Bioluminescence Resonance Energy Transfer Experiments Research Articles

Carfentanil is a β-arrestin-biased agonist at the μ opioid receptor.

The illicit use of fentanyl-like drugs (fentanyls), which are μ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls. For agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with μ opioid receptors, to assess Gi protein activation and β-arrestin 2 recruitment. Agonist-induced cell surface receptor loss was assessed using an enzyme-linked immunosorbent assay, whilst agonist-induced G protein-coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the μ opioid receptor were determined in silico using molecular dynamics simulations. Relative to the reference ligand DAMGO, carfentanil was β-arrestin-biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein-coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias. Carfentanil is a β-arrestin-biased opioid drug at the μ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

Heterobivalent Ligand for the Adenosine A2A–Dopamine D2 Receptor Heteromer

A G protein-coupled receptor heteromer that fulfills the established criteria for its existence in vivo is the complex between adenosine A2A (A2AR) and dopamine D2 (D2R) receptors. Here, we have designed and synthesized heterobivalent ligands for the A2AR-D2R heteromer with various spacer lengths. The indispensable simultaneous binding of these ligands to the two different orthosteric sites of the heteromer has been evaluated by radioligand competition-binding assays in the absence and presence of specific peptides that disrupt the formation of the heteromer, label-free dynamic mass redistribution assays in living cells, and molecular dynamic simulations. This combination of techniques has permitted us to identify compound 26 [KDB1 (A2AR) = 2.1 nM, KDB1 (D2R) = 0.13 nM], with a spacer length of 43-atoms, as a true bivalent ligand that simultaneously binds to the two different orthosteric sites. Moreover, bioluminescence resonance energy transfer experiments indicate that 26 favors the stabilization of the A2AR-D2R heteromer.

In Vivo Assessment of Protein-Protein Interactions Using BRET Assay.

Proteins play an important part in almost all life activities and across all organisms. Proteins occasionally act on their own but rather fulfill most of their biological tasks by cooperating with other proteins or ligand molecules. The bioluminescence resonance energy transfer (BRET) assay serves to measure dynamic events such as protein-protein or protein-ligand interactions in vitro or in-vivo. With several inherent attributes such as rapid and fairly sensitive ratio-metric measurements, assessment of interactions irrespective of protein location within the cellular compartment, cost-effectiveness consenting to high-throughput screening compatibility, makes BRET a popular genetic reporter-based assay system for protein-protein interaction (PPI) studies. Based on the Förster principle, BRET allows to judge if the proximity has been achieved between the interacting partners. In recent years, the BRET application has emerged as a significantly versatile assay format by using multiple detection devices such as a plate reader or in-vivo optical imaging platform, or even a bioluminescence microscope has expanded its scope for advancing PPI studies. Beyond the scope of quantitative measurement of PPIs, molecular optical imaging applications based on BRET assay have expanded the scope for screening pharmacological compounds by unifying live cell and in-vivo animal-/plant-based experiments using the same platform technology. In this chapter, we have given intricate methodological details for performing in-vitro and in-vivo BRET experiments, primarily by using donor/acceptor reporter protein combinations.

Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, β, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1β, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

The GTPase-activating protein-related domain of neurofibromin interacts with MC1R and regulates pigmentation-mediated signaling in human melanocytes

The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor (GPCR) which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signaling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signaling, two pathways involved in MC1R signaling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R. In particular, the GAP domain of Nf1 directly and constitutively interacts with MC1R in melanocytes. Pharmacologic and genetic approaches revealed that the GAP activity of Nf1 is important to regulate intracellular signaling pathways involved in melanogenesis and, consequently, melanogenic enzyme expression and melanin production. These finding shed new light on the understanding and cure of skin pigmentation disorders.

Mapping the Interface of a GPCR Dimer: A Structural Model of the A2A Adenosine and D2 Dopamine Receptor Heteromer.

