RESUMO
Despite the observation of synergistic interactions between the urotensinergic and angiotensinergic systems, the interplay between the urotensin II receptor (hUT) and the angiotensin II type 1 receptor (hAT1R) in regulating cellular signaling remains incompletely understood. Notably, the putative interaction between hUT and hAT1R could engender reciprocal allosteric modulation of their signaling signatures, defining a unique role for these complexes in cardiovascular physiology and pathophysiology. Using a combination of co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and FlAsH BRET-based conformational biosensors, we first demonstrated the physical interaction between hUT and hAT1R. Next, to analyze how this functional interaction regulated proximal and distal hUT- and hAT1R-associated signaling pathways, we used BRET-based signaling biosensors and western blots to profile pathway-specific signaling in HEK 293 cells expressing hUT, hAT1R or both. We observed that hUT-hAT1R heterodimers triggered distinct signaling outcomes compared to their respective parent receptors alone. Notably, co-transfection of hUT and hAT1R has no impact on hUII-induced Gq activation but significantly reduced the potency and efficacy of Ang II to mediate Gq activation. Interestingly, URP, the second hUT endogenous ligand, produce a distinct signaling signature compared to hUII at hUT-hAT1R. Our results therefore suggest that assembly of hUT with hAT1R might be important for allosteric modulation of outcomes associated with specific hardwired signaling complexes in healthy and disease states. Altogether, our work, which potentially explains the interplay observed in native cells and tissues, validates such complexes as potential targets to promote the design of compounds that can modulate heterodimer function selectively.
Assuntos
Receptor Tipo 1 de Angiotensina , Urotensinas , Humanos , Angiotensina II , Células HEK293RESUMO
Initially identified as monomers, G protein-coupled receptors (GPCRs) can also form functional homo- and heterodimers that act as distinct signaling hubs for cellular signal integration. We previously found that the angiotensin II (Ang II) type 1 receptor (AT1R) and the prostaglandin F2α (PGF2α) receptor (FP), both important in the control of smooth muscle contractility, form such a functional heterodimeric complex in HEK 293 and vascular smooth muscle cells. Here, we hypothesize that both Ang II- and PGF2α-induced activation of the AT1R/FP dimer, or the parent receptors alone, differentially regulate signaling by distinct patterns of ß-arrestin recruitment. Using BRET-based biosensors, we assessed the recruitment kinetics of ß-arrestin1/2 to the AT1R/FP dimer, or the parent receptors alone, when stimulated by either Ang II or PGF2α. Using cell lines with CRISPR/Cas9-mediated gene deletion, we also examined the role of G proteins in such recruitment. We observed that Ang II induced a rapid, robust, and sustained recruitment of ß-arrestin1/2 to AT1R and, to a lesser extent, the heterodimer, as expected, since AT1R is a strong recruiter of both ß-arrestin subtypes. However, PGF2α did not induce such recruitment to FP alone, although it did when the AT1R is present as a heterodimer. ß-arrestins were likely recruited to the AT1R partner of the dimer. Gαq, Gα11, Gα12, and Gα13 were all involved to some extent in PGF2α-induced ß-arrestin1/2 recruitment to the dimer as their combined absence abrogated the response, and their separate re-expression was sufficient to partially restore it. Taken together, our data sheds light on a new mechanism whereby PGF2α specifically recruits and signals through ß-arrestin but only in the context of the AT1R/FP dimer, suggesting that this may be a new allosteric signaling entity.
RESUMO
How G protein-coupled receptors (GPCR) interact with one another remains an area of active investigation. Obligate dimers of class C GPCRs such as metabotropic GABA and glutamate receptors are well accepted, although whether this is a general feature of other GPCRs is still strongly debated. In this review, we focus on the idea that GPCR dimers and oligomers are better imagined as parts of larger metastable signalling complexes. We discuss the nature of functional oligomeric entities, their stabilities and kinetic features and how structural and functional asymmetries of such metastable entities might have implications for drug discovery. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.
Assuntos
Regulação Alostérica/fisiologia , Multimerização Proteica/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Descoberta de Drogas , Ligação Proteica/fisiologia , Receptor Cross-Talk , Receptores Acoplados a Proteínas G/fisiologia , Transdução de SinaisRESUMO
The functional importance of G protein-coupled receptor (GPCR) oligomerization remains controversial. Although obligate dimers of class C GPCRs are well accepted, the generalizability of this phenomenon is still strongly debated with respect to other classes of GPCRs. In this review, we focus on understanding the organization and dynamics between receptor equivalents and their signaling partners in oligomeric receptor complexes, with a view toward integrating disparate viewpoints into a unified understanding. We discuss the nature of functional oligomeric entities, and how asymmetries in receptor structure and function created by oligomers might have implications for receptor function as allosteric machines and for future drug discovery.
