RESUMO
Signal transducing heterotrimeric G proteins are responsible for coupling a large number of cell surface receptors to the appropriate effector(s). Of the three subunits, 16 alpha, 4 beta, and 5 gamma subunits have been characterized, indicating a potential for over 300 unique combinations of heterotrimeric G proteins. To begin deciphering the unique G protein combinations that couple specific receptors with effectors, we examined the subcellular localization of the gamma subunits. Using anti-peptide antibodies specific for each of the known gamma subunits, neonatal cardiac fibroblasts were screened by standard immunocytochemistry. The anti-gamma 5 subunit antibody yielded a highly distinctive pattern of intensely fluorescent regions near the periphery of the cell that tended to protrude into the cell in a fibrous pattern. Dual staining with anti-vinculin antibody showed co-localization of the gamma 5 subunit with vinculin. In addition, the gamma 5 subunit staining extended a short distance out from the vinculin pattern along the protruding stress fiber, as revealed by double staining with phalloidin. These data indicated that the gamma 5 subunit was localized to areas of focal adhesion. Dual staining of rat aortic smooth muscle cells and Schwann cells also indicated co-localization of the gamma 5 subunit and vinculin, suggesting that the association of the gamma 5 subunit with areas of focal adhesion was wide-spread.
Assuntos
Adesão Celular , Proteínas de Ligação ao GTP/análise , Animais , Western Blotting , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Membrana Celular/química , Células Cultivadas , Matriz Extracelular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Microscopia de Fluorescência , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Vinculina/análiseRESUMO
A complementary DNA clone encoding the alpha subunit of the adenylate cyclase stimulatory G protein (Gs) was isolated and identified. A bovine brain complementary DNA library was screened with an oligonucleotide probe derived from amino acid sequence common to known G proteins. The only clone that was obtained with this probe has a complementary DNA insert of approximately 1670 base pairs. An antibody to a peptide synthesized according to deduced amino acid sequence reacts specifically with the alpha subunit of Gs. In addition, RNA that hybridizes with probes made from the clone is detected in wild-type S49 cells; however, cyc- S49 cells, which are deficient in Gs alpha activity, are devoid of this messenger RNA.
Assuntos
Adenilil Ciclases/metabolismo , Clonagem Molecular , DNA/análise , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Córtex Cerebral , Ativação Enzimática , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RetinaRESUMO
The guanosine triphosphate-binding proteins (G proteins) found in a variety of tissues transduce signals generated by ligand binding to cell surface receptors into changes in intracellular metabolism. Amino acid sequences of peptides prepared by partial proteolysis of the alpha subunit of a bovine brain G protein and the alpha subunit of rod outer-segment transducin were determined. The two proteins show regions of sequence identity as well as regions of diversity. A portion of the amino-terminal peptide sequence of each protein is highly homologous with the corresponding region in the ras protein (a protooncogene product). These similarities suggest that G proteins and ras proteins may have analogous functions.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Oncogenes , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras) , Transdução GenéticaRESUMO
Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2',7'-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPgammaS or GDPbetaS, which maintain the alpha subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPgammaS. However, injection of GDPbetaS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, alpha or betagamma transducin subunits were purified and microinjected as a sink for betagamma or alpha subunits in the erythroblast, respectively. Transducin betagamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Gi(alpha)2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G-proteins in hematopoietic cells and show that Gi(alpha)2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.
Assuntos
Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Humanos , Microinjeções , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Heterotrimeric G-proteins are components of the signal transduction pathways for the soluble and cell-contact signals that regulate normal growth and differentiation. There is now a greater appreciation of the role of the Gbetagamma-dimer in the regulation of a variety of intracellular effectors, including ion channels, adenylyl cyclase, and phospholipase Cbeta. In many cases, Gbetagamma-dimers are required for the activation of mitogen activated protein kinase (MAPK) pathways that promote cellular proliferation, although the underlying mechanisms have yet to be fully elucidated. Activation of phosphotidylinositol-3-kinase (PI3K) is a critical step in the intracellular transduction of survival signals. Gbetagamma-dimers directly activate PI3Kgamma as well as the more widely distributed PI3Kbeta. The activation of PI3Kgamma by Gbetagamma-dimers likely involves direct binding of specific Gbetagamma-dimers to both subunits of PI3Kgamma. Thus, Gbetagamma-dimers transmit signals from numerous receptors to a variety of intracellular effectors in distinct cellular contexts. Five distinct Gbeta-subunits and 12 distinct Ggamma-subunits have been identified. New experimental approaches are needed to elucidate the specific roles of individual Gbetagamma-dimers in the pathways that transduce signals for proliferation and survival.
