RESUMO
As the properties of more and more isoforms of the molecules involved in G-protein-mediated signal transduction pathways are unravelled, surprising diversity and versatility are being revealed. The path from receptor to effector is not dictated exclusively by the alpha subunits of heterotrimetric G proteins. The nature of the beta lambda subunit complex probably controls interactions of G(alpha) with receptors. In addition, dissociation of G(alpha)-GTP from G(beta lambda)provides two signalling complexes, and these proteins regulate effectors independently or synergistically. Synergistic or conditional regulation of effectors by G(alpha) and G(beta lambda)can provide a molecular signal that records the association of independent events.
RESUMO
Various heterotrimeric guanine nucleotide-binding proteins have been identified on the basis of the individual subtypes of their alpha subunits. The beta gamma complexes, composed of beta and gamma subunits, remain tightly associated under physiological conditions and have been assumed to constitute a common pool shared among various guanosine triphosphate (GTP)-binding (G) protein heterotrimers. Particular alpha and beta subunit subtypes participate in the signal transduction processes between somatostatin or muscarinic receptors and the voltage-sensitive L-type calcium channel in rat pituitary GH3 cells. Among gamma subunits the gamma 3 subtype was found to be required for coupling of the somatostatin receptor to voltage-sensitive calcium channels, whereas the gamma 4 subtype was found to be required for coupling of the muscarinic receptor to those channels.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais/fisiologia , Animais , Sequência de Bases , Cálcio/metabolismo , Carbacol/farmacologia , Proteínas de Ligação ao GTP/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Oligonucleotídeos Antissenso/farmacologia , Neoplasias Hipofisárias , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Somatostatina/farmacologia , Células Tumorais CultivadasAssuntos
Proteínas de Ligação ao GTP/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Transporte Biológico , Células Cultivadas , Desenho de Fármacos , Feminino , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/efeitos dos fármacos , Substâncias Macromoleculares , Microinjeções/métodos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacocinética , Oócitos/metabolismo , RNA Mensageiro/metabolismoAssuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Radioisótopos de Carbono , Linhagem Celular , Membrana Celular/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Citosol/metabolismo , Indicadores e Reagentes , Cinética , Substâncias Macromoleculares , Mamíferos , Dados de Sequência Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Ácido Palmítico , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Spodoptera , Transfecção/métodos , TrítioRESUMO
In rat pituitary GH3 cells Ca2+ current through L-type channels is reduced by somatostatin. This modulation of channel activity by somatostatin receptors is mediated by a guanine nucleotide-binding regulatory protein (G protein). It is sensitive to pertussis toxin, indicating the involvement of a G(o)- or Gi-type G protein in this pathway. The identity of this G protein was determined by suppressing the expression of endogenous G proteins individually via intranuclear injection of antisense oligonucleotides. This method was applied to GH3 cells to screen several G protein alpha, beta and gamma subunits for their roles in the defined signal transduction pathway. The loss of somatostatin's modulating activity on the voltage-dependent Ca2+ channel after oligonucleotide injection revealed the involvement of G(o) alpha 2 beta 1 gamma 3 to the exclusion of other closely related subtypes.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hipófise/metabolismo , Somatostatina/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Mutação , Ratos , Transdução de Sinais , Somatostatina/farmacologiaRESUMO
Many G protein alpha subunits are dually acylated with myristate and palmitate or are palmitoylated on more than one cysteine residue near their N termini. The Galpha protein that activates adenylyl cyclase, alphas, is not myristoylated but can be reversibly palmitoylated. It appears that alphas contains another, as-yet-unidentified covalent modification that decreases its apparent dissociation constant for adenylyl cyclase from 50 nM to <0. 5 nM. This modification is at or near the N terminus of the protein and is hydrophobic. Palmitoylation of native alphas does not account for its high affinity for adenylyl cyclase.
Assuntos
Adenilil Ciclases/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Linhagem Celular , Membrana Celular/enzimologia , DNA Complementar , Ativação Enzimática , Éxons , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/química , Cinética , Fígado/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sitios de Sequências Rotuladas , Spodoptera , TransfecçãoRESUMO
Mammalian particulate adenylyl cyclases contain two transmembrane regions (M(1) and M(2)) and two cytosolic domains (C(1) and C(2)) forming the catalytic core. The cytosolic domains are subdivided into a highly conserved region (part a) and a region with lower similarity (part b). Hypothetical models exist that account for the mechanism by which Galpha(s) and forskolin stimulate mammalian adenylyl cyclase. In contrast, little is known about how Gbetagamma dimers regulate catalysis. The so-called QEHA region located in the C(2a) domain of type II adenylyl cyclase has been proposed to represent a site of interaction. Here we show (i) that the QEHA region directly interacts with Gbetagamma but (ii) that it is of minor importance for the stimulation of type II adenylyl cyclase because it can be replaced by corresponding, nonidentical regions of other adenylyl cyclase isoforms without altering the stimulatory effect of Gbetagamma and (iii) that the C(1b) region is necessary for Gbetagamma to exert a stimulatory effect on adenylyl cyclase type II as in a C(1b) deletion mutant the Gbetagamma regulation was specifically impeded whereas the Galpha(s)- and forskolin-mediated stimulation was maintained.
