Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

Expression of active, secreted human prostate-specific antigen by recombinant baculovirus-infected insect cells on a pilot-scale

We have expressed human prostate-specific antigen (PSA) on a pilot-scale in Spodoptera frugiperda Sf9 insect cells using recombinant baculovirus system. Infected cells secreted PSA into culture medium at a concentration of 2-4 mg per liter. PSA was expressed both in active and inactive forms which were separated in a final purification step using cation-exchange chromatography eluted with a low salt gradient. The N-terminus of active PSA was correctly cleaved; two amino acids of the propeptide remained, however, at the N-terminus of the inactive PSA. Purified recombinant PSA showed a chymotrypsin-like activity with the synthetic substrate MeO-Suc-Arg-Pro-Tyr-pNA, but did not have a trypsin-like activity when Pro-Phe-Arg-pNA was used. The molecular mass of active PSA was 31.0 kDa in reduced SDS-PAGE, 26.0 kDa in nonreduced SDS-PAGE and 26.5 kDa in ion spray mass spectrometry. The active protein formed complexes with alpha 1-antichymotrypsin (ACT) and alpha 2-macroglobulin (alpha 2M) in vitro similar to the commercial PSA purified from human seminal fluid.     

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.     

Identification of a structural element in phospholipase C beta2 that interacts with G protein betagamma subunits

To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.     

Differential coupling of rat D2 dopamine receptor isoforms expressed in Spodoptera frugiperda insect cells

By using a baculovirus expression system, the two isoforms of the rat D2 dopamine receptor were expressed at densities ranging up to 15 pmol/mg of protein. D2L and D2S dopamine receptors expressed in aline of Spodoptera frugiperda (Sf9) insect cells Sf9cells, displayed high affinity for the antagonists spiroperidol and (+)-butaclamol and the agonist N-propylnorapomorphine. Antisera raised against the D2 receptor immunoprecipitated binding sites for a radiolabeled D2 antagonist from solubilized extracts of infected Sf9cells. In immunoblots of Sf9cells infected with recombinant D2 baculovirus, these antisera recognized a major species of protein of approximately 46 kDa. Photoaffinity-labeling of infected Sf9cells using N-(p-azido-m-[125I]iodophenethyl)spiperone also identified a protein of this size, suggesting that D2 receptors expressed in Sf9cells are largely unglycosylated. In cells expressing receptors at a density greater than 1 pmol/mg, GTP-sensitive, high-affinity binding of agonists was not detected in studies of the inhibition of the binding of a radiolabeled D2 antagonist. When expression levels were under 1 pmol/mg, the binding of agonists was sensitive to the addition of guanine nucleotides, indicating that D2 receptors were coupled to endogenous G proteins. Endogenous G proteins enable both isoforms of D2 receptors to couple to the inhibition of adenylyl cyclase activity. The high-affinity state of the D2 receptor was directly measured using a radiolabeled agonist. Although the density of receptors increased with longer times after infection, the density of high-affinity sites reached a maximum of approximately 40 fmol/mg 30 to 36 hr after infection. Coexpression of D2 receptors and G protein subunits in Sf9cells dramatically increased the density of high-affinity sites, whereas the total density of receptors was unchanged, confirming that D2 receptors in Sf9 cells can exist in the high-affinity-coupled state, but that appropriate G proteins are expressed at relatively low levels. The density of D2S receptors converted to a coupled, agonist-preferring state when coexpressed with G proteins subunits (alpha i1, beta 1 and gamma 2) was 5 times greater than that of D2L receptors expressed under the same conditions, consistent with the hypothesis that D2 dopamine receptor isoforms differentially couple to alpha i1.     

Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits

In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.     

Interactions between the human RNA polymerase II subunits

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.     

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

A chromatographic analysis of capsid protein isolated from alfalfa mosaic virus: zinc binding and proteolysis cause distinct charge heterogeneity

