Nucleoside diphosphate kinase and GTP-binding proteins. Possible mechanisms of coupling

The results of numerous investigations during the last 20 years show that nucleoside diphosphate kinase (NDP kinase) is a multifunctional protein. In this paper, the current data are analyzed indicating that one of the possible mechanisms by which NDP kinase manifests its multifunctional role is its participation in the activation (or regulation) of heterotrimeric GTP-binding proteins (G proteins). We demonstrate that one of the NDP kinase isoforms dynamically interacts with the retinal rod G protein transducin (Gt) and phosphorylates its β-subunit at histidine residue (His 266). It is also shown that it leads to the consecutive transfer of the phosphate group to GDP in the active center of G protein α-subunit and G protein activation. The advantages of this mechanism are considered as compared to the classic G protein activation mechanism, GDP/GTP exchange.     

Identification of heterotrimeric G proteins in human sperm tail membranes

Heterotrimeric G proteins play important roles as signal transducing components in various mammalian sperm functions. We were interested in the distribution of G proteins in human sperm tails. Prior to membrane preparation, spermatozoa were separated from contaminating cells which are frequently present in human ejaculates. Enriched human sperm tail membranes were generated by using hypoosmotic swelling and homogenization procedures. Antisera against synthetic peptides were used to identify G proteins in immunoblots. AS 8, an antiserum directed against an amino acid sequence that is found in most G protein α-subunits, and A 86, which detects all known pertussis toxin-sensitive α-subunits, reacted specifically with a 40-kDa protein. Antisera against individual G protein α-subunits failed to detect any specific antigens in enriched tail membranes AS 36, recognizing the ã2-subunit of G proteins, identified a 35-kDa protein in sperm tail membranes. Antisera against the 36-kDa β₁-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-subunits were found in the cytosol. ADP ribosylation of spermatozoal membrane or cytosolic proteins revealed no pertussis toxin-sensitive α-subunits. However, membrane preparations of nonpurified human spermatozoa contained α₂ subunits, as shown immunologically and by ADP ribosylation; they most probably derived from somatic cells which are frequently present in human ejaculates. Our results stress the fact that spermatozoa need to be purified before sperm membrane preparation to avoid misinterpretations caused by contaminating cells. Furthermore, we suggest that G proteins in membranes of human sperm tails belong to a novel subtype of G protein α-subunits; the putative β-subunit was identified as a β₂-subunit. © 1995 Wiley-Liss, Inc.     

High energy phosphate transfer by NDPK B/Gβγcomplexes - an alternative signaling pathway involved in the regulation of basal cAMP production

The activation of heterotrimeric G proteins induced by G protein coupled receptors (GPCR) is generally believed to occur by a GDP/GTP exchange at the G protein α -subunit. Nevertheless, nucleoside diphosphate kinase (NDPK) and the β-subunit of G proteins (Gβ) participate in G protein activation by phosphate transfer reactions leading to the formation of GTP from GDP. Recent work elucidated the role of these reactions. Apparently, the NDPK isoform B (NDPK B) forms a complex with β; γ; dimers in which NDPK B acts as a histidine kinase phosphorylating G#x03B2; at His266. Out of this high energetic phosphoamidate bond the phosphate can be transferred specifically onto GDP. The formed GTP binds to the G protein α -subunit and thus activates the respective G protein. Evidence is presented, that this process occurs independent of the classical GPCR-induced GTP/GTP exchange und thus contributes, e.g. to the regulation of basal cAMP synthesis in cells.     

Suppression of the rice heterotrimeric G protein β-subunit gene, RGB1, causes dwarfism and browning of internodes and lamina joint regions

In the present study, we investigated the function of the heterotrimeric G protein β-subunit (Gβ) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gβγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.     

Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires beta-subunit

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel alpha(1C)-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel beta(1a)- or beta(2a)-subunit with the alpha(1C)-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in alpha(1C) + beta(1a)-expressing HEK-293 cells was rapid after a depolarization to +100 mV (tau = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5'-O-(2-thiodiphosphate) and guanosine 5'-O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A(1) receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel alpha(1C)-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary beta-subunit and that this facilitation is independent of G protein pathways.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.     

