RESUMO
The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.
Assuntos
Citoesqueleto de Actina/imunologia , Forma Celular/imunologia , Complemento C5a/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Quimiotaxia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Complemento C5a/imunologia , Humanos , Ativação de Neutrófilo , Neutrófilos/patologia , Receptor da Anafilatoxina C5a/metabolismo , Receptores Purinérgicos P2X/metabolismo , Transdução de SinaisRESUMO
The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.
Assuntos
Adenilil Ciclases/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Adenilil Ciclases/química , Adenilil Ciclases/genética , Animais , Linhagem Celular , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/farmacologia , Proteínas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Immune complexes (IC) bound to the primate erythrocyte (E) complement receptor (CR1) are cleared from the circulation of primates and localized to phagocytic cells in the liver and spleen without E destruction. IC can be bound to E CRI either via C3b opsonization or with cross-linked mAb complexes (heteropolymers, HP) which contain a mAb specific for CRI and a mAb specific for an antigen. The long-term goal of our work is to apply the HP system to the treatment of human diseases associated with blood-borne pathogens. This review discusses the mechanism by which the E-bound IC are transferred to acceptor cells. Our studies in animal models as well as our in vitro investigations indicate that IC transfer is rapid (usually >90% in 10 min) and does not lead to lysis or phagocytosis of the E. Experiments with specific inhibitors and the use of IC prepared with Fab' fragments suggest that transfer depends mainly upon recognition by Fc receptors on the acceptor cell. Moreover, we find that IC release from the E is associated with a concerted loss of CR1, and is followed by uptake and internalization of the IC by the acceptor cell. We suggest that recognition and binding of the E-bound IC substrates by Fc receptors allows close contact between the E and acceptor cells, which in turn facilitates proteolysis of E CR1, presumably by a macrophage-associated protease. After proteolysis, the released IC are internalized by the macrophages.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Eritrócitos/imunologia , Fagócitos/imunologia , Receptores de Complemento 3b/sangue , Animais , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , Transporte Biológico Ativo , Plaquetas/imunologia , Complemento C3b/metabolismo , Humanos , Primatas , Receptores de Complemento 3b/metabolismo , Receptores Fc/metabolismoRESUMO
A protocol has been worked out for separating all thirteen different polypeptides in the beef heart cytochrome c oxidase complex from a single aliquot of enzyme. This involves an initial separation of polypeptides by gel filtration on a Biogel P-60 column in SDS, a step which purifies subunits CIV and CVIII and gives mixtures of CV + CVI, ASA, AED and STA, as well as CVII, CIX and IHQ. These mixtures are then resolved by reverse-phase high-performance liquid chromatography. The separation procedures have been applied to fetal heart cytochrome c oxidase of gestation between 100 and 200 days. No differences were found in the N-terminal sequences of any of the cytoplasmically made subunits or in the entire sequence of CIX between late fetal and adult forms of the enzyme.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Miocárdio/enzimologia , Animais , Bovinos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Coração/embriologiaRESUMO
High avidity anti-dsDNA IgG antibodies are believed to play an important role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) and therefore attempts have been made to reduce the concentration of these antibodies in the bloodstream of SLE patients. Previously we reported the development of an antigen based heteropolymer (AHP), a bispecific complex prepared by using the avidin-biotin system to crosslink dsDNA to a mAb specific for the human erythrocyte (E) complement receptor. Our studies indicated that this AHP could bind anti-dsDNA antibodies to E and facilitate clearance of these autoantibodies from the circulation of a monkey without E destruction. Here we report an improved covalent crosslinking procedure and purification scheme in which the AHP construct is isolated by precipitation in 50% saturated ammonium sulfate. We used a dsDNA binding dye, PicoGreen, to demonstrate specificity of binding of dsDNA to E via the AHP. The efficacy of the AHP in binding IgG anti-dsDNA antibodies to E was demonstrated in a sensitive and quantitative assay, based on the time resolved fluorescence properties of europium-labeled anti-human IgG mAbs used to probe the E. We also used this assay to screen SLE patient and normal plasmas for levels of anti-dsDNA IgG. The results of this assay correlate very well with the Farr assay, and therefore this approach may be useful in the development of informative and specific assays for a variety of autoantibodies. Treatment of SLE plasmas with E-AHP under conditions close to physiological led to substantial reductions (> or = 90%) in anti-dsDNA titers. It should be possible to test these new AHP for their ability to target and safely remove IgG anti-dsDNA antibodies from the circulation in animal models.
