RESUMO
Scavenger receptor class B, type I (SR-BI) binds HDL and mediates selective delivery of cholesteryl esters (CEs) to the liver, adrenals, and gonads for product formation (bile acids and steroids). Because relatively little is known about SR-BI posttranslational regulation in steroidogenic cells, we examined the roles of Na(+)/H(+) exchanger regulatory factors (NHERFs) in regulating SR-BI expression, SR-BI-mediated selective CE uptake, and steroidogenesis. NHERF1 and NHERF2 mRNA and protein are expressed at varying levels in model steroidogenic cell lines and the adrenal, with only low expression of PDZK1 (NHERF3) and NHERF4. Dibutyryl cyclic AMP decreased NHERF1 and NHERF2 and increased SR-BI mRNA expression in primary rat granulosa cells and MLTC-1 cells, whereas ACTH had no effect on NHERF1 and NHERF2 mRNA levels but decreased their protein levels in rat adrenals. Co-immunoprecipitation, colocalization, bimolecular fluorescence complementation, and mutational analysis indicated that SR-BI associates with NHERF1 and NHERF2. NHERF1 and NHERF2 down-regulated SR-BI protein expression through inhibition of its de novo synthesis. NHERF1 and NHERF2 also inhibited SR-BI-mediated selective CE transport and steroidogenesis, which were markedly attenuated by partial deletions of the PDZ1 or PDZ2 domain of NHERF1, the PDZ2 domain of NHERF2, or the MERM domains of NHERF1/2 or by gene silencing of NHERF1/2. Moreover, an intact COOH-terminal PDZ recognition motif (EAKL) in SR-BI is needed. Transient transfection of hepatic cell lines with NHERF1 or NHERF2 caused a significant reduction in endogenous protein levels of SR-BI. Collectively, these data establish NHERF1 and NHERF2 as SR-BI protein binding partners that play a negative role in the regulation of SR-BI expression, selective CE transport, and steroidogenesis.
Assuntos
Ésteres do Colesterol/metabolismo , Regulação para Baixo/fisiologia , Células da Granulosa/metabolismo , Fosfoproteínas/metabolismo , RNA Mensageiro/biossíntese , Receptores Depuradores Classe B/biossíntese , Trocadores de Sódio-Hidrogênio/metabolismo , Motivos de Aminoácidos , Animais , Transporte Biológico Ativo/fisiologia , Células CHO , Células COS , Chlorocebus aethiops , Ésteres do Colesterol/genética , Cricetinae , Cricetulus , Feminino , Células da Granulosa/citologia , Masculino , Fosfoproteínas/genética , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores Depuradores Classe B/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3)-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.
Assuntos
Mutação Puntual/genética , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Acetilcolina/farmacocinética , Acetilcolina/farmacologia , Asparagina/genética , Carbacol/farmacocinética , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligação Proteica/genética , Receptor Muscarínico M3/agonistas , Tirosina/genéticaRESUMO
Historically, it has been assumed that the functional roles of G proteins in receptor recognition and effector regulation are specified by their diverse alpha subunits. However, the discovery of similarly diverse betagamma subunits that participate in both of these functional processes has called this assumption into question; recent work suggests that G proteins function as heterotrimers whose roles in particular receptor signaling pathways are determined by their specific alphabetagamma subunit combinations. Although much remains to be learned, the assembly of specific alphabetagamma subunit combinations seems to involve both structural and spatial factors.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Transdução de Sinais/genética , Animais , Compartimento Celular/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Membranas Intracelulares/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Prófase/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Cultivadas , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/fisiologiaRESUMO
We have applied multicolor BiFC to study the association preferences of G protein beta and gamma subunits in living cells. Cells co-express multiple isoforms of beta and gamma subunits, most of which can form complexes. Although many betagamma complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate betagamma complex formation with functionality in intact cells by comparing the amounts of fluorescent betagamma complexes with their abilities to modulate effector proteins. The relative predominance of specific betagamma complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of beta and gamma subunits by simultaneously visualizing the two fluorescent complexes formed when beta or gamma subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common gamma or beta subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Bioensaio/métodos , Biomarcadores/análise , Linhagem Celular Transformada , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Plasmídeos , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Espectrometria de Fluorescência/métodos , TransfecçãoRESUMO