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.     

Changes in sodium pump transport activity and Na+, K+-ATPase expression level following lymphocytes activation in humans

Ouabain-inhibitable rubidium influxes, intracellular sodium content (Nai), and alpha 1-subunit abundance have been studied in human blood lymphocytes, stimulated by phytohemagglutinin (PHA) or by the phorbol 12,13-dibutyrate (PDBu), and calcium ionophore--ionomycin. It is shown that at early stages of PHA-induced activation, the Na/K pump expression (as determined by Wesrn blots of alpha 1 protein in membrane fractions of total cell lysates) does not change, and the increase in Rb influx is due to the increase in Nai and results from the enhanced transport activity of Na/K pumps present in plasma membrane. During the late stages of G0-->G1-->S transit (16-48 h), the increase in Rb influx occurs without changes in Nai, and monensin increases both Nai, and the Rb influx via the Na/K pump. To the end of the first day of mitogen activation, the alpha 1 protein content was found to increase by 5-7 times. A correlation was revealed between changes in ouabain-inhibitable Rb influxes, alpha 1 protein abundance, and the proliferation rate. It is concluded that blasttransformathion of normal human lymphocytes is accompanied by the increase in membrane-associated pool of alpha 1-subunit of Na+,K(+)-ATPase, and the enhanced activity of sodium pump during the G0-->G1-->S progression is provided by increased number of Na+,K(+)-ATPase pumps in plasma membrane.     

Production of antibodies to the alpha7-subunit of human acetylcholine nicotinic receptor with the use of immunoactive synthetic peptides

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the alpha7-subunit of human acetylcholine receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four alpha7-subunit regions containing 16-23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR alpha7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR alpha7-subunit and do not react with the analogous domain of the alpha1-subunit of the ray Torpedo californica AChR.     

Activation of the heterotrimeric G protein α-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 ( thf1 ), indicating that inactivation or activation of the endogenous G protein α-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Gα-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1 . Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2 . Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1 . This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.     

Glycogen debranching enzyme association with beta-subunit regulates AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) regulates both glycogen and lipid metabolism functioning as an intracellular energy sensor. In this study, we identified a 160-kDa protein in mouse skeletal muscle lysate by using a glutathione-S-transferase (GST)-AMPK fusion protein pull-down assay. Mass spectrometry and a Mascot search revealed this protein to be a glycogen debranching enzyme (GDE). The association between AMPK and GDE was observed not only in the overexpression system but also endogenously. Next, we showed the beta1-subunit of AMPK to be responsible for the association with GDE. Furthermore, experiments using deletion mutants of the beta1-subunit of AMPK revealed amino acids 68-123 of the beta1-subunit to be sufficient for GDE binding. W100G and K128Q, both beta1-subunit mutants, are reportedly incapable of binding to glycogen, but both bound GDE, indicating that the association between AMPK and GDE does not involve glycogen. Rather, the AMPK-GDE association is likely to be direct. Overexpression of amino acids 68-123 of the beta1-subunit inhibited the association between endogenous AMPK and GDE. Although GDE activity was unaffected, basal phosphorylation and kinase activity of AMPK, as well as phosphorylation of acetyl-CoA carboxylase, were significantly increased. Thus it is likely that the AMPK-GDE association is a novel mechanism regulating AMPK activity and the resultant fatty acid oxidation and glucose uptake.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Amino acid sequence of a region on the glycogen-binding subunit of protein phosphatase-1 phosphorylated by cyclic AMP-dependent protein kinase

The amino acid sequence of a region on the glycogen-binding (G)-subunit of protein phosphatase-1G that is phosphorylated by cyclic AMP-dependent protein kinase has been determined. The sequence is: (formula see text) This finding will facilitate studies of the effects of hormones on the phosphorylation state of the G-subunit in vivo.     