The A2A adenosine (A2AR) and D2 dopamine (D2R) receptors form oligomers in the cell membrane and allosteric interactions across the A2AR–D2R heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of A2AR–D2R heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the A2AR–D2R heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on A2AR–D2R receptor–receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the A2AR blocked heterodimer interactions and disrupted the allosteric effect of A2AR activation on D2R agonist binding. Protein–protein docking was used to construct a model of the A2AR–D2R heterodimer with a TM-IV/V interface, which was refined using molecular dynamics simulations. Mutations in the predicted interface reduced A2AR–D2R interactions in BRET experiments and altered the allosteric modulation. The heterodimer model provided insights into the structural basis of allosteric modulation and the technique developed to characterize the A2AR–D2R interface can be extended to study the many other G protein-coupled receptors that engage in heteroreceptor complexes.

The proximity-labeling technique BioID identifies sorting nexin 6 as a member of the insulin-like growth factor 1 (IGF1)–IGF1 receptor pathway

The insulin-like growth factor 1 receptor (IGF1R) is a receptor tyrosine kinase with critical roles in various biological processes. Recent results from clinical trials targeting IGF1R indicate that IGF1R signaling pathways are more complex than previously thought. Moreover, it has become increasingly clear that the function of many proteins can be understood only in the context of a network of interactions. To that end, we sought to profile IGF1R-protein interactions with the proximity-labeling technique BioID. We applied BioID by generating a HEK293A cell line that stably expressed the BirA* biotin ligase fused to the IGF1R. Following stimulation by IGF1, biotinylated proteins were analyzed by MS. This screen identified both known and previously unknown interactors of IGF1R. One of the novel interactors was sorting nexin 6 (SNX6), a protein that forms part of the retromer complex, which is involved in intracellular protein sorting. Using co-immunoprecipitation, we confirmed that IGF1R and SNX6 physically interact. SNX6 knockdown resulted in a dramatic diminution of IGF1-mediated ERK1/2 phosphorylation, but did not affect IGF1R internalization. Bioluminescence resonance energy transfer experiments indicated that the SNX6 knockdown perturbed the association between IGF1R and the key adaptor proteins insulin receptor substrate 1 (IRS1) and SHC adaptor protein 1 (SHC1). Intriguingly, even in the absence of stimuli, SNX6 overexpression significantly increased Akt phosphorylation. Our study confirms the utility of proximity-labeling methods, such as BioID, to screen for interactors of cell-surface receptors and has uncovered a role of one of these interactors, SNX6, in the IGF1R signaling cascade.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle.

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

Identification of insulin-sensitizing molecules acting by disrupting the interaction between the Insulin Receptor and Grb14

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.

Development of an Acrylate Derivative Targeting the NLRP3 Inflammasome for the Treatment of Inflammatory Bowel Disease.

Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1β release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.

Use of BRET to Study Protein-Protein Interactions In Vitro and In Vivo.

Application of bioluminescence resonance energy transfer (BRET) assay has been of special value in measuring dynamic events such as protein-protein interactions (PPIs) in vitro or in vivo. It was only in the late 1990s the BRET assay using RLuc-YFP was introduced for biological research showing its use in determining interaction of two proteins involved in circadian rhythm. Several inherent attributes such as rapid and fairly sensitive ratiometric measurements, assessment of PPI irrespective of protein location in cellular compartment, and cost-effectiveness consenting to high-throughput assay development make BRET a popular genetic reporter-based assay for PPI studies. In BRET-based screening, within a defined proximity range of 10-100 Å, excited state energy of the luminescence molecule can excite the acceptor fluorophore in the form of resonance energy transfer, causing it to emit at its characteristic emission wavelength. Based on this principle, several such donor-acceptor pairs, using the Renilla luciferase or its mutants as donor and either GFP2, YFP, mOrange, TagRFP, or TurboFP as acceptor, have been reported for use.In recent years, BRET-related research has become significantly versatile in the assay format and its applicability by adopting the assay on multiple detection devices such as small-animal optical imaging platform or bioluminescence microscope. Beyond the scope of quantitative measurement of PPIs and protein dimerization, molecular optical imaging applications based on BRET assays have broadened its scope for screening of pharmacological compounds by unifying in vitro, live cell, and in vivo animal/plant measurement all on one platform. Taking examples from the literature, this chapter contributes to in-depth methodological details on how to perform in vitro and in vivo BRET experiments, and illustrates its advantages as a single-format assay.