Assuntos
Anticorpos Antinucleares/análise , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , DNA/imunologia , Imunoglobulina G/análise , Lúpus Eritematoso Sistêmico/terapia , Receptores de Complemento 3b/imunologia , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/metabolismo , Anticorpos Biespecíficos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Relação Dose-Resposta Imunológica , Eritrócitos/metabolismo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Lúpus Eritematoso Sistêmico/imunologiaRESUMO
Beef heart mitochondrial complex III was separated into 12 polypeptide bands representing 11 different subunits by using the electrophoresis conditions described by Schägger et al. [(1986) Methods Enzymol. 126, 224-237]. Eight of the 12 polypeptide bands were identified from their NH2-terminal sequences as obtained by electroblotting directly from the NaDodSO4-polyacrylamide gel onto a solid support. The topology of the subunits in complex III was explored by three different approaches. (1) Protease digestion experiments of submitochrondrial particles in the presence and absence of detergent showed that subunits II and VI are on the M side of the inner membrane and subunits V and XI on the C side. (2) Labeling experiments with the membrane-intercalated probes [125I]TID and arylazidoPE indicated that cytochrome b is the predominant bilayer embedded subunit of complex III, while the non-heme iron protein appears to be peripherally located. (3) Cross-linking studies with carbodiimides and homobifunctional cleavable reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and reagents demonstrated that near-neighbor pairs include subunits I+II, II+VI, III+VI, IV+V, V+X, and VI+VII. The cytochrome c binding site was found to include subunits IV, VIII, and X. The combined data are used to provide an updated model for the topology of beef heart complex III.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons , Mitocôndrias Cardíacas/enzimologia , Sequência de Aminoácidos , Animais , Bovinos , Reagentes de Ligações Cruzadas , Complexo III da Cadeia de Transporte de Elétrons/isolamento & purificação , Substâncias Intercalantes , Ferro , Bicamadas Lipídicas , Modelos Químicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Partículas Submitocôndricas/enzimologiaRESUMO
The topology of several of the cytoplasmically made subunits of beef heart cytochrome c oxidase has been determined by protease digestion of oriented membrane preparations, using subunit-specific antibodies to identify cleavage products. Reconstituted vesicles of cytochrome c oxidase and asolectin were used as a vesicle preparation with the C domain of the enzyme available for protease digestion. Submitochondrial particles were used as vesicles with the M domain outermost. Trypsin and/or proteinase K cleaved polypeptides CIV, ASA, AED, STA, and IHQ. Cleavage of CIV, STA, and IHQ was from the M domains only and involved the removal of a fragment from the N-terminus in each case. Polypeptide AED was cleaved from the C side in the N-terminal part, while ASA was cleaved from both the C and M domains. Polypeptide fragments were electroblotted from polyacrylamide gels onto derivatized glass paper and sites of proteolytic cleavage determined by N-terminal sequence analysis.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Mitocôndrias Cardíacas/enzimologia , Miocárdio/enzimologia , Partículas Submitocôndricas/enzimologia , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo , Bovinos , Citoplasma/enzimologia , Cinética , Lipossomos , Substâncias Macromoleculares , Peso Molecular , Fragmentos de Peptídeos/análise , Peptídeo Hidrolases/metabolismo , Fosfatidilcolinas , Fosfolipídeos , Conformação ProteicaRESUMO
Opsonization of particulate pathogens by antibodies and complement can lead to their binding to the complement receptor (CR1), specific for C3b, on primate erythrocytes (E). This process of immune adherence may play a role in immunologic defense by immobilizing bacteria and viruses, thus preventing them from leaving the bloodstream to invade susceptible tissue and organs. Immune adherence of C3b-opsonized and immune complexed pathogens to E may also facilitate their transfer to, and destruction by, fixed tissue macrophages. We have used mAbs specific for CR1 crosslinked with pathogen specific mAbs to generate heteropolymers (HP) which can bind a wide range of substrates to primate erythrocytes. Both prototype and bonafide pathogens bound to primate E via HP are handled in the circulation of non-human primates in a manner which appears to be virtually identical to the mechanism by which C3b-opsonized substrates bound to E CR1 are cleared. In this process of focused phagocytosis, Fc receptors on the phagocytic cell engage the E-bound complex, CR1 is removed by proteolysis, and the entire immune complex and CR1 are internalized while sparing the E. It may be possible to use HP to target pathogens in the bloodstream in a wide range of therapeutic applications.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Eritrócitos/imunologia , Receptores de Complemento 3b/imunologia , Animais , Biopolímeros/imunologia , Humanos , Reação de Imunoaderência , Modelos ImunológicosRESUMO
Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.
Assuntos
Gramicidina/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , EstereoisomerismoRESUMO
Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.