Identification of three in vivo phosphorylation sites on the glycogen-binding subunit of protein phosphatase 1 from rabbit skeletal muscle, and their response to adrenaline

The in vivo phosphorylation stoichiometries of 4 serines on the glycogen-binding (G)-subunit of protein phosphatase 1 (PP1) have been determined. In fed rabbits injected with propranolol stoichiometries (mol/mol) were: site 1 (0.67 +/- 0.09), site 2 (0.20 +/- 0.07), site 3a (0.23 +/- 0.01) and site 3b (0). After injection with adrenalin they became: site 1 (0.90 +/- 0.02), site 2 (0.72 +/- 0.01), site 3a (0.23 +/- 0.02) and site 3b (0). These results, together with other data, establish that site 2 phosphorylation by cyclic AMP-dependent protein kinase triggers dissociation of PP1 from the G-subunit in vivo. They also demonstrate that a residue phosphorylated in vitro by glycogen synthase kinase 3 (site 3a) is phosphorylated in vivo.     

Exogenous methionine depresses level of mRNA for a soybean storage protein

$\text{In vitro}$ translation experiments indicate that absence of the β-subunit of 7S storage protein in soybean ($\text{Glycine max}$ L. Merr. cv. Provar) cotyledons cultured on methionine-supplemented medium is due to lack of functional mRNA for that polypeptide. Relative amounts of functional mRNA for the 7S α′- and α-subunits were unaffected by methionine in the cotyledon culture medium. Measurements of β-subunit accumulation in cotyledons transferred from basal medium to methionine-supplemented medium show that methionine inhibits continued accumulation of the β-subunit after synthesis of the β-subunit has begun, and that methionine does not promote degradation of existing β-subunit.     

Production of antibodies to the α7-subunit of human acetylcholine receptor with the use of immunoactive synthetic peptides

Potential B epitopes and T-helper epitopes in the N-terminal extracellular domain of the α7-subunit of human acetylchloline receptor (AChR) were theoretically calculated in order to reveal peptides that can induce the formation of specific antibodies to this domain. Four peptides structurally corresponding to four α7-subunit regions containing 16–23 aa and three of their truncated analogues were synthesized. Rabbits were immunized with both free peptides and protein conjugates of their truncated analogues, and a panel of antibodies to various exposed regions of the N-terminal extracellular domain of the AChR α7-subunit was obtained. All of the four predicted peptides were shown to induce the production of antipeptide antibodies in free form, without conjugation with any protein carrier. The free peptides and the protein conjugates of truncated analogues induced the formation of almost equal levels of antibodies. Most of the obtained antisera contained antibodies that bind to the recombinant extracellular N-terminal domain of the rat AChR α7-subunit and do not react with the analogous domain of the α1-subunit of the ray Torpedo californica AChR.     

Constitutive Gαi Coupling Activity of Very Large G Protein-coupled Receptor 1 (VLGR1) and Its Regulation by PDZD7 Protein

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gα_i coupling. Co-expression of the Gα_iq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gα_i protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gα_i coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gα_i signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome.     

The Characterization of the α-Subunits of 7S Nerve Growth Factor

Abstract: The molecular weight of the α-subunit from 7S Nerve Growth Factor was found to be 26,500 by sedimentation equilibrium and by electrophoretic analysis under denaturing conditions of the native and dimethylsuberimidate cross-linked α-subunit. The α-subunit, and each of the individual α-subunits in this preparation, contained one heavy peptide chain of molecular weight 17,200 and one light peptide chain of molecular weight 9500, held together by disulfide bridges. It is likely that the charge differences between the individual α-subunits arise from posttranslational modifications of a parent α-subunit.     

Synaptic localization and presynaptic function of calcium channel beta 4-subunits in cultured hippocampal neurons

Neurotransmitter release is triggered by the influx of Ca(2+) into the presynaptic terminal through voltage gated Ca(2+)-channels. The shape of the presynaptic Ca(2+) signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca(2+)-channel function is modulated in particular by the auxiliary beta-subunits that interact intracellularly with the pore-forming alpha(1)-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-beta(4) constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that beta(4)-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the beta(4)-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the beta(4)-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-beta(4) revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-beta(4) mutant, GFP-beta(4)(Delta50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca(2+)-channel beta(4)-subunit in synaptic transmission.