Assuntos
Multimerização Proteica , Receptores Acoplados a Proteínas G/metabolismo , Regulação Alostérica , Animais , Descoberta de Drogas , Humanos , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) are conformationally dynamic proteins transmitting ligand-encoded signals in multiple ways. This transmission is highly complex and achieved through induction of distinct GPCR conformations, which preferentially drive specific receptor-mediated signaling events. This conformational capacity can be further enlarged via allosteric effects between dimers, warranting further study of these effects. Using GPCR conformation-sensitive biosensors, we investigated allosterically induced conformational changes in the recently reported F prostanoid (FP)/angiotensin II type 1 receptor (AT1R) heterodimer. Ligand occupancy of the AT1R induced distinct conformational changes in FP compared with those driven by PGF2α in bioluminescence resonance energy transfer (BRET)-based FP biosensors engineered with Renilla luciferase (RLuc) as an energy donor in the C-tail and fluorescein arsenical hairpin binder (FlAsH)-labeled acceptors at different positions in the intracellular loops. We also found that this allosteric communication is mediated through Gαq and may also involve proximal (phospholipase C) but not distal (protein kinase C) signaling partners. Interestingly, ß-arrestin-biased AT1R agonists could also transmit a Gαq-dependent signal to FP without activation of downstream Gαq signaling. This transmission of information was specific to the AT1R/FP complex, as activation of Gαq by the oxytocin receptor did not recapitulate the same phenomenon. Finally, information flow was asymmetric in the sense that FP activation had negligible effects on AT1R-based conformational biosensors. The identification of partner-induced GPCR conformations may help identify novel allosteric effects when investigating multiprotein receptor signaling complexes.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Modelos Moleculares , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Prostaglandina/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Regulação Alostérica , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Técnicas Biossensoriais , Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Ligantes , Luciferases de Renilla/química , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/metabolismo , Multimerização Proteica , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/genética , Receptores de Ocitocina/agonistas , Receptores de Ocitocina/química , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/química , Receptores de Prostaglandina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A number of studies have profiled G protein-coupled receptor (GPCR) conformation using fluorescent biaresenical hairpin binders (FlAsH) as acceptors for BRET or FRET. These conformation-sensitive biosensors allow reporting of movements occurring on the intracellular surface of a receptor to investigate mechanisms of receptor activation and function. Here, we generated eight FlAsH-BRET-based biosensors within the sequence of the ß2-adrenergic receptor (ß2AR) and compared agonist-induced responses to the angiotensin II receptor type I (AT1R) and the prostaglandin F2α receptor (FP). Although all three receptors had FlAsH-binding sequences engineered into the third intracellular loops and carboxyl-terminal domain, both the magnitude and kinetics of the BRET responses to ligand were receptor-specific. Biosensors in ICL3 of both the AT1R and FP responded robustly when stimulated with their respective full agonists as opposed to the ß2AR where responses in the third intracellular loop were weak and transient when engaged by isoproterenol. C-tail sensors responses were more robust in the ß2AR and AT1R but not in FP. Even though GPCRs share the heptahelical topology and are expressed in the same cellular background, different receptors have unique conformational fingerprints.
RESUMO
Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or ß-arrestins, we showed that Gαq and Gα11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of ß-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or ß-arrestin.