Assuntos
Proteínas Fúngicas/metabolismo , Processamento de Proteína Pós-Traducional , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas Fúngicas/química , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Precursores de Proteínas/metabolismo , Deleção de SequênciaRESUMO
The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains. At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa polypeptides (Kane, P.M., Yamashiro, C.T., and Stevens, T.H. (1989a) J. Biol. Chem. 264, 19236-19244). We took a reverse genetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6, vma6 null mutants displayed growth characteristics typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+. Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparations contained no detectable V-ATPase activity. The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 39.8-kDa). We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is required for V-ATPase complex assembly. Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides. In yeast cells lacking the integral membrane portion of the V-ATPase complex, Vma6p was unable to stably associate with vacuolar membranes. Conversely, in mutants specifically lacking Vma6p, components of the V-ATPase integral membrane domain were destabilized, and peripheral subunits failed to assemble onto vacuolar membranes. These results are discussed in the context of a developing model for V-ATPase assembly in yeast.
Assuntos
Genes Fúngicos , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Vacúolos/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Southern Blotting , Western Blotting , Bovinos , Códon , DNA Fúngico , DNA Recombinante/metabolismo , Eletroforese em Gel de Poliacrilamida , Substâncias Macromoleculares , Modelos Estruturais , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , AMP Cíclico/metabolismo , Dimerização , Humanos , Isoenzimas/metabolismo , Dados de Sequência Molecular , Fosfolipase C beta , Fosforilação , Conformação Proteica , Proteína Quinase C/metabolismo , Relação Estrutura-Atividade , Fosfolipases Tipo C/metabolismoRESUMO
The vacuolar membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae is a multisubunit enzyme complex composed of an integral membrane V0 sector, and a peripherally associated V1 sector. Deletion of one of several structural genes for vacuolar H(+)-ATPase subunits was previously demonstrated to prevent proper assembly of the remaining V1 subunits onto the vacuolar membrane (Kane, P.M., Kuehn, M.C., Howald-Stevenson, I., and Stevens, T.H. (1992) J. Biol. Chem. 267, 447-454). A genetic screen was designed to identify new genes whose products were essential for the synthesis, assembly, and/or function of the yeast vacuolar H(+)-ATPase. Mutants were identified based on phenotypes associated with vacuolar membrane H(+)-ATPase loss of function (vma), including an inability to grow on media buffered at neutral pH. Representatives in five complementation groups were identified, including four novel mutant vma5, vma21, vma22, and vma23, all of which were defective in vacuolar ATPase enzyme activity. We report here the characterization of two genes, VMA4 and VMA5, that encode peripheral subunits of the vacuolar H(+)-ATPase. We determined that VMA5 encodes the 42-kDa subunit of the vacuolar H(+)-ATPase. The VMA4 gene, originally described by Foury (Foury, F. (1990) J. Biol. Chem. 265, 18554-18560), was determined to encode the 27-kDa subunit of the purified yeast vacuolar H(+)-ATPase. Characterization of the vma5 and vma4 mutants revealed that the 42- and 27-kDa subunits are essential for the assembly of the peripheral membrane portion of the H(+)-ATPase onto the vacuolar membrane.
Assuntos
Genes Fúngicos , Mutação , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Vacúolos/enzimologia , Sequência de Aminoácidos , Western Blotting , Clonagem Molecular , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Teste de Complementação Genética , Membranas Intracelulares/enzimologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Fenótipo , ATPases Translocadoras de Prótons/biossíntese , ATPases Translocadoras de Prótons/metabolismo , Mapeamento por RestriçãoRESUMO
We have studied the interactions of purified A1 adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the betagamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A1 adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with pure recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alphai2 and beta1gamma2 or beta1gamma3 and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta1gamma1. This difference is not overcome by increasing the concentration of betagamma subunits. Receptor-G protein complexes containing beta1gamma1 also bind less of the agonist, [125I]-iodoaminobenzyladenosine (125I-ABA), than do complexes containing beta1gamma3. Kinetic experiments show that 125I-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta1gamma1 than from complexes containing the other betagamma subunits. The affinity of the interaction between immobilized Galphai2 subunits and beta1gamma1 or beta1gamma2 measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A1 adenosine receptor and G proteins.
Assuntos
Adenosina/análogos & derivados , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Iodobenzenos/metabolismo , Lipossomos/metabolismo , Fenilisopropiladenosina/metabolismo , Receptores Purinérgicos P1/metabolismo , Adenosina/metabolismo , Amidoidrolases/metabolismo , Animais , Azidas/metabolismo , Técnicas Biossensoriais , Biotinilação , Bovinos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/química , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cinética , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Fenilisopropiladenosina/farmacologia , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica , Agonistas do Receptor Purinérgico P1 , Receptores Purinérgicos P1/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xantinas/metabolismoRESUMO
When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of Gi/Go holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant Gi2 alpha subunit purified from Sf9 cells. The Gi3 alpha subunit was less effective, and the Gi1 alpha subunit had no effect. Purified recombinant beta1gamma2 subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against Gi2 alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.
