Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Purification and characterization of a new GTP-binding protein of Mr 24,000 in bovine brain membranes

A GTP-binding protein with an Mr of 24,000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported alpha beta gamma-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of alpha beta gamma-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg(2+)-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg(2+)-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do alpha beta gamma-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized alpha beta gamma-trimeric G proteins.     

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

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

Baculovirus expression of mammalian G protein alpha subunits.

Complementary DNAs encoding three subtypes of the alpha subunit (alpha i-1, alpha o and alpha s) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high-level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different alpha chains, and co-migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive alpha chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of alpha i-1 and alpha o were myristoylated; in contrast, alpha s did not incorporate myristate. The soluble fractions from cells expressing alpha chains showed high levels of GTP-binding activity over that observed in uninfected cells, or in cells infected with wild-type virus. The peak expression levels observed at 72 h post-infection were highest for alpha o at ca. 400 pmol of GTP-gamma-35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high-level production of mammalian G protein alpha chains which retain GTP-binding activity and are appropriately modified by myristoylation.     

Isolation of GTP-binding proteins from myeloid HL-60 cells. Identification of two pertussis toxin substrates

We have isolated the major GTP-binding proteins from myeloid HL-60 cell plasma membranes. Two pertussis toxin substrates with similar apparent molecular masses of 40 and 41 kDa, respectively, are contained in these preparations, with both proteins being ADP-ribosylated to a similar extent. Partial chymotryptic proteolysis of fractions containing the [32P]ADP-ribosylated 40-kDa GTP-binding protein alpha subunit demonstrated production of 32P-labeled peptides of 28 and 16 kDa which were not observed after partial proteolysis of fractions containing solely the 41-kDa protein. Similarly, mild acid hydrolysis produced an additional 28-kDa fragment only from fractions containing the 40-kDa protein. The results presented here indicate the presence of two distinct pertussis toxin substrates in myeloid cells. The 41-kDa pertussis toxin substrate is likely to represent the alpha subunit of the inhibitory GTP-binding regulatory protein of adenylate cyclase, whereas the 40-kDa substrate may represent the alpha subunit of the GTP-binding protein which is coupled to chemoattractant receptors. In addition to the pertussis toxin substrates, an additional major peak of guanosine 5'-(3-O-thio)triphosphate-binding activity closely corresponded to the appearance of a 23-kDa protein.     

The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtAC) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtAC, we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtAC is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtAC gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtAC is necessary for DNA replication and progression through the cell cycle.     

Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus.

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39- and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofold-higher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.     

Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein (or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.     

Expression of polyomavirus large T antigen by using a baculovirus vector.

A gene encoding the large T antigen of polyomavirus was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus so that gene expression was under the control of the strong, very late polyhedrin gene promoter. Significantly more large T antigen was produced in recombinant virus-infected insect cells than was observed in polyomavirus-transformed mouse cells. The insect-derived T antigen exhibited polyomavirus origin-specific DNA binding. The baculovirus expression system provides a convenient source of T antigen for in vitro studies.     

Cellular proteins that associate with the middle and small T antigens of polyomavirus.

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.     

Rhes: a GTP-binding protein integral to striatal physiology and pathology

Rhes, the Ras Homolog Enriched in Striatum, is a GTP-binding protein whose gene was discovered during a screen for mRNAs preferentially expressed in rodent striatum. This 266 amino acid protein is intermediate in size between small Ras-like GTP-binding proteins and α-subunits of heterotrimeric G proteins. It is most closely related to another Ras-like GTP-binding protein termed Dexras1 or AGS1. Although subsequent studies have shown that the rhes gene is expressed in other brain areas in addition to striatum, the striatal expression level is relatively high, and Rhes protein is likely to play a vital role in striatal physiology and pathology. Indeed, it has recently been shown to interact with the Huntingtin protein and play a pivotal role in the selective vulnerability of striatum in Huntington's disease (HD). Not surprisingly, Rhes can interact with multiple proteins to affect striatal physiology at multiple levels. Functional studies have indicated that Rhes plays a role in signaling by striatal G protein-coupled receptors (GPCR), although the details of the mechanism remain to be determined. Rhes has been shown to bind to both α- and β-subunits of heterotrimeric G proteins and to affect signaling by both Gi/o- and Gs/olf-coupled receptors. In this context, Rhes can be classified as a member of the family of accessory proteins to GPCR signaling. With documented effects in dopamine- and opioid-mediated behaviors, an interaction with thyroid hormone systems and a role in HD pathology, Rhes is emerging as an important protein in striatal physiology and pathology.     

Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.     

Octyl-beta-D-glucopyranoside extracts polyomavirus receptor moieties from the surfaces of mouse kidney cells.

Polyomavirus receptor moieties were extracted from the surfaces of mouse kidney cells with the nonionic detergent octyl-beta-D-glucopyranoside. Following extraction with this detergent, mouse kidney cells were refractory to polyomavirus infection. Binding studies demonstrated that this loss of susceptibility resulted from extraction of a peripheral membrane protein or proteins required for proper virus attachment to and infection of mouse kidney cells. Infection of extracted mouse kidney cells returned following a 2-h recovery period. However, the presence of cycloheximide or tunicamycin in the recovery media interfered with recovery from infection. Cells could be infected immediately after extraction by supplying them with the extracted moieties prior to or concomitant with infection. A complex of polyomavirus and the extracted receptor protein was formed by in vitro incubation and was stable in sucrose gradient analysis. Functional receptor moieties were prepared in the form of liposomes from the detergent extract. The virus-receptor complex was immunoprecipitated with anti-polyomavirus immunoglobulin G, and the portion of the complex contributed by the cell was identified. Immunoblot analysis of the mouse kidney cell detergent extract with a receptor-specific 125I-labeled anti-idiotypic antibody or 125I-labeled polyomavirus demonstrated several reactive proteins. Attachment of polyomavirus to mouse kidney cells, followed by extraction of the virus-receptor complex, identified polyomavirus-binding proteins similar to those observed in in vitro binding. Proteins with molecular weights of approximately 95,000, 50,000 and 25,000 to 30,000 were consistently observed in all receptor assays. The relationship between these proteins and their possible involvement as the cell receptor for polyomavirus are discussed.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.     

Superinfection rescue of an integrated defective polyomavirus genome.

We have characterized the viral sequences integrated in a polyomavirus-transformed mouse cell line, Py-3T3 (clone Py-6), and followed their excision and packaging upon superinfection. The polyomavirus sequences contained in Py-6 cells are present as a single insert of nonidentical tandem copies which includes, in addition to a normal middle T-antigen-coding region, some very rearranged sequences. Infection of Py-6 cells with polyomavirus strains encoding a normal large T antigen leads to the reproducible recovery in the resulting viral stock of specific defective viral genomes. The defective genomes contain a wild-type coding region for middle and small T antigens and intact viral origin and enhancer sequences. The remainder of the viral genome is rearranged or lost, so that there is no capacity to code for large T antigen or viral capsid proteins. The recovered defective sequences are also found integrated in Py-6 genomic DNA. Presumably, in infections of Py-6 cells, large T antigen, provided by the superinfecting virus, amplifies and excises the integrated viral sequences. The superinfecting helper virus must also produce viral capsids for packaging of the defective viral DNA and thus provides a means to shuttle the defective sequences from the mouse cells into other hosts, such as rat cells. In the latter host, the defective sequences are able to induce transformation.     

Trimeric G proteins and vesicle formation

Among the proteins regulating vesicular traffic, the small, Ras-like GTPases have received particular attention. Several recent reports indicate that another class of GTP-binding (G) protein, the heterotrimeric G proteins, also participates in the regulation of vesicular traffic. Thus, studies using transfected cells and cell-free systems show that a pertussis toxin-sensitive trimeric G protein, G(i3), is involved in the formation of secretory vesicles from the Golgi complex. These results raise the intriguing possibility that signal transduction processes across intracellular membranes play a role in vesicle formation, and provide important clues about the molecular machinery involved in this process.     

Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.     

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.     

Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex

ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.