Assuntos
Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Bovinos , Linhagem Celular , Membrana Celular/enzimologia , Sequência Conservada , Citosol/enzimologia , DNA Complementar , Dimerização , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Spodoptera , TransfecçãoRESUMO
Many alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are palmitoylated. Exposure of cells to the beta-adrenergic agonist isoproterenol increased incorporation of [3H]palmitate specifically into alpha s, the alpha subunit that mediates stimulation of adenylyl cyclase. Pulse-chase experiments suggested that isoproterenol increased turnover of alpha s-bound palmitate. Mutagenesis of Cys-3 in alpha s or alpha o (a homologous alpha subunit) prevented palmitoylation of these proteins. Differing results were obtained when mutations of Cys-3 in alpha s or alpha o were expressed in cells and assayed for their distribution between soluble and membrane fractions. Some alpha subunits, including alpha o, are myristoylated at the amino-terminal glycine residue. Mutation of this glycine prevented both myristoylation and palmitoylation of alpha o, indicating that myristoylation precedes palmitoylation of dually acylated alpha subunits. The amino-terminal sequences and fatty acylation properties of dually acylated alpha subunits are strikingly similar to those of some members of the Src family of protein-tyrosine kinases. The amino-terminal sequence Met-Gly-Cys-Xaa-Xaa-Ser/Cys shared by these proteins may represent a motif for cotranslational and posttranslational processing that includes myristoylation of the glycine residue and reversible palmitoylation of the cysteine residue.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta/metabolismo , Acilação , Sequência de Aminoácidos , Animais , Sequência de Bases , Análise Mutacional de DNA , Proteínas de Ligação ao GTP/genética , Isoproterenol/farmacologia , Dados de Sequência Molecular , Ácido Palmítico , Ratos , Receptores Adrenérgicos beta/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
Adenylyl and guanylyl cyclases synthesize second messenger molecules by intramolecular esterification of purine nucleotides, i.e., cAMP from ATP and cGMP from GTP, respectively. Despite their sequence homology, both families of mammalian cyclases show remarkably different regulatory patterns. In an attempt to define the functional domains in adenylyl cyclase responsible for their isotypic-common activation by Galphas or forskolin, dimeric chimeras were constructed from soluble guanylyl cyclase alpha1 subunit and the C-terminal halves of adenylyl cyclases type I, II, or V. The cyclase-hybrid generated cAMP and was inhibited by P-site ligands. The data establish structural equivalence and the ability of functional complement at the catalytic sites in both cyclases. Detailed enzymatic characterization of the chimeric cyclase revealed a crucial role of the N-terminal adenylyl cyclase half for stimulatory actions, and a major importance of the C-terminal part for nucleotide specificity.
Assuntos
Adenilil Ciclases/genética , Guanilato Ciclase/genética , Proteínas Recombinantes de Fusão/fisiologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/biossíntese , Adenilil Ciclases/metabolismo , Animais , Baculoviridae/genética , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva/genética , Membrana Celular/enzimologia , Colforsina/farmacologia , Difosfatos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Glicosilação , Guanilato Ciclase/metabolismo , Guanilato Ciclase/fisiologia , Ligantes , Proteínas de Membrana/metabolismo , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/genética , Especificidade por Substrato/genéticaRESUMO
Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification.