The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

A proteomic analysis reveals the interaction of GluK1 ionotropic kainate receptor subunits with Go proteins.

Kainate receptors (KARs) are found ubiquitously in the CNS and are present presynaptically and postsynaptically regulating synaptic transmission and excitability. Functional studies have proven that KARs act as ion channels as well as potentially activating G-proteins, thus indicating the existance of a dual signaling system for KARs. Nevertheless, it is not clear how these ion channels activate G-proteins and which of the KAR subunits is involved. Here we performed a proteomic analysis to define proteins that interact with the C-terminal domain of GluK1 and we identified a variety of proteins with many different functions, including a Go α subunit. These interactions were verified through distinct in vitro and in vivo assays, and the activation of the Go protein by GluK1 was validated in bioluminescence resonance energy transfer experiments, while the specificity of this association was confirmed in GluK1-deficient mice. These data reveal components of the KAR interactome, and they show that GluK1 and Go proteins are natural partners, accounting for the metabotropic effects of KARs.

Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.

Application of BRET for studying G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute one of the largest classes of cell surface receptors. GPCR biology has been a subject of widespread interest owing to the functional relevance of these receptors and their potential importance in the development of new drugs. At present, over 30% of all launched drugs target these receptors. GPCRs have been considered for a long time to function as monomeric entities and the idea of GPCR dimerization and oligomerization was initially accepted with disbelief. However, a significant amount of experimental and molecular modeling evidence accumulated during the last several years suggests that the process of GPCRs dimer or oligomer formation is a general phenomenon, in some cases even essential for receptor function. Among the many methods to study GPCR dimerization and oligomerization, modern biophysical techniques such as those based on resonance energy transfer (RET) and particularly bioluminescence resonance energy transfer (BRET) have played a leading role. RET methods are commonly applied as non-destructive indicators of specific protein-protein interactions (PPIs) in living cells. Data from numerous BRET experiments support the idea that the process of GPCR oligomerization may be relevant in many physiological and pathological conditions. The application of BRET to the study of GPCRs is not only limited to the assessment of receptor oligomerization but also expands to the investigation of the interactions of GPCRs with other proteins, including G proteins, G protein-coupled receptor kinases, β-arrestins or receptor tyrosine kinases, as well as to the characterization of GPCR activation and signaling. In this review, we briefly summarize the fundaments of BRET, discuss new trends in this technology and describe the wide range of applications of BRET to study GPCRs.

Label-free monitoring of μ-opioid receptor-mediated signaling.

In this study, we used a combination of traditional signaling investigation approaches, bioluminescence resonance energy transfer (BRET) biosensors, and the label-free approach surface plasmon resonance (SPR) spectroscopy to monitor the signaling cascades of the μ-opioid receptor (MOP). In human embryonic kidney cells stably expressing a Flag-tagged version of human MOP, we compared the signals triggered by the noninternalizing and internalizing MOP agonists morphine and DAMGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), respectively. We studied three major and well described components of MOP signaling: receptor internalization, G protein coupling, and activation of extracellular signal-regulated kinase ERK1/ERK2. Our results show that morphine and DAMGO display different profiles of receptor internalization and a similar ability to trigger the phosphorylation of ERK1/ERK2. Our SPR analyses revealed that morphine and DAMGO evoke similar SPR signatures and that Gαi, cAMP-dependent pathways, and ERK1/ERK2 have key roles in morphine- and DAMGO-mediated signaling. Most interestingly, we found that the so-called MOP neutral antagonists CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2)), naloxone, and naltrexone behave like partial agonists. Even more intriguing, BRET experiments indicate that CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) induces similar conformational changes as naltrexone at the Gαi-βγ interface, whereas it appears as an inverse agonist based on its SPR response thus indicating distinct signaling mechanisms for the two ligands. Taken together, our results support the usefulness of label-free methods such as SPR to study whole-cell responses and signaling cascades triggered by G protein-coupled receptors and complement the conventional approaches by revealing cellular responses that would have been otherwise undetectable.

