RESUMO
ß-Adrenergic receptors (ßAR) and D(2)-like dopamine receptors (which include D(2)-, D(3)- and D(4)-dopamine receptors) activate G(s) and G(i), the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). ß(2)-Adrenergic receptors (ß(2)AR) and D(4)-dopamine receptors (D(4)DR) co-immunoprecipitated when co-expressed in HEK 293 cells, suggesting the existence of a signaling complex containing both receptors. In order to determine if these receptors are closely associated with each other, and with other components involved in G protein-mediated signal transduction, ß(2)AR, D(4)DR, G protein subunits (Gα(i1) and the Gß(1)γ(2) heterodimer) and AC were tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions. All of the tagged proteins retained biological function. For the first time, FlAsH-labeled proteins were used in BRET experiments as fluorescent acceptors for the energy transferred from Renilla luciferase-tagged donor proteins. Our experiments revealed that ß(2)AR, D(4)DR, G proteins and AC were closely associated in a functional signaling complex in cellulo. Furthermore, BRET experiments indicated that although activation of G(i) caused a conformational change within the heterotrimeric protein, it did not cause the Gßγ heterodimer to dissociate from the Gα(i1) subunit. Evidence for the presence of a signaling complex in vivo was obtained by purifying ßAR from detergent extracts of mouse brain with alprenolol-Sepharose and showing that the precipitate also contained both D(2)-like dopamine receptors and AC.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Dopamina D4/metabolismo , Animais , Encéfalo/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Camundongos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores de Dopamina D4/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The role of Gbetagamma subunits in cellular signaling has become well established in the past 20 years. Not only do they regulate effectors once thought to be the sole targets of Galpha subunits, but it has become clear that they also have a unique set of binding partners and regulate signaling pathways that are not always localized to the plasma membrane. However, this may be only the beginning of the story. Gbetagamma subunits interact with G protein-coupled receptors, Galpha subunits, and several different effector molecules during assembly and trafficking of receptor-based signaling complexes and not simply in response to ligand stimulation at sites of receptor cellular activity. Gbetagamma assembly itself seems to be tightly regulated via the action of molecular chaperones and in turn may serve a similar role in the assembly of specific signaling complexes. We propose that specific Gbetagamma subunits have a broader role in controlling the architecture, assembly, and activity of cellular signaling pathways.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Animais , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Humanos , Modelos Teóricos , Receptores Acoplados a Proteínas G/químicaRESUMO
A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Complexos Multiproteicos/análise , Proteínas Recombinantes de Fusão/análise , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bioensaio/métodos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/análise , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Plasmídeos , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Projetos de Pesquisa , Espectrometria de Fluorescência/métodos , TransfecçãoRESUMO
We have previously demonstrated that adenylyl cyclase II (ACII) interacts with beta2-adrenergic receptors and heterotrimeric G proteins as part of a pre-assembled signalling complex. In this study, we further show that AC interacts with these proteins before it is targetted to the cell surface. Using a combination of approaches including bioluminescence resonance energy transfer (BRET) in concert with subcellular fractionation, we show that ACII and beta2AR initially interact in the ER. Further, dominant-negative Rab1 and Sar1 GTPases which block anterograde trafficking out of the ER have no effect on either ACII/receptor or ACII/Gbetagamma protein interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Galpha subunits in ACII signalling complexes suggesting it assembles into the complex at a slightly later stage. Thus, like Kir3.1 inwardly rectifying potassium channels, signalosomes containing ACII are formed during their biosynthesis and not in response to agonist at the cell surface.
Assuntos
Adenilil Ciclases/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/biossíntese , Subunidades beta da Proteína de Ligação ao GTP/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Transdução de Sinais , Sítios de Ligação , Linhagem Celular , Retículo Endoplasmático/metabolismo , Transferência de Energia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/metabolismo , Luminescência , Microscopia de Fluorescência , Ligação Proteica , Receptores Adrenérgicos beta 2/metabolismo , Frações SubcelularesRESUMO
Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Galpha(s), Galpha(i), Gbeta(1) and Ggamma(2)) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gbeta(1)-YFP(1-158) and Ggamma(2)-YFP(159-238), which heterodimerize to produce fluorescent YFP-Gbeta(1)gamma(2)). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Ggamma(2), GFP-Gbeta(1) or YFP-Gbeta(1)gamma(2). Galpha subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gbetagamma was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.