The capsid protein (CP) of alfalfa mosaic virus (AIMV) is required for viral replication when susceptible plants are inoculated with purified viral genomic RNA. The discovery of AIMV CP in the zinc activated RNA-dependent RNA polymerase complex prompted our further investigation of AIMV virions and the potential involvement of AIMV CP in metal binding. AIMV CP, isolated from nucleoprotein components, fractionated into four distinct ionic species when purified by cation exchange fast protein liquid chromatography. The CP existed as zinc complexed homodimers, metal-free homodimers, and two forms of proteolyzed heterodimers, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography, amino-terminal sequencing, and atomic absorption spectroscopy. Although the relative amounts of proteolyzed heterodimers varied, the ratio of zinc complexed homodimers to metal-free homodimers (1:10) was constant between virus and protein isolations for the strains 425 and WISC14. Purified metal-free and zinc-complexed homodimers could be interconverted in vitro by incubation with zinc chloride or with the metal chelator, sodium diethyldithiocarbamate (NaDDC). The potential role of zinc in AIMV nucleoprotein structure and infectivity was investigated by treatment of the virions with NaDDC. Electron microscopy and sucrose density gradient studies failed to detect any gross structural changes for zinc depleted virus; however, a decrease in infectivity was observed with local lesion leaf assays, suggesting a functional role for zinc in viral replication.     

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').     

The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3

A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate. Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection. The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants. Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3. The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein. Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein. Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.     

Phosphorylation of the G protein gamma12 subunit regulates effector specificity

Although the G protein betagamma dimer is an important mediator in cell signaling, the mechanisms regulating its activity have not been widely investigated. The gamma12 subunit is a known substrate for protein kinase C, suggesting phosphorylation as a potential regulatory mechanism. Therefore, recombinant beta1 gamma12 dimers were overexpressed using the baculovirus/Sf9 insect cell system, purified, and phosphorylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the Gi1 alpha subunit to the A1 adenosine receptor and to activate type II adenylyl cyclase or phospholipase C-beta was examined. Phosphorylation of the beta1 gamma12 dimer increased its potency in the receptor coupling assay from 6.4 to 1 nM, changed the Kact for stimulation of type II adenylyl cyclase from 14 to 37 nM, and decreased its maximal efficacy by 50%. In contrast, phosphorylation of the dimer had no effect on its ability to activate phospholipase C-beta. The native beta1gamma10 dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphorylated by protein kinase C alpha. Creation of a phosphorylation site in the N terminus of the protein (Gly4 --> Lys) resulted in a beta1 gamma10G4K dimer which could be phosphorylated. The activities of this beta gamma dimer were similar to those of the phosphorylated beta1 gamma12 dimer. Thus, phosphorylation of the beta1 gamma12 dimer on the gamma subunit with protein kinase C alpha regulates its activity in an effector-specific fashion. Because the gamma12 subunit is widely expressed, phosphorylation may be an important mechanism for integration of the multiple signals generated by receptor activation.     

The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex?

Elongation factor 1 (EF-1) regulates the specific interaction of aminoacyl-tRNA with the ribosome during the elongation phase of protein biosynthesis. Although individual functions of its separate chains have been well defined, to date there is hardly information about the structure and function of the whole complex. We describe here the complete subunit structure of elongation factor 1, and discuss its change during development of Artemia. Elongation factor 1 consists of a pentameric complex, composed of four different subunits alpha, beta, gamma, and delta in a molar ratio of 2:1:1:1. Although one molecule of EF-1 alpha dissociates easily from the complex EF-1 alpha 2 beta gamma delta under the influence of aminoacyl-tRNA and GTP, the second molecule of EF-1 alpha was found to remain firmly attached. Thus, in eukaryotic protein synthesis, movement of transfer RNAs to the ribosome seems under the influence of two distinct molecules of EF-1 alpha, a result possibly related to the presumed consumption of two molecules of GTP by EF-Tu during the elongation step of prokaryotic protein synthesis.     

Alpha-melanocyte stimulating hormone-induced pigmentation is blocked by depletion of protein kinase C

Partial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a kinetoplastid protozoan, Trypanosoma cruzi, were amplified by means of polymerase chain reaction and their primary structures were analyzed. The deduced amino acid sequence was aligned with those of other eukaryotic and archaebacterial EF-2s, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) method. ML analyses of EF-2 phylogeny using six different stochastic models of amino acid substitutions consistently suggested that the phylogenetic position of T. cruzi is likely to be closer to higher eukaryotes than that inferred from the phylogeny of small subunit ribosomal RNA (SrRNA). These results are consistent with those for the elongation factor 1alpha (EF-1alpha) phylogeny. When the EF-1alpha and EF-2 phylogenies were totally evaluated, it became much clearer that the divergence of T. cruzi occurred later than that of a mitochondrion-lacking protozoan, Entamoeba histolytica, although this is not conclusive.