Assuntos
Transformação Celular Neoplásica , Transdução de Sinais , Dimerização , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Receptores de Superfície Celular/metabolismoRESUMO
Activation of alpha 1-adrenergic receptors in neonatal cardiac myocytes results in changes in contractile activity and the induction of hypertrophic growth. The biochemical mechanisms responsible for these diverse effects are not yet established, but presumably involve the associated alpha 1-adrenergic stimulation of phosphatidylinositol (PI) hydrolysis, with concomitant generation of Ins 1,4,5-P3 and diacylglycerol. This study examined whether alpha 1-adrenergic generation of Ins 1,4,5-P3 in intact, quiescent, neonatal cardiac myocytes resulted in a Ca2+ signal. Stimulation of myocytes with norepinephrine in the presence of propranolol caused accumulation of inositol mono-, bis and trisphosphates. However, alpha 1-adrenergic stimulation did not alter cytosolic free Ca2+ levels in 85% of the myocytes examined. Direct generation of Ins 1,4,5-P3, by photolysis of microinjected caged Ins 1,4,5-P3, was also unable to alter cytosolic free Ca2+ levels, despite the presence of Ins 1,4,5-P3 receptors. Taken together, these data indicated that alpha 1-adrenergic stimulation did not initiate Ca2+ signaling because Ins 1,4,5-P3-induced Ca2+ mobilization was not operative in quiescent neonatal cardiac myocytes. Normal excitation-contraction Ca2+ handling mechanisms were present in these cells, as illustrated by depolarization- and caffeine-induced Ca2+ transients. Analysis of these same myocytes following 48 h in the presence of norepinephrine and propranolol showed a 40% increase in the ratio of protein to DNA and a 350% increase in release of atrial naturietic factor, compared to control cells, indicating the normal operation of alpha 1-adrenergic-induced hypertrophic growth. Therefore, the assumption that Ca(2+)-dependent processes will be activated by receptor signaling pathways coupled to enhanced phosphatidylinositol turnover in cardiac cells must be avoided. In addition, the data presented in this study clearly indicated that an increase in cytosolic free Ca2+ was not necessary for the induction of alpha 1-adrenergic-mediated cardiac hypertrophy.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Divisão Celular/fisiologia , Células Cultivadas , Hidrólise , Miocárdio/citologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/fisiologiaRESUMO
The membrane-delimited activation of muscarinic K+ channels by G protein beta gamma subunits plays a prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying K+ channels. Here, we demonstrate that muscarinic K+ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel, which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of Gbeta gamma subunits to the channel was tested, using recombinant Gbeta1 gamma5. Gbeta1 gamma5 was able to control the equilibrium between the four gating modes of the channel in a manner consistent with binding of Gbeta gamma to four equivalent and independent sites in the protein complex. Surprisingly, however, Gbeta1 gamma5 lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K+ channel gating by Gbeta gamma provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.
Assuntos
Ativação do Canal Iônico/fisiologia , Miocárdio/química , Canais de Potássio/fisiologia , Receptores Muscarínicos/fisiologia , Adenosina/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/farmacologia , Átrios do Coração/química , Átrios do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Fibras Musculares Esqueléticas/química , Miocárdio/citologia , Técnicas de Patch-Clamp , Canais de Potássio/química , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , SpodopteraRESUMO
A family of G proteins, composed of α, ß, and γ subunits, plays a central role in coupling receptors to a variety of enzymes and ion channels. In the cardiovascular system, G proteins are involved in coupling receptors for epinephrine, norepinephrine, acetylcholine, adenosine, angiotensin II, and endothelin to regulation of adenylyl cyclases, phospholipases, and ion channels. For many years, the classic view has been that G protein α subunits provide the requisite specificity for receptor and effector interactions. Recent advances, however, have revealed that the ß and γ subunits also play prominent roles in transducing information from receptors to the appropriate effectors. With the identification of multiple subtypes of ß and γ subunits in the heart, questions are raised regarding their respective roles in signal transduction processes regulating cardiac function.