BACKGROUND: Inhibition of G-protein ßγ (Gßγ) signaling was found previously to enhance T cell receptor (TCR)-stimulated increases in interleukin 2 (IL-2) mRNA in CD4(+) T helper cells, suggesting that Gßγ might be a useful drug target for treating autoimmune diseases, as low dose IL-2 therapy can suppress autoimmune responses. Because IL-2 may counteract autoimmunity in part by shifting CD4(+) T helper cells away from the Type 1 T helper cell (TH1) and TH17 subtypes towards the TH2 subtype, the purpose of this study was to determine if blocking Gßγ signaling affected the balance of TH1, TH17, and TH2 cytokine mRNAs produced by CD4(+) T helper cells. METHODS: Gallein, a small molecule inhibitor of Gßγ, and siRNA-mediated silencing of the G-protein ß1 subunit (Gß1) were used to test the effect of blocking Gßγ on mRNA levels of cytokines in primary human TCR-stimulated CD4(+) T helper cells. RESULTS: Gallein and Gß1 siRNA decreased interferon-γ (IFN-γ) and IL-17A mRNA levels in TCR-stimulated CD4(+) T cells grown under TH1-promoting conditions. Inhibiting Gßγ also decreased mRNA levels of STAT4, which plays a positive role in TH1 differentiation and IL-17A production. Moreover, mRNA levels of the STAT4-regulated TH1-associated proteins, IL-18 receptor ß chain (IL-18Rß), mitogen-activated protein kinase kinase kinase 8 (MAP3K8), lymphocyte activation gene 3 (LAG-3), natural killer cell group 7 sequence (NKG7), and oncostatin M (OSM) were also decreased upon Gßγ inhibition. Gallein also increased IL-4, IL-5, IL-9, and IL-13 mRNA levels in TCR-stimulated memory CD4(+) T cells grown in TH2-promoting conditions. CONCLUSIONS: Inhibiting Gßγ to produce these shifts in cytokine mRNA production might be beneficial for patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), psoriasis, multiple sclerosis (MS), and Hashimoto's thyroiditis (HT), in which both IFN-γ and IL-17A are elevated.
RESUMO
G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein ßγ (Gßγ) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of Gßγ, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. Gß1 and Gß2 mRNA accounted for >99% of Gß mRNA, and small interfering RNA (siRNA)-mediated silencing of Gß1 but not Gß2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking Gßγ enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking Gßγ also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous Gßγ inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking Gßγ in CD4+ T helper cells could have applications for autoimmune diseases.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Interleucina-2/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/metabolismo , Transcrição Gênica , Cálcio/metabolismo , Linhagem Celular , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Fatores de Tempo , Xantenos/farmacologiaRESUMO
BACKGROUND: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. METHODS: The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. RESULTS: ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gßγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. CONCLUSIONS: GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.
RESUMO
BACKGROUND: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. RESULTS: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. CONCLUSION: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas Luminescentes/genética , Mutagênese Insercional/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Rim/citologia , Rim/metabolismo , Dobramento de Proteína , Subunidades Proteicas/genética , Receptores de AMPA/genética , Proteínas Recombinantes de Fusão/química , TransfecçãoRESUMO
Heterotrimeric G proteins transmit signals from a wide range of cell surface G protein-coupled receptors (GPCRs) to mediate multiple cellular events. Within the plasma membrane, G proteins interact with GPCRs and effector proteins such as adenylyl cyclase (AC) and phospholipase C (PLC). Plasma membrane subdomains (e.g., lipid rafts and caveolae) may organize and regulate these interactions. G protein subunits have been reported to be in additional cellular regions, such as the Golgi apparatus and the cytoskeleton, and G protein alpha subunits may move within the cell during the activation cycle. Changes in the cellular localization of alpha subunits could be important for interactions with effectors that are not in the plasma membrane and/or could be a means for terminating G protein signaling. However, until recently, the topic of G protein alpha subunit localization under basal and activated conditions has been controversial, partly because of spatial and temporal limitations inherent to procedures like cell fractionation and immunohistochemistry. Green fluorescent protein (GFP)-tagging is a useful way to enable real-time visualization of proteins in living cells. This chapter describes how to produce and visualize functional GFP-tagged alpha subunits and to investigate whether activation affects their subcellular localization.