Assuntos
Proteínas de Ligação ao GTP/análise , Soros Imunes/imunologia , Proteínas de Membrana/imunologia , Animais , Bovinos , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Proteínas de Ligação ao GTP/imunologia , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Imunização , Fosforilação , Coelhos , TransducinaRESUMO
In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Hipófise/metabolismo , Proteína Quinase C/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Animais , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Dados de Sequência Molecular , Fosforilação , Ratos , Transdução de SinaisRESUMO
Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man. A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis. Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Trifosfato/metabolismo , Compostos de Alumínio/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , DNA/química , Escherichia coli , Fluoretos/metabolismo , Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Hidrólise , Cinética , Substâncias Macromoleculares , Magnésio/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Radioisótopos de Fósforo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/análogos & derivados , Proteínas de Membrana/isolamento & purificação , Animais , Bovinos , Cromatografia por Troca Iônica , Guanosina Trifosfato/análogos & derivados , Retina/análise , Sefarose/análogos & derivados , Radioisótopos de Enxofre , Tionucleotídeos , TransducinaRESUMO
Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Sequência de Bases , Cálcio/fisiologia , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Técnicas In Vitro , Ativação do Canal Iônico , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , RNA Mensageiro/genética , Ratos , Receptores Muscarínicos/fisiologia , Receptores de Neurotransmissores/fisiologia , Receptores de Somatostatina , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Receptores Muscarínicos/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Sequência de Bases , Carbacol/farmacologia , Linhagem Celular , Membrana Celular/fisiologia , DNA Antissenso/genética , Condutividade Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/genética , Microinjeções , Dados de Sequência Molecular , Toxina Pertussis , Hipófise/fisiologia , RNA Mensageiro/genética , Ratos , Receptores de Somatostatina , Transdução de Sinais , Somatostatina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Forskolin- and Gsalpha-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized approximately 25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native Mr approximately 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 micromol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-Gsalpha is responsible for the observed enzymatic activity and can be isolated. In addition, GTPgammaS-Gsalpha interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsalpha and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsalpha can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsalpha, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Sítios de Ligação , Centrifugação , Cromatografia em Gel , Colforsina/farmacologia , Citosol/enzimologia , Ativação Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Peso Molecular , Conformação ProteicaRESUMO
RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Proteínas , Proteínas RGS/fisiologia , Adenilil Ciclases/fisiologia , Animais , Células COS , Canais de Cálcio/fisiologia , Feminino , Proteínas de Ligação ao GTP/química , Proteínas Ativadoras de GTPase/fisiologia , Guanosina Trifosfato/metabolismo , Mutação , Proteínas RGS/química , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 2/fisiologia , Relação Estrutura-AtividadeRESUMO
Galphao, the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Galphao1 and Galphao2, derived by alternative splicing of the mRNA. A third Galphao1-related isoform, Galphao3, has been purified, representing about 30% of total Go in brain. Initial studies revealed distinct biochemical properties of Galphao3 as compared with other Galphao isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Galphao1 and Galphao3, C-terminal peptides showed a difference of +1 Da for Galphao3. Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Galphao3. Gel electrophoretic analysis of recombinant Galphao3 showed the same mobility as native Galphao3 but distinct to Galphao1. The conversion of 346Asn-->Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5'-[gamma-thio]triphosphate binding. No cDNA coding for Galphao3 was detected, suggesting an enzymatic deamidation of Galphao1 by a yet-unidentified activity. Therefore, Galpha heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of Galphao1 and Galphao3 differed from cell type to cell type, indicating an additional principle of G protein regulation.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Processamento de Proteína Pós-Traducional , Transcrição Gênica , Sequência de Aminoácidos , Animais , Asparagina , Ácido Aspártico , Sítios de Ligação , Bovinos , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TripsinaRESUMO
The pyrophosphate (PP(i)) analog foscarnet inhibits viral DNA-polymerases and is used to treat cytomegalovirus and human immunodeficiency vius infections. Nucleotide cyclases and DNA-polymerases catalyze analogous reactions, i.e. a phosphodiester bond formation, and have similar topologies in their active sites. Inhibition by foscarnet of adenylyl cyclase isoforms was therefore tested with (i) purified catalytic domains C1 and C2 of types I and VII (IC1 and VIIC1) and of type II (IIC2) and (ii) membrane-bound holoenzymes (from mammalian tissues and types I, II, and V heterologously expressed in Sf9 cell membranes). Foscarnet was more potent than PP(i) in suppressing forskolin-stimulated catalysis by both, IC1/IIC2 and VIIC1/IIC2. Stimulation of VIIC1/IIC2 by Galpha(s) relieved the inhibition by foscarnet but not that by PP(i). The IC(50) of foscarnet on membrane-bound adenylyl cyclases also depended on their mode of regulation. These findings predict that receptor-dependent cAMP formation is sensitive to inhibition by foscarnet in some, but not all, cells. This was verified with two cell lines; foscarnet blocked cAMP accumulation after A(2A)-adenosine receptor stimulation in PC12 but not in HEK-A(2A) cells. Foscarnet also inhibited soluble and, to a lesser extent, particulate guanylyl cylase. Thus, foscarnet interferes with the generation of cyclic nucleotides, an effect which may give rise to clinical side effects. The extent of inhibition varies with the enzyme isoform and with the regulatory input.
Assuntos
Inibidores de Adenilil Ciclases , Antivirais/farmacologia , Foscarnet/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ciclofosfamida , Dimerização , Difosfatos/química , Difosfatos/metabolismo , Difosfatos/farmacologia , Doxorrubicina , Inibidores Enzimáticos/farmacologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteínas de Membrana/metabolismo , VincristinaRESUMO
Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.