RESUMO
A cDNA (pJ5) encoding a protein with significant sequence identity to the previously known beta 1, beta 2, beta 3 and beta 4 subunits of guanine-nucleotide-binding proteins (G proteins) has been cloned from a 8-12-week-old rat heart cDNA library. Its predicted amino acid (aa) sequence of 340 aa most closely resembles that of the human beta 3 subunit, showing 96% identity. Northern blot analysis revealed that the major mRNA corresponding to the rat pJ5 cDNA is 2.0 kb in length. It is expressed at a high level in the heart and at a much lower level in the brain.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Hominidae/genética , Miocárdio/metabolismo , Ratos/genética , Retinaldeído/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular/métodos , DNA Complementar/biossíntese , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Homologia de Sequência de AminoácidosRESUMO
Activation of cardiac alpha1-adrenoreceptors has a number of physiological effects. Ascribing these effects to a specific alpha1-adrenoreceptor subtype first requires the elucidation of the subtypes that are present in the tissue of interest. In the present study, mRNA transcripts for the alpha1A, alpha1B and alpha1D-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using reverse transcriptase-polymerase chain reaction analysis. However, binding sites for only the alpha1A and alpha1B-adrenoreceptor subtypes were detected in cultured neonatal rat cardiac myocytes, using competition binding analysis with a variety of alpha1 selective receptor antagonists. Phenylephrine-stimulated phosphatidylinositol hydrolysis was inhibited by alpha1 selective receptor antagonists with affinities consistent with the alpha1A-adrenoreceptor subtype, whereas phenylephrine-induced activation of the mitogen activated protein kinase cascade was inhibited by these same antagonists with affinities more closely resembling the alpha1B-adrenoreceptor subtype. In the case of both signaling pathways, the alpha1D selective receptor antagonist, BMY 7378, exhibited affinities suggestive of the relative absence of a alpha1D-adrenoreceptor subtype. Thus, despite the presence of mRNA transcripts for all three alpha1-adrenoreceptor subtypes, only the alpha1A and alpha1B-adrenoreceptor subtypes were expressed and functionally coupled at detectable levels in neonatal rat cardiac myocytes. Of particular interest, phenylephrine-induced activation of the mitogen activated protein kinase cascade appears to be mediated by a subtype resembling most closely the pharmacological profile of the alpha1B-adrenoreceptor subtype.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Miocárdio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Animais Recém-Nascidos , Ligação Competitiva , Células Cultivadas , Hidrólise , Miocárdio/citologia , Fosfatidilinositóis/metabolismo , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyAssuntos
Anticorpos , Proteínas de Ligação ao GTP/análise , Peptídeos/síntese química , Sequência de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Encéfalo/metabolismo , Bovinos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/imunologia , Soros Imunes , Immunoblotting/métodos , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos/imunologia , Coelhos/imunologiaRESUMO
The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue and X is any amino acid). This domain represents the site of Cys thioether modification by isoprenoids in p21ras, nuclear lamins, and fungal mating factors. We now show that G gamma 6, translated in reticulocyte lysate, is efficiently labeled with the isoprenoid precursor, [3H]mevalonate. Alteration of the sequence of G gamma 6 so that a Gly was substituted for Cys in the C-terminal Cys-A-A-X element rendered the protein incapable of undergoing isoprenoid modification. In contrast to G gamma 6, the Gi alpha 1 subunit did not appear to undergo isoprenylation when translated in reticulocyte lysate. Transient expression of the protein in COS cells, which were able to isoprenylate the p21 product of transfected H-ras, also failed to demonstrate isoprenylation of Gi alpha 1. The modification of the gamma subunit by a hydrophobic moiety may have important implications for the assembly of the brain G-protein beta gamma complexes into the cell membrane.