Assuntos
Receptor Tipo 1 de Angiotensina/química , Angiotensina II/metabolismo , Engenharia Celular/métodos , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Métodos , Conformação Proteica , Transdução de Sinais , beta-Arrestinas/metabolismoRESUMO
Ligand-biased signaling is starting to have significant impact on drug discovery programs in the pharmaceutical industry and has reinvigorated our understanding of pharmacological efficacy. As such, many investigators and screening campaigns are now being directed at a larger section of the signaling responses downstream of an individual G protein-coupled receptor. Many biosensor-based platforms have been developed to capture signaling signatures. Despite our growing ability to use such signaling signatures, we remain hampered by the fact that signaling signatures may be particular to an individual cell type and thus our platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. Here, we provide a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin arsenical dyes engineered into different positions in intracellular loop 3 of FP, the receptor for PGF2α. We discuss the design and optimization of such sensors for orthosteric and allosteric ligands.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Receptores Acoplados a Proteínas G/química , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/metabolismo , Sequência de Aminoácidos , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Luciferases de Renilla/síntese química , Luciferases de Renilla/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptor A2A de Adenosina/análise , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle we have worked with Kir3 neuronal channels producing 10 different constructs which were combined into 17 donor-acceptor BRET pairs. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the interfacial helix (N70) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation with direct channel ligands or via stimulation of G protein-coupled receptors. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH-BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Conformational biosensors as the ones reported herein should prove a valuable complement to other methodologies currently used in channel drug discovery.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Desenho de Fármacos , Fluoresceína/síntese química , Fluoresceína/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Analgésicos Opioides/síntese química , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacologia , Animais , Relação Dose-Resposta a Droga , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Camundongos , Conformação ProteicaRESUMO
The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure.
Assuntos
Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Prostaglandina/metabolismo , Regulação Alostérica/fisiologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Ratos , Transdução de Sinais/fisiologiaRESUMO
The role of Gßγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gßγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gßγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gß1γ2, apart from its role in channel opening that it shares with other Gßγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gß1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gßγ effects on Kir3 maturation and trafficking.
RESUMO
Gßγ subunits from heterotrimeric G proteins perform a vast array of functions in cells with respect to signaling, often independently as well as in concert with Gα subunits. However, the eponymous term "Gßγ" does not do justice to the fact that 5 Gß and 12 Gγ isoforms have evolved in mammals to serve much broader roles beyond their canonical roles in cellular signaling. We explore the phylogenetic diversity of Gßγ subunits with a view toward understanding these expanded roles in different cellular organelles. We suggest that the particular content of distinct Gßγ subunits regulates cellular activity, and that the granularity of individual Gß and Gγ action is only beginning to be understood. Given the therapeutic potential of targeting Gßγ action, this larger view serves as a prelude to more specific development of drugs aimed at individual isoforms.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas , Animais , Sítios de Ligação , Descoberta de Drogas , Subunidades beta da Proteína de Ligação ao GTP/química , 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/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Modelos Moleculares , Organelas/efeitos dos fármacos , Organelas/metabolismo , Filogenia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade da EspécieRESUMO
Kir3 channels are essential regulators of cellular excitability, maintaining cells at resting membrane potentials. While much research has been dedicated to elucidating the mechanisms regulating Kir3 channel gating, little is known regarding the channel's early associations with signaling partners, its stability at the plasma membrane or mechanisms regulating its internalization and degradation. To address these issues we have established an inducible Kir3.1 cell line that allows monitoring of a discrete "pulse" of channel as it progresses along the biosynthetic pathway. Using this system, we have been able to track Kir3 maturation and the influence of partner subunits on Kir3 lifetime and stability. Of note, we show that Kir3.1, in the absence of trafficking partner subunits, can exit the endoplasmic reticulum (ER) and reach the Golgi (though not the plasma membrane), and that expression of Kir3.3 subunits drastically reduced levels of Kir3.1 in the cell. We also show that interfering with trafficking from the ER to Golgi has a pronounced inhibitory effect on Kir3.1-Kir3.2 interactions, suggesting that this complex is stabilized either en route to the Golgi or in the Golgi itself. Finally, we showed that the Kir3 channel can reach the cell surface as early as 6 h post-induction and that removal of cell surface-localized channel occurs within 48 h. This system can be adapted to study the life cycle of any cellular protein without the confounds associated with radioactive labeling or the complications noted with expressing supraphysiological levels of proteins.
Assuntos
Retículo Endoplasmático/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais da Membrana/genética , Transdução de Sinais , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Regulação da Expressão Gênica , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/genética , Subunidades Proteicas/metabolismo , Transporte ProteicoRESUMO
This study assessed how conformational information encoded by ligand binding to δ-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gßγ, and Kir3 subunits constitutively interacted with one another. GαoA associated with DORs and Gßγ, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GαoA, DOR-Gßγ, GαoA-Gßγ, and Gßγ-Kir3 interfaces. Ligand-induced BRET changes assessing Gßγ-Kir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GαoA-Gßγ interface, 2) displayed the same order of efficacy as those observed at the DOR-Gßγ interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gßγ/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gßγ-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gßγ with respect to DORs, GαoA, and channel subunits. Further, the fact that BRET changes at the Gßγ-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.