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/análise , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Microscopia de Fluorescência/métodos , Biologia Molecular/métodos , Sequência de Aminoácidos , Linhagem Celular , Subunidades alfa de Proteínas de Ligação ao GTP/química , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes/metabolismo , Rim/citologia , Proteínas Luminescentes/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Cells co-express multiple G protein ß and γ subunit isoforms, but the extent to which individual subunits associate to form particular ßγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific ßγ complexes may have unique functions despite the fact that most complexes exhibit similar properties when assayed in reconstituted systems. This chapter describes how multicolor bimolecular fluorescence complementation (BiFC) can be used in living cells to study the association preferences of ß and γ subunits. Multicolor BiFC determines the association preferences of these subunits by quantifying the two fluorescent complexes formed when ß or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete for interaction with limiting amounts of a common γ or ß subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). One means by which ßγ complexes may differ from each other and thereby mediate unique functions in vivo is in the kinetics and patterns of their internalization responses to stimulation of G protein-coupled receptors (GPCRs). Methods are described for imaging and quantifying the internalization of pairs of ßγ complexes in response to GPCR stimulation in living cells.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/análise , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/análise , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Proteínas Luminescentes/análise , Microscopia de Fluorescência/métodos , Proteínas Recombinantes de Fusão/análise , Espectrometria de Fluorescência/métodos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Estruturas Celulares/metabolismo , Estruturas Celulares/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoAssuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Fracionamento Celular/métodos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , AMP Cíclico/metabolismo , Vetores Genéticos , Proteínas Heterotriméricas de Ligação ao GTP/genética , Isoproterenol/farmacologia , Cinética , Linfoma de Células T , Camundongos , Camundongos Endogâmicos BALB C , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologia , Proteínas Recombinantes/metabolismo , Retroviridae , Transfecção/métodos , Células Tumorais CultivadasAssuntos
Proteínas Heterotriméricas de Ligação ao GTP/genética , Alanina , Sequência de Aminoácidos , Linhagem Celular , Escherichia coli/genética , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Rim , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Subunidades Proteicas , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Transformação BacterianaRESUMO
The G protein beta(5) subunit differs from other beta subunits in having divergent sequence and subcellular localization patterns. Although beta(5)gamma(2) modulates effectors, beta(5) associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate beta(5) complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 gamma subunits and RGS7 to compete for interaction with beta(5). Among the gamma subunits, beta(5) interacted preferentially with gamma(2), followed by gamma(7), and efficacy of phospholipase C-beta2 activation correlated with amount of beta(5)gamma complex formation. beta(5) also slightly preferred gamma(2) over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), beta(5) interacted similarly with gamma(2) and RGS7. Moreover, gamma(2) interacted preferentially with beta(1) rather than beta(5). These results suggest that multiple coexpressed proteins influence beta(5) complex formation. Fluorescent beta(5)gamma(2) labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas beta(5)RGS7 stained the cytoplasm diffusely. Coexpression of alpha(o) targeted both beta(5) complexes to the plasma membrane, and alpha(q) also targeted beta(5)gamma(2) to the plasma membrane. The constitutively activated alpha(o) mutant, alpha(o)R179C, produced greater targeting of beta(5)RGS7 and less of beta(5)gamma(2) than did alpha(o). These results suggest that alpha(o) may cycle between interactions with beta(5)gamma(2) or other betagamma complexes when inactive, and beta(5)RGS7 when active. Moreover, the ability of beta(5)gamma(2) to be targeted to the plasma membrane by alpha subunits suggests that functional beta(5)gamma(2) complexes can form in intact cells and mediate signaling by G protein-coupled receptors.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Ativação Enzimática , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Fosfolipase C beta/metabolismo , Ligação Proteica , Transdução de Sinais , Frações Subcelulares/metabolismoRESUMO
The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Medições Luminescentes/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Dimerização , Fluorescência , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
To analyze individual steps of G(S)-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor G(S) activation in living cells. This was done by coexpressing a Galpha(s) mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gbetagamma subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the G(S) signaling cascade. When A(2A)-adenosine or beta(1)-adrenergic receptors are coexpressed with G(S) in HEK293T cells, the receptor-G(S) interaction was on the same time scale as A(2A) receptor activation with a time constant of <50 ms. G(S) activation was markedly slower and around 450 ms with similar kinetics following activation of A(2A)- or beta(1)-receptors. Taken together, our kinetic measurements demonstrate that the rate of G(S) activation limits initiation of G(S)-coupled receptor signaling.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Transdução de Sinais , Linhagem Celular , AMP Cíclico/metabolismo , Ativação Enzimática , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Cinética , Ligação Proteica , Receptor A2A de Adenosina/genética , Receptores Adrenérgicos beta 1/genética , Fatores de TempoRESUMO