Assuntos
Cisteína/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Livre de Células , Clonagem Molecular , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , Coelhos , Reticulócitos/metabolismo , TerpenosRESUMO
The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein gamma subunits in a suitable state for immunodetection. We describe a high temperature (70 degrees C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein gamma subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellulose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.
Assuntos
Proteínas de Ligação ao GTP/análise , Immunoblotting/métodos , Sequência de Aminoácidos , Animais , Anticorpos , Colódio , Epitopos/química , Estudos de Avaliação como Assunto , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/imunologia , Temperatura Alta , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Desnaturação Proteica , Dodecilsulfato de SódioRESUMO
Culturing neonatal rat cardiac myocytes in 50 mM KCl inhibits the accumulation of Go alpha that occurs when myocytes are placed in culture. The mechanism by which high extracellular K+ inhibits Go alpha accumulation in myocytes was investigated by measurement of the concentration of intracellular Ca2+ ([Ca2+]) and adenosine 3',5'-cyclic monophosphate concentration ([cAMP]) of control and K(+)-depolarized myocytes. Although intracellular [Ca2+] in K(+)-depolarized myocytes was twofold higher than basal intracellular [Ca2+] in control cells, the mean intracellular [Ca2+] in contracting control myocytes was comparable to that of K(+)-depolarized myocytes. Furthermore, myocytes cultured in low Ca2+ plus high K+ exhibited an inhibition of Go alpha accumulation, even though intracellular [Ca2+] was 10-fold lower than that of cells cultured in normal Ca2+ plus high K+. In addition, intracellular [cAMP] of K(+)-depolarized myocytes was comparable to that of control cells. Moreover, dibutyryl cAMP inhibited Go alpha accumulation in myocytes to the same extent as high K+, even though intracellular [cAMP] differed 10-fold. Thus neither intracellular Ca2+ nor cAMP appear to mediate the inhibitory effect of high K+ on Go alpha accumulation. However, cAMP has an inhibitory effect on Go alpha expression that is independent of K+.
Assuntos
Cálcio/farmacologia , AMP Cíclico/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Bucladesina/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Espaço Extracelular/metabolismo , Membranas Intracelulares/metabolismo , Concentração Osmolar , Potássio/metabolismo , RatosRESUMO
A precise description of the involvement of G proteins in regulation of the cardiovascular system is not possible at the present time although it is clear that they do have important regulatory roles. The cardiovascular system is composed of a variety of cell types, which are subject to control by several different hormones, as well as by hormones that have several different effects in the same cell type. Although, historically, variations in the type and number of receptors located on each cell have been used to explain this diversity of hormonal responses, we must now consider the large number and diversity of G proteins in any effort to understand the coordinated hormonal regulation of cellular functions. Given that there are eight known G proteins and several others have been speculated, each of which is composed of three subunits, each of which has several different forms, the possible combinations of subunits into functionally distinct G proteins is enormous. To place this newly described family of G proteins into the appropriate hormone signaling pathways will require a continued research effort. However, with recent progress in producing specific antibodies to each of the G protein subunits, it may now be possible to determine the specific receptor-effector functions of each G protein and their individual subunits.
Assuntos
Fenômenos Fisiológicos Cardiovasculares , Proteínas de Ligação ao GTP/fisiologia , Animais , Fenômenos Químicos , Química , Proteínas de Ligação ao GTP/classificação , Coração/fisiologia , Humanos , Neurotransmissores/fisiologia , Receptores Adrenérgicos alfa/fisiologia , Receptores Adrenérgicos beta/fisiologia , Receptores Muscarínicos/fisiologiaRESUMO
The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.