Assuntos
Antígenos CD20/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Células 3T3 , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adenilil Imidodifosfato/farmacologia , Animais , Antígenos CD20/efeitos dos fármacos , Encéfalo/metabolismo , Bovinos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/isolamento & purificação , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Cinética , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Patch-Clamp , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Receptores Purinérgicos P1/metabolismo , Animais , Baculoviridae/genética , Bovinos , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Técnicas In Vitro , Cinética , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Prenilação de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
The diversity in the heterotrimeric G protein alpha, beta, and gamma subunits may allow selective protein-protein interactions and provide specificity for signaling pathways. We examined the ability of five alpha subunits (alphai1, alphai2, alphao, alphas, and alphaq) to associate with three beta subunits (beta1, beta2, and beta5) dimerized to a gamma2 subunit containing an amino-terminal hexahistidine-FLAG affinity tag (gamma2HF). Sf9 insect cells were used to overexpress the recombinant proteins. The hexahistidine-FLAG sequence does not hinder the function of the beta1gamma2HF dimer as it can be specifically eluted from an alphai1-agarose column with GDP and AlF4-, and purified beta1gamma2HF dimer stimulates type II adenylyl cyclase. The beta1gamma2HF and beta2gamma2HF dimers immobilized on an anti-FLAG affinity column bound all five alpha subunits tested, whereas the beta5gamma2HF dimer bound only alphaq. The ability of other alpha subunits to compete with the alphaq subunit for binding to the beta5gamma2HF dimer was tested. Addition of increasing amounts of purified, recombinant alphai1 to the alphaq in a Sf9 cell extract did not decrease the amount of alphaq bound to the beta5gamma2HF column. When G proteins in an extract of brain membranes were activated with GDP and AlF4- and deactivated in the presence of equal amounts of the beta1gamma2HF or beta5gamma2HF dimers, only alphaq bound to the beta5gamma2HF dimer. The alphaq-beta5gamma2HF interaction on the column was functional as GDP, and AlF4- specifically eluted alphaq from the column. These results indicate that although the beta1 and beta2 subunits interact with alpha subunits from the alphai, alphas, and alphaq families, the structurally divergent beta5 subunit only interacts with alphaq.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Animais , Baculoviridae/genética , Encéfalo/metabolismo , Bovinos , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
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.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Receptores de Endotelina/fisiologia , Receptores Muscarínicos/fisiologia , Fosfolipases Tipo C/metabolismo , Animais , Baculoviridae , Linhagem Celular , Membrana Celular/metabolismo , Dimerização , Ativação Enzimática , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Cinética , Substâncias Macromoleculares , Fosfolipase C beta , Processamento de Proteína Pós-Traducional , Receptor de Endotelina B , Receptor Muscarínico M1 , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Spodoptera , TransfecçãoRESUMO
We investigated the coupling of A1 adenosine receptors to recombinant G proteins. Recombinant baculoviruses were used to express bovine A1 adenosine receptors in Sf9 insect cells that lack endogenous adenosine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [125I]BW-A844U ([125I]8-cyclopentyl-3-iodoaminophenethyl-1-propylxanthine) are Bmax = 2-5 pmol/mg of protein and K(D) = 0.53 +/- 0.12 nM. In competition assays, the potency order of agonists is (R)-phenylisopropyladenosine > (S)-phenylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties characteristic of native bovine A1 adenosine receptors. The agonist radioligand 125I-N6-4-aminobenzyladenosine binds to two affinity states of the recombinant A1 adenosine receptors with K(D) values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total sites and is increased 7-fold on reconstitution with both alpha and betagamma G protein subunits but not with either subunit alone; thus, exogenous alpha and betagamma subunits do not functionally interact with endogenous Sf9 betagamma and alpha subunits, respectively. Four different alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta1gamma1, beta1gamma2, beta1gamma3, beta2gamma2, beta2gamma3, and bovine brain betagamma)) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A1 adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with betagamma subunits containing the beta1 or beta2 subunits and gamma2 or gamma3 subunits. G protein heterotrimers that contain the beta1gamma1 dimer couple with similar potency but reduced efficacy to A1 adenosine receptors.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Purinérgicos P1/metabolismo , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , Ensaio Radioligante , Receptores Purinérgicos P1/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.