To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative alpha(s) mutant containing substitutions that alter distinct functions was produced and tested for its effects on G(s)-coupled receptor activity in HEK-293 cells. Mutations in the alpha3beta5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for betagamma, and A366S, which decreases affinity for GDP. This triple alpha(s) mutant can inhibit signaling to G(s) from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this alpha(s) mutant blocks all signaling from the calcitonin receptor to G(q). These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein alpha subunits can compete with each other for binding to the same receptor. This dominant negative alpha(s) construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Linhagem Celular , Guanosina Difosfato/metabolismo , Humanos , Mutagênese , Receptores da Calcitonina/antagonistas & inibidores , Receptores da Calcitonina/metabolismo , Receptores de Superfície Celular/genéticaRESUMO
Short-term activation of Galpha(i/o)-coupled receptors inhibits adenylyl cyclase, whereas persistent activation of Galpha(i/o)-coupled receptors results in a compensatory sensitization of adenylyl cyclase activity after subsequent activation by Galpha(s) or forskolin. Several indirect observations have suggested the involvement of increased Galpha(s)-adenylyl cyclase interactions in the expression of sensitization; however, evidence supporting a direct role for Galpha(s) has not been well established. In the present report, we used two genetic approaches to further examine the role of Galpha(s) in heterologous sensitization of Ca(2+)-sensitive type 1 adenylyl cyclase (AC1). In the first approach, we constructed Galpha(s)-insensitive mutants of AC1 (F293L and Y973S) that retained sensitivity to Ca2+ and forskolin activation. Persistent (2 h) activation of the D2 dopamine receptor resulted in a significant augmentation of basal or Ca(2+)- and forskolin-stimulated AC1 activity; however, sensitization of Galpha(s)-insensitive mutants of AC1 was markedly reduced compared with wild-type AC1. In the second strategy, we examined the requirement of an intact receptor-Galpha(s) signaling pathway for the expression of sensitization using dominant-negative Galpha(s) mutants (alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S) to disrupt D1 dopamine receptor activation of recombinant AC1. D1 dopamine receptor-Galpha(s) signaling was attenuated in the presence of alpha3beta5 G226A/A366S or alpha3beta5 G226A/E268A/A366S, but D2 agonist-induced sensitization of Ca(2+)-stimulated AC1 activity was not altered. Together, the present findings directly support the hypothesis that the expression of sensitization of AC1 involves Galpha(s)-adenylyl cyclase interactions.
Assuntos
Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Receptores de Dopamina D2/fisiologia , Substituição de Aminoácidos , Linhagem Celular , Humanos , Rim , Quimpirol/farmacologia , Receptores de Dopamina D2/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Processamento Alternativo , Animais , Proteínas de Bactérias/genética , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Genes Reporter , Isoproterenol/farmacologia , Cinética , Proteínas Luminescentes/genética , Ratos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
To investigate the role of subcellular localization in regulating the specificity of G protein betagamma signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize betagamma dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to beta and a carboxyl-terminal yellow fluorescent protein fragment to gamma. When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on betagamma assembly in that it was not obtained using beta2 and gamma1, which do not form a functional dimer. In addition to assembly, BiFC betagamma complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent beta and gamma subunits and by their abilities to potentiate activation of adenylyl cyclase by alpha(s) in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different beta subunits with three different gamma subunits were compared. BiFC betagamma complexes containing either beta1 or beta2 localized to the plasma membrane, whereas those containing beta5 accumulated in the cytosol or on intracellular membranes. These results indicate that the beta subunit can direct trafficking of the gamma subunit. Taken together with previous observations, these results show that the G protein alpha, beta, and gamma subunits all play roles in targeting each other. This method of specifically visualizing betagamma dimers will have many applications in sorting out roles for particular betagamma complexes in a wide variety of cell types.