Assuntos
Adenilil Ciclases/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Bovinos , Células Cultivadas , Dimerização , Transferência Ressonante de Energia de Fluorescência , Reguladores de Proteínas de Ligação ao GTP/agonistas , Proteínas de Ligação ao GTP/agonistas , Humanos , Imunoprecipitação , Proteínas Luminescentes/análise , Oócitos , Ligação Proteica , Subunidades Proteicas/metabolismo , Ratos , Proteínas Recombinantes/análise , Transdução de Sinais/efeitos dos fármacos , XenopusRESUMO
An important goal in cell biology has been to observe dynamic interactions between protein molecules within a living cell as they execute the reactions of a particular biochemical pathway. An important step toward achieving this goal has been the development of noninvasive fluorescence-based detection and imaging techniques for determining whether and when specific biomolecules in a cell become associated with one another. Furthermore, these techniques, which take advantage of phenomena known as bioluminescence- and fluorescence resonance energy transfer (BRET and FRET, respectively) as well as bimolecular fluorescence complementation (BiFC), can provide information about where and when protein-protein interactions occur in the cell. Increasingly BRET, FRET, and BiFC are being used to probe interactions between components involved in G protein- mediated signal transduction. Heptahelical (7TM) receptors, heterotrimeric guanine nucleotide binding proteins (G proteins) and their proximal downstream effectors constitute the core components of these ubiquitous signaling pathways. Signal transduction is initiated by the binding of agonist to heptahelical (7TM) receptors that in turn activate their cognate G proteins. The activated G protein subsequently regulates the activity of specific effectors. 7TM receptors, G proteins, and effectors are all membrane-associated proteins, and for decades two opposing hypotheses have vied for acceptance. The predominant hypothesis has been that these proteins move about independently of one another in membranes and that signal transduction occurs when they encounter each other as the result of random collisions. The contending hypothesis is that signaling is propagated by organized complexes of these proteins. Until recently, the data supporting these hypotheses came from studying signaling proteins in solution, in isolated membranes, or in fixed cells. Although the former hypothesis has been favored, recent studies using BRET and FRET have generally supported the latter hypothesis as being the most likely scenario operating in living cells. In addition to the core components, there are many other proteins involved in G protein signaling, and BRET and FRET studies have been used to investigate their interactions as well. This review describes various BRET, FRET, and BiFC techniques, how they have been or can be applied to the study of G protein signaling, what caveats are involved in interpreting the results, and what has been learned about G protein signaling from the published studies.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Fluorescência , Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Fenômenos Fisiológicos Celulares , Luciferases/metabolismo , Medições Luminescentes , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
G protein-coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Galpha-, Gbeta- and Ggamma-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Galphabetagamma-complexes (t(1/2) approximately 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gbetagamma interaction was highly dependent on the identity of the Galpha-subunit present in the complex. Therefore, this G protein-activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Ligação ao GTP/metabolismo , Rim/metabolismo , Medições Luminescentes/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Sistemas Computacionais , Corantes Fluorescentes , Humanos , Proteínas LuminescentesRESUMO
Signal transduction mediated by heterotrimeric G proteins that couple to heptahelical receptors requires the involvement of many different proteins. Although some of the early evidence suggested that signal transduction components were assembled into complexes, much of the data supported an alternative hypothesis positing that the process involved transient interactions driven by random collision events. However, recent data indicate that many of the components involved in signal transduction do indeed form complexes. Here we review the evidence for these complexes and how they contribute to the specificity and efficiency of signaling in cells that must manage numerous signal transduction pathways.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/fisiologia , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.