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

A novel approach to quantify G-protein-coupled receptor dimerization equilibrium using bioluminescence resonance energy transfer.

Along with other resonance energy transfer techniques, bioluminescence resonance energy transfer (BRET) has emerged as an important method for demonstrating protein-protein interactions in cells. In the field of G-protein-coupled receptors, including chemokine receptors, BRET has been widely used to investigate homo- and heterodimerization, a feature of their interactions that is emerging as integral to function and regulation. While demonstrating the existence of dimers for a given receptor proved to be fairly straightforward, quantitative comparisons of different receptors or mutants are nontrivial because of inevitable variations in the expression of receptor constructs. The uncontrollable parameters of the cellular expression machinery make amounts of transfected DNA extremely poor predictors for the expression levels of BRET donor and acceptor receptor constructs, even in relative terms. In this chapter, we show that properly accounting for receptor expression levels is critical for quantitative interpretation of BRET data. We also provide a comprehensive account of expected responses in all types of BRET experiments and propose a framework for uniform and accurate quantitative treatment of these responses. The framework allows analysis of both homodimer and heterodimer BRET data. The important caveats and obstacles for quantitative treatment are outlined, and the utility of the approach is illustrated by its application to the homodimerization of wild-type (WT) and mutant forms of the chemokine receptor CXCR4.

D2-like dopamine and β-adrenergic receptors form a signaling complex that integrates Gs- and Gi-mediated regulation of adenylyl cyclase

β-Adrenergic receptors (βAR) and D2-like dopamine receptors (which include D2-, D3- and D4-dopamine receptors) activate Gs and Gi, the stimulatory and inhibitory heterotrimeric G proteins, respectively, which in turn regulate the activity of adenylyl cyclase (AC). β2-Adrenergic receptors (β2AR) and D4-dopamine receptors (D4DR) 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, β2AR, D4DR, G protein subunits (Gαi1 and the Gβ1γ2 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 β2AR, D4DR, G proteins and AC were closely associated in a functional signaling complex in cellulo. Furthermore, BRET experiments indicated that although activation of Gi caused a conformational change within the heterotrimeric protein, it did not cause the Gβγ heterodimer to dissociate from the Gαi1 subunit. Evidence for the presence of a signaling complex in vivo was obtained by purifying βAR from detergent extracts of mouse brain with alprenolol–Sepharose and showing that the precipitate also contained both D2-like dopamine receptors and AC.

Mapping Human Protease-activated Receptor 4 (PAR4) Homodimer Interface to Transmembrane Helix 4

Thrombin activates platelets by binding and cleaving protease-activated receptors 1 and 4 (PAR1 and PAR4). Because of the importance of PAR4 activation on platelets in humans and mice and emerging roles for PAR4 in other tissues, experiments were done to characterize the interaction between PAR4 homodimers. Bimolecular fluorescence complementation and bioluminescence resonance energy transfer (BRET) were used to examine the PAR4 homodimer interface. In bimolecular fluorescence complementation experiments, PAR4 formed homodimers that were disrupted by unlabeled PAR4 in a concentration-dependent manner, but not by rhodopsin. In BRET experiments, the PAR4 homodimers showed a specific interaction as indicated by a hyperbolic BRET signal in response to increasing PAR4-GFP expression. PAR4 did not interact with rhodopsin in BRET assays. The threshold maximum BRET signal was disrupted in a concentration-dependent manner by unlabeled PAR4. In contrast, rhodopsin was unable to disrupt the BRET signal, indicating that the disruption of the PAR4 homodimer is not due to nonspecific interactions. A panel of rho-PAR4 chimeras and PAR4 point mutants has mapped the dimer interface to hydrophobic residues in transmembrane helix 4. Finally, mutations that disrupted dimer formation had reduced calcium mobilization in response to the PAR4 agonist peptide. These results link the loss of dimer formation to a loss of PAR4 signaling.