-mediated signaling events.