Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.     

G protein-coupled receptor kinase 4gamma interacts with inactive Galpha(s) and Galpha13

G protein-coupled receptors (GPCRs) are regulated by multiple families of kinases including GPCR kinases (GRKs). GRK4 is constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to hypertension. We examined, through co-immunoprecipitation, the interactions between GRK4γ and the Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gα_s-coupled GPCR signaling and lacks a PH domain, we hypothesized that GRK4γ would interact with active Gα_s, but not Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gα_s and Gβ to a greater extent than active Gα_s. GRK4γ also interacts with inactive Gα₁₃ and Gβ. Functional studies demonstrate that wild-type GRK4γ, but not kinase-dead GRK4γ, ablates isoproterenol-mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that binding to inactive Gα_s and Gβ may explain the constitutive activity of GRK4γ towards Gα_s-coupled receptors.     

Poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Specific antibodies to poly(ADP-ribose) were obtained and characterized. Using these antibodies, the tissue specificity of poly(ADP-ribose) modified nuclear proteins from rat thymocytes and hepatocytes was studied. The differences in the levels of poly(ADP-ribosylation) of nuclear proteins from both tissues were found to be quantitative rather than qualitative. Analysis of intranuclear distribution of poly(ADP-ribose) acceptor proteins revealed that the bulk of them is localized in the nuclear sap and matrix. A comparison of spectral properties of poly(ADP-ribosylated) proteins, using specific antibodies and label incorporation from [14C]NAD showed the existence of two protein groups. Some of those were modified in a great degree but exchange poly(ADP-ribose) at a slow rate, whereas others (e.g., histones and HMG proteins) modified in a small degree exchanged poly(ADP-ribose) at a much higher rate. The results obtained by different methods are discussed.     

Peroxiredoxin-4 interacts with and regulates the thromboxane A(2) receptor

We identified peroxiredoxin-4 (Prx-4) as a protein interacting with the beta isoform of the thromboxane A(2) receptor (TPbeta) by yeast two-hybrid analysis. Prx-4 co-immunoprecipitated constitutively with TPbeta in HEK293 cells. The second and third intracellular loops as well as the C-terminus of TPbeta interacted directly with Prx-4. Co-expression of Prx-4 caused a 60% decrease in cell surface expression of TPbeta. Prx-4 and TPbeta predominantly co-localized in the endoplasmic reticulum. Co-expression of Prx-4 in cells treated with H(2)O(2) targeted TPbeta for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx-4, an anti-oxidative enzyme.     

GTP-binding proteins in etiolated epicotyls of Pisum sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.     

Ethylene rapidly up-regulates the activities of both monomeric GTP-binding proteins and protein kinase(s) in epicotyls of pea

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants.     

Comparison of the early chromatin reaction of the mouse thymocytes incubated with phytohemagglutinin and Concanavalin A

Summary The proventriculus, gizzard and pyloric antrum (region between the gizzard and the duodenum) of 18-day Black Australorp chick embryos and of chicks within 30 h of hatching have been studied by electron microscopy. D and EC cells, and putative G, D₁ and A-like cells were identified (terminology of Solcia et al., 1973) but no ECL cells. No endocrine cells of any kind were revealed in the gizzard.Supported by grants from the Senyte Research Comittee of the University of the Witwaterstrand, Johannesburg     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

Paip1 interacts with poly(A) binding protein through two independent binding motifs

The 3' poly(A) tail of eukaryotic mRNAs plays an important role in the regulation of translation. The poly(A) binding protein (PABP) interacts with eukaryotic initiation factor 4G (eIF4G), a component of the eIF4F complex, which binds to the 5' cap structure. The PABP-eIF4G interaction brings about the circularization of the mRNA by joining its 5' and 3' termini, thereby stimulating mRNA translation. The activity of PABP is regulated by two interacting proteins, Paip1 and Paip2. To study the mechanism of the Paip1-PABP interaction, far-Western, glutathione S-transferase pull-down, and surface plasmon resonance experiments were performed. Paip1 contains two binding sites for PABP, PAM1 and PAM2 (for PABP-interacting motifs 1 and 2). PAM2 consists of a 15-amino-acid stretch residing in the N terminus, and PAM1 encompasses a larger C-terminal acidic-amino-acid-rich region. PABP also contains two Paip1 binding sites, one located in RNA recognition motifs 1 and 2 and the other located in the C-terminal domain. Paip1 binds to PABP with a 1:1 stoichiometry and an apparent K(d) of 1.9 nM.     

The cytosolic HinT protein of Mycoplasma hominis interacts with two membrane proteins

Histidine triad nucleotide-binding (HinT) proteins are dimeric proteins that bind to purines and are found in all three kingdoms: the eukarya, bacteria and archaea. In eukaryotes, HinT proteins have been detected intracellularly, but their function is unknown. Until now, knowledge about HinT proteins in prokaryotes was restricted to sequence similarities and nucleotide-binding studies. In this study, we provide evidence that, in the cell wall-less prokaryote, Mycoplasma hominis, the gene encoding the HinT protein forms an operon with two other genes. These genes encode the species-specific membrane proteins, P60 and P80, which are associated within the mycoplasma membrane. The finding that HinT interacts with this complex by binding to P80 provides novel insight into the organization of bacterial HinT proteins.     

The Streptomyces coelicolor A3(2) bldB region contains at least two genes involved in morphological development

Streptomyces coelicolor A3(2) bldB mutants are blocked in the formation of aerial hyphae. A phage library of wild-type S. coelicolor DNA was used to isolate recombinant phages which restore wild-type morphological development to several bldB mutants. Of several mutations, one, bld-28, previously mapped at bldB was not complemented by the cloned region, indicating that the bldB locus is composed of at least two distinct genes. Partial localization of bldB-complementing activity showed that a 1.5 kb fragment is sufficient for complementation of the bld-15 mutation whereas bld-17 requires the same region as well as additional sequences. Under stringent conditions, genomic DNA hybridizing to the cloned sequences was absent from other Streptomyces species, including the closely related Streptomyces lividans 66. DNA sequences causing marked plasmid structural instability in S. coelicolor, but not in S. lividans, are also located in this region.     

A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA-DRB-genes

Electrophoretic mobility shift assays reveal that HeLa neuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)_n(ga)_m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA bindinlg capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into head sensitive, water soluble and temporary stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase 1 footpoint analyses yield four different protein binding regions only on the (gt)_n(ga)_m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5′ of the (gt)_n part. Hence at lealst two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)_n(ga)_m-containing strand intron 2 in HLA-DRB genes     

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)     

Histatins, a family of salivary histidine-rich proteins, are encoded by at least two loci (HIS1 and HIS2)

We screened a human parotid gland cDNA library with mixed synthetic oligonucleotide probes representing a central coding region common to histatins 1 and 3. Sequence analysis of 12 histatin cDNA clones strongly suggests that the histatin protein family is encoded by at least two closely related loci (HIS1 and HIS2) such that histatins 1 and 3 are primary products of HIS1(1) and HIS2(1) alleles, respectively, and that histatins 4-6 are derived from histatin 3 by proteolysis. We present additional data indicating that histatin 2 may represent the non-phosphorylated form of histatin 1.