Assuntos
Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Difosfato/farmacologia , Substâncias Macromoleculares , Ácido Mevalônico/metabolismo , Mariposas , Mutagênese , Mapeamento de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transfecção , Tripsina/metabolismoRESUMO
In view of the expanding roles of the betagamma subunits of the G proteins in signaling, the possibility was raised that the rich diversity of betagamma subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant alpha2A-adrenergic receptor were used to assess the contribution of the betagamma subunit composition. Reconstituted coupling between the receptor and heterotrimeric Gi protein was assayed by high affinity, guanine nucleotide-sensitive binding of the alpha2-adrenergic agonist, [3H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various betagamma dimers, including those containing the beta3 subtype and the newly described gamma4, gamma10, and gamma11 subtypes, to promote interaction of the same alphai subunit with the alpha2A-adrenergic receptor.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacologia , Sequência de Aminoácidos , Animais , Biopolímeros , Tartarato de Brimonidina , Membrana Celular/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Quinoxalinas/farmacologia , SpodopteraRESUMO
The metabolism of coenzyme A and control of its synthesis are reviewed. Pantothenate kinase is an important rate-controlling enzyme in the synthetic pathway of all tissues studied and appears to catalyze the flux-generating reaction of the pathway in cardiac muscle. This enzyme is strongly inhibited by coenzyme A and all of its acyl esters. The cytosolic concentrations of coenzyme A and acetyl coenzyme A in both liver and heart are high enough to totally inhibit pantothenate kinase under all conditions. Free carnitine, but not acetyl carnitine, deinhibits the coenzyme A-inhibited enzyme. Carnitine alone does not increase enzyme activity. Thus changes in the acetyl carnitine-to-carnitine ratio that occur with nutritional states provides a mechanism for regulation of coenzyme A synthetic rates. Changes in the rate of coenzyme A synthesis in liver and heart occurs with fasting, refeeding, and diabetes and in heart muscle with hypertrophy. The pathway and regulation of coenzyme A degradation are not understood.
Assuntos
Coenzima A/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Acetilcoenzima A/metabolismo , Animais , Coenzima A/biossíntese , Dieta , Cinética , Fígado/metabolismo , Miocárdio/metabolismo , Ácido Pantotênico/metabolismo , Fosfotransferases/metabolismo , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Control of coenzyme A (CoA) synthesis was studied in isolated perfused rat hearts. The data obtained support the hypothesis that phosphorylation of pantothenic acid by pantothenate kinase is the flux-generating reaction in the pathway of CoA synthesis. This reaction operated in the cell far removed from its thermodynamic equilibrium; it was saturated with substrates under all conditions studied; and the concentration of substrate changed in the opposite direction to flux when flux was altered. The reaction was subject to control by external factors associated with oxidation of glucose, pyruvate, or palmitate. CoA synthesis from 4'-phosphopantothenic acid was not inhibited by glucose and pyruvate, suggesting that pantothenate kinase is the only reaction in the pathway that is controlled in isolated hearts. Maximum rates of CoA synthesis in perfused hearts with pantothenate kinase stimulation were dependent on a supply of exogenous cysteine. Perfusate [14C]cysteine was incorporated into intermediates of this pathway and CoA. When protected from oxidation to cystine by low concentrations of dithiothreitol, 0.1 mM cysteine in the perfusate resulted in maximum rates of CoA synthesis. Evidence was obtained that indicates that addition of cysteine relieves a substrate limitation at the 4'-phosphopantothenyl cysteine synthase reaction.
Assuntos
Coenzima A/biossíntese , Miocárdio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/metabolismo , Animais , Cisteína/farmacologia , Glucose/farmacologia , Masculino , Ácido Pantotênico/metabolismo , Fosforilação , Piruvatos/farmacologia , Ácido Pirúvico , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Membranes from bovine brain bind relatively large quantities of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinity. The two proteins responsible for most of this activity were purified; they account for 1.5% of the membrane protein. The two proteins contain alpha subunits of either 39,000 or 41,000 Da, beta subunits of 36,000 or 35,000 Da, and a potential gamma subunit (11,000 Da). These structures are the same as a family of proteins that includes transducin and the regulatory proteins, GS and GI, of adenylate cyclase. The 41,000- and 39,000-Da polypeptides can be ADP-ribosylated with islet-activating protein from Bordetella pertussis, bind guanine nucleotides specifically, and migrate through polyacrylamide gels with rates similar to the alpha subunits of GI and transducin, respectively. The 36,000- and 35,000-Da polypeptides are similar to the beta subunits of GI and GS. The gamma subunit is found whenever beta subunits are present. The 41,000- and 39,000-Da polypeptides (with beta and gamma) are designated, respectively, GI and GO from brain. The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins. GO alpha binds GTP gamma S reversibly in the absence of Mg2+ and is relatively stable in cholate. This isolated alpha subunit should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.