Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.     

Chronic Membrane Depolarization Regulates the Level of the Guanine Nucleotide Binding Protein Goα in Cultured Neuronal Cells

Chronic membrane depolarization results in an increase in muscarinic acetylcholine receptor (mAChR) number in N1E-115 neuroblastoma cells. Because the mAChR interacts with the guanine nucleotide binding regulatory (G) proteins, Gi and Go, the effect of chronic membrane depolarization on the levels of subunits of these G proteins was examined. Quantitation of G protein subunit levels was performed using affinity-purified, monospecific antibodies in a quantitative immunoblot assay. Incubation with 50 microM veratridine (VTN), an activator of voltage-sensitive Na+ channels, induced a 48 +/- 15% increase in the level of the alpha subunit of Go. The effect of VTN was blocked by tetrodotoxin. On removal of VTN, the level of Go alpha decreased to control levels within 24 h. The levels of the alpha subunit of Gi and the common beta subunit were not affected by VTN treatment. These results show that in N1E-115 cells, the level of the alpha subunit of Go is regulated in a manner similar to the level of mAChR in response to chronic membrane depolarization.     

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.     

Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane.

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5'-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Treatment of intact striatal neurones with cholera toxin or 8-bromoadenosine 3',5'-(cyclic)phosphate decreases the ability of pertussis toxin to ADP-ribosylate the alpha-subunits of inhibitory and other guanine-nucleotide-binding regulatory proteins, Gi and Go. Evidence for two distinct mechanisms.

Using primary cultures of striatal neurones from the mouse embryo, we showed that treatment of intact cells with cholera toxin (5 micrograms/ml, 22 h) decreases the subsequent ADP-ribosylation of the alpha subunit of the guanine-nucleotide-binding regulatory protein Go (Go alpha) and the alpha subunit of the inhibitory guanine-nucleotide-binding regulatory protein (Gi alpha) of adenylate cyclase, which is catalyzed in vitro on neuronal membranes by pertussis toxin. The inhibitory effect of cholera toxin could not only be attributed to an increased production of cAMP in neurones. Treatment of cells with 0.1 microM 8-bromoadenosine 3',5'-(cyclic)phosphate (BrcAMP) for 16 h, or with 0.1 mM BrcAMP for 5 min, mimicked the effect of cholera toxin on the ADP-ribosylation of Go alpha and Gi alpha in vitro. However, the two agents seem to act through distinct mechanisms. The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine prevented the action of Br8cAMP but not that of cholera toxin. In addition, measurements of the pI of the Go alpha deduced from immunoblots of two-dimensional gels performed using a specific antibody directed against Go alpha suggest that treatment of neurones with cholera toxin induces ADP-ribosylation of Go alpha in intact cells, while BrcAMP does not.     

Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP

The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp)-GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta-phosphorus atom of GTP. These results lead to the following conclusions: The pro-R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.     

The adipocyte Go alpha-immunoreactive polypeptide is different from the alpha subunit of the brain Go protein.

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.     

Ultrastructural localization of the GTP-binding protein Go in neurons

The ultrastructural localization of Go, a GTP-binding protein (G protein) highly expressed in nervous tissues, was performed in cultured fetal and adult murine neurons, using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha). These antibodies recognized denatured Go alpha and both the native Go alpha-subunit and the Go alpha beta gamma heterotrimer. At the ultrastructural level, the positive immunoreactivity detected in cultured cells as well as in thin frozen sections, showed that Go was largely distributed in cell bodies and neuritic cytoplasm. Labelling was principally noted on the cytoplasmic face of the plasma membrane lining the cell body and the neurites, especially in 'cell-cell' contacts, but also in the cytoplasmic matrix, between endoplasmic reticulum and Golgi cisternae. No immunoreactivity was observed on the inner face of the pre- or postsynaptic membranes in both adult brain and in cultured neurons. This last finding strongly suggests that the Go protein is not involved in transducing chemical signals at the level of synapses, but more probably modulates the synaptic functions by controlling the activity of effectors localized outside of the synaptic densities.     

Go, a GTP-Binding Protein: Immunochemical and Immunohistochemical Localization in the Rat

The tissue and cellular distribution of a GTP-binding protein, Go, was investigated in the rat by immunochemical and immunohistochemical methods. Because the specific antibody for the alpha subunit of bovine Go (Go alpha) cross-reacted with rat Go alpha, an enzyme immunoassay method developed for bovine Go alpha was applied for measuring the tissue concentration of Go alpha in the rat. Go alpha was detected in all tissues examined except blood cells. The concentration of Go alpha was highest in the CNS (approximately 7.7 and 4.4 nmol/g in the cerebrum and cerebellum, respectively), followed by the pituitary gland and sciatic nerve. Among the other peripheral tissues, relatively high concentrations of Go alpha were observed in the urinary bladder, stomach, and intestines; however, these values were less than 2% of the concentration in the cerebrum. Go alpha in the intestine was located mostly in the muscle layer. Immunohistochemical study showed that Go alpha was associated mostly with the neural elements but not with cells particular to each peripheral organ. Go alpha was also present in the membranes of neuroendocrine cells, including glandular cells in the anterior lobe of the pituitary gland, chromaffin cells in the medulla of the adrenal gland, islets cells in the pancreas, and parafollicular cells in the thyroid. These results indicate that Go is localized exclusively in the nervous tissues and neuroendocrine cells.     

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.     

Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain

Choleragen (cholera toxin) activates adenylate cyclase by catalyzing ADP-ribosylation of Gs alpha, the stimulatory guanine nucleotide-binding protein. It was recently found (Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142) that a bovine brain membrane protein known as ADP-ribosylation factor or ARF, which enhances ADP-ribosylation of Gs alpha, also increases the GTP-dependent NAD:arginine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase, and auto-ADP-ribosylation activities of choleragen. We report here the purification and characterization of two soluble proteins from bovine brain that similarly enhance the Gs alpha-dependent and independent ADP-ribose transfer reactions catalyzed by toxin. Like membrane ARF, both soluble factors are 19-kDA proteins dependent on GTP or GTP analogues for activity. Maximal ARF effects were observed at a molar ratio of less than 2:1, ARF/toxin A subunit. Dimyristoyl phosphatidylcholine was necessary for optimal ADP-ribosylation of Gs alpha but inhibited auto-ADP-ribosylation of the choleragen A1 subunit and NAD:agmatine ADP-ribosyltransferase activity. It appears that the soluble factors directly activate choleragen in a GTP-dependent fashion. The relationships of the ARF proteins to the ras oncogene products and to the family of guanine nucleotide-binding regulatory proteins that includes Gs alpha remains to be determined.     

Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma

Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P. O., Devine, C.S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S.A. 87, 1506-1510). Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose. Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus. Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid. The second form of the protein was not myristoylated. Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits. The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions. Thus, myristoylation increases the affinity of alpha subunits for beta gamma. We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.     

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.     

Expression of Go alpha mRNA and protein in bovine tissues

Go alpha is a 39-kDa guanine nucleotide-binding protein (G protein) similar in structure and function to Gs alpha and Gi alpha of the adenylate cyclase complex and to transducin (Gt alpha) of the retinal photon receptor system. Although expression of Go alpha protein has been reported to be tissue-specific, other workers have found Go alpha mRNA in all rat tissues examined. In order to clarify this contradiction, studies to verify the distribution of Go alpha mRNA and protein in bovine and rat tissues were performed. Tissues were screened for the presence of Go alpha mRNA by use of a series of restriction fragments of a bovine retinal cDNA clone, lambda GO9, and oligonucleotide probes complementary to sequences specific among G alpha subunits for the 5' untranslated and coding regions of Go alpha. These probes hybridized predominantly with mRNA of 4.0 and 3.0 kb in bovine brain and retina. A 2.0-kb mRNA in retina also hybridized strongly with the cDNA but weakly with the oligonucleotide probes. In bovine lung, two mRNAs of 1.6 and 1.8 kb hybridized with the cDNA while only the 1.6-kb species hybridized with the coding-region oligonucleotide. In bovine heart, only a 4.0-kb mRNA was detected and in amounts much less than those in the other tissues. A similar distribution of Go alpha mRNAs was seen in rat tissues. In bovine tissues, Go alpha protein was identified with rabbit polyclonal antibodies directed against purified bovine brain Go alpha. An immunoreactive 39-kDa membrane protein was found principally in retina and brain, and in a lesser amount in heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.     

Turnover of the catalytic subunit of Na,K-ATPase in HTC cells

A polyclonal antibody to the catalytic subunit of rat kidney Na,K-ATPase has been raised in rabbits and used to analyze the turnover of the subunit in the rat hepatoma cell line HTC. It had been shown previously (Baumann, H., and Doyle, D. (1978) J. Biol. Chem. 253, 4408-4418) that the membrane proteins of these cells displayed multicomponent turnover kinetics, the minority of the surface proteins turning over with a half-time of about 20 h and the remainder with a half-time of about 100 h. That the antibody precipitated both the alpha (catalytic) and beta (glycosylated) subunits of the Na,K-ATPase from Triton extracts of HTC cells could be demonstrated following metabolic labeling of the cells with either [3H]leucine or a mixture of [3H] mannose and [3H]fucose, but following labeling with [35S]methionine radioactivity was found only in the alpha subunit of the precipitates. Incorporation of [35S]methionine into the alpha subunit could be detected 2 min after addition of the isotope to the cell suspension. Then and at all times thereafter the label was recoverable only from the particulate fraction of a 150,000 X g 60-min centrifugation; no labeled alpha subunit was ever detected in the supernatant fraction. By quantitative densitometry of radioautographs of sodium dodecyl sulfate-polyacrylamide gels of labeled antibody precipitates, it could be shown in pulse-chase experiments that the specific activity of the alpha subunit remained unchanged for 3-4 h (transit time) after the pulse was initiated and that the activity subsequently decayed exponentially with a half-time of 18 h. In a population growing with a generation time (tG) of 33 h, this decay corresponds to a turnover rate constant of 0.49/tG. The catalytic subunit is among those membrane proteins with a rapid turnover rate.     

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.     

Subunit interactions of the Go protein.

The monoclonal antibody, MONO, recognizes an epitope on the G protein alpha o-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTP gamma S, all beta gamma-subunits are released from the alpha o-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTP gamma S or GTP gamma S-induced subunit dissociation. However, we have been unable to induce a similar dissociation of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent Kd of alpha o-GDP for binding beta gamma of 340-390 nM has been reported [(1989) J. Biol. Chem. 264, 20688-20696] our results would suggest that binding of MONO to the alpha o-subunit induces an increased affinity of alpha o-GDP for beta gamma. Alternatively, these results could be explained if, under the conditions used, the Kd of alpha o-GDP for beta gamma were at least two orders of magnitude lower than estimated previously.