Membrane attachment of recombinant G-protein alpha-subunits in excess of beta gamma subunits in a eukaryotic expression system

Recombinant cDNAs encoding the alpha-subunits of Gi1, Gi2, Gi3, Go and Gs were transfected into COS cells with the pCD-PS mammalian expression vector. Expression of each G alpha was verified using subtype-specific peptide antisera on immunoblots. Quantitative immunoblotting of alpha and beta subunits indicated: i) that there was no change in expression of endogenous beta subunits, and ii) overexpression of alpha subunits could achieve a ratio of alpha:beta greater than 25:1. Despite the excess of alpha over beta, the G alpha subunits were found predominantly in the membrane fraction. The results demonstrate that G alpha subunits can attach to the membrane independently of beta gamma subunits.     

Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics

The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel.     

Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain

Antisera AS/6 and 7, raised against a synthetic peptide KENLKDCGLF corresponding to the carboxyl-terminal decapeptide of transducin-alpha, react on immunoblots with purified transducin-alpha and with proteins of 40-41 kDa in all tissues tested. The latter represent one or more forms of Gi alpha but not Go alpha, since a synthetic peptide, KNNLKDCGLF, corresponding to the carboxyl-terminal decapeptide of two forms of Gi alpha blocks AS/6 and 7 reactivity with transducin-alpha and Gi alpha on immunoblots, whereas the corresponding Go-related peptide, ANNLRGCGLY, does not. Antisera LE/2 and 3, raised against the synthetic peptide LERIAQSDYI, corresponding to an internal sequence predicted by one form of Gi alpha cDNA (Gi alpha-2) and differing by 3 residues from the sequence of another form, Gi alpha-1, react strongly with a 40-kDa protein abundant in neutrophil membranes and with the major pertussis toxin substrate purified from bovine neutrophils. LE/2 and 3 reveal a relatively faint 40-kDa band on immunoblots of crude brain membranes or of purified brain Gi/Go. LE/2 and 3 do not react with transducin-alpha or Go alpha nor with the 41-kDa form of pertussis toxin substrate in brain, Gi alpha-1. These antisera distinguish between the major pertussis toxin substrates of brain and neutrophil and tentatively identify the latter as Gi alpha-2.     

Identification of the GTP-binding protein encoded by Gi3 complementary DNA

Three closely related, but distinct, GTP-binding proteins (G-proteins) are encoded by cDNAs arbitrarily designated Gi1, Gi2, and Gi3. The in vitro translated products of mRNAs prepared from Gi1, Gi2, and Gi3 cDNAs migrate as 41-, 40-, and 41-kDa proteins, respectively, on sodium dodecyl sulfate-polyacrylamide gels. Antisera were raised against synthetic decapeptides corresponding to a divergent sequence (residues 159-168 for Gi1 and Gi3; 160-169 for Gi2) of the three cDNAs and tested on immunoblots for reactivity with three purified G-proteins, G41 and G40 from brain and G41 from HL-60 cells. LD antisera (Gi1 peptide) react only with brain G41. LE antisera (Gi2 peptide) react only with brain G40, and SQ antisera (Gi3 peptide) react exclusively with HL-60 G41. The results indicate that the 41-kDa G-protein purified from HL-60 cells differs from the purified brain 41-kDa protein and suggest that the HL-60 cell protein corresponds to that encoded by Gi3 cDNA.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Chromatographic resolution and immunologic identification of the alpha 40 and alpha 41 subunits of guanine nucleotide-binding regulatory proteins from bovine brain

A guanine nucleotide-binding regulatory protein (G protein), with subunits designated as alpha 40 beta gamma, was identified and partially resolved from two other purified G proteins, Go (alpha 39 beta gamma) and Gi (alpha 41 beta gamma), found in bovine brain. The alpha 40 G protein subunit served as a substrate for ADP-ribosylation catalyzed by Bordetella pertussis toxin, as did alpha 39 and alpha 41. alpha 40 was shown to be closely related to, but distinct from, alpha 41 by reaction with various peptide antisera. An antiserum generated against a peptide derived from the sequence of a Gi alpha clone isolated from a rat C6 glioma cDNA library (Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K., and Kaziro, Y. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3776-3780) reacted with alpha 40 to the exclusion of all other alpha subunits tested. Another antiserum generated against a peptide derived from an analogous region of a different Gi alpha clone from a bovine brain cDNA library (Nukuda, T., Tanabe, T., Takahashi, H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa, K., Hiranaga, M., Matsuo, H., and Numa, S. (1986) FEBS Lett. 197, 305-310) reacted exclusively with alpha 41. Evidence is given for the existence of another form of alpha 41 that did not react with either of these two peptide antisera. The antisera were used to survey various rat tissues for the expression of alpha 40 and alpha 41.     

Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets.

Noradrenaline (NA) stimulated the release of arachidonic acid (AA) from the [3H]AA-labelled rabbit platelets via alpha 2-adrenergic receptors, since the effect of NA was inhibited by yohimbine. The stimulatory effect of NA in digitonin-permeabilized platelets was completely dependent on the simultaneous presence of GTP and Ca2+. The NA- and thrombin-stimulated releases of AA were markedly decreased by the prior ADP-ribosylation of the permeabilized platelets with pertussis toxin. Antiserum directed against the pig brain Go (a GTP-binding protein of unknown function), recognizing both alpha 39 and beta 35,36 subunits, but not alpha 41, of pig brain, reacted with 41 kDa and 40 kDa bands, with not one of 39 kDa, in rabbit platelet membranes. Anti-Go antiserum inhibited guanosine 5'-[gamma-thio]triphosphate-, A1F4(-)-, NA- and thrombin-stimulated AA releases in the membranes. Although the effect of thrombin was inhibited by low concentrations of anti-Go antiserum, high concentrations of the antiserum was needed for inhibition of the NA effect. Antiserum directed against the pig brain G1 (inhibitory G-protein), recognizing both alpha 41 and beta 35,36 subunits, but not alpha 39, of pig brain, reacted with the 41 kDa band in platelets. Anti-G1 antiserum inhibited only the effect of NA. Reconstitution of the platelet membranes ADP-ribosylated by pertussis toxin with Go, not Gi, purified from pig brain restored the thrombin-stimulated release of AA. In contrast, reconstitution of those membranes with Gi, not Go, restored the NA-stimulated release of AA. These results indicate that different GTP-binding proteins, Gi- and Go-like proteins, may be involved in the mechanism of signal transduction from alpha 2-adrenergic receptors and thrombin receptors to phospholipase A2 in rabbit platelets.     

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.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and identification of two pertussis-toxin-sensitive GTP-binding proteins of bovine spleen

Two alpha subunits of GTP-binding proteins were purified from bovine spleen membranes. Both proteins were ADP-ribosylated by pertussis toxin in the presence of beta gamma subunits. The major protein had a molecular mass of 40 kDa and its immunological reactivity and fragmentation pattern by limited proteolysis were identical with those of the alpha subunit of Gi2. The minor protein had a molecular mass of 41 kDa and its partial amino acid sequences completely matched with those predicted from human and rat Gi3 alpha cDNAs.     

Antisera against the 3-17 sequence of rat G alpha i recognize only a 40 kDa G-protein in brain

To obtain antisera specific for the GTP-binding protein Gi alpha we immunized rabbits against a synthetic peptide derived from the N-terminal (3-17) sequence predicted from the rat Gi alpha cDNA clone published by Itoh et al. (1986) (Proc. Natl. Acad. Sci. USA 83, 3776-3780). Western-blot analysis of bovine brain G-proteins purified and resolved by hydrophobic chromatography and of rat striatal membranes, indicate that this antiserum does not recognize 41 kDa alpha i or 39 kDa alpha o. However, it reacts with a 40 kDa alpha-subunit. The data suggest that the sequence deduced from the rat G alpha i cDNA corresponds to a G40 alpha protein and that N-terminus directed antisera are useful tools to discriminate between two different G alpha i-like types of G-proteins present in mammalian brain.     

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.     

Differential tissue expression and developmental regulation of guanine nucleotide binding regulatory proteins and their messenger RNAs in rat heart

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.     

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.     

Characterization of partially purified prostaglandin E2 receptor from the canine renal medulla: evidence for physical association of the receptor protein with the inhibitory guanine nucleotide-binding protein.

Prostaglandin (PG) E2 binding protein, a putative PGE2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE2, but lost most of the sensitivity to guanosine-5'-(gamma-thio)triphosphate (GTP gamma S). An antibody against alpha subunit of the inhibitory guanine nucleotide-binding protein (alpha Gi)1 and alpha Gi2 or that against common sequences of alpha subunit of guanine nucleotide-binding proteins (alpha G(common)) reacted at 41 kDa protein in the sample of each step of purification, but failed to do so in the final preparation. An antibody against alpha Gi3 or alpha Go had no effect. In fact, peaks of the binding activity and immunoreactivity for alpha Gi1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against alpha G(common) or alpha Gi1,2, but not that against alpha Gi3 and alpha Go, precipitated PGE2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE2 receptor is an acidic glycoprotein and that Gi1 or Gi2 is physically associated with the PGE2 receptor and dissociates from the receptor protein during purification procedures.     

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.