Bioluminescence Resonance Energy Transfer Research Articles

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Ready-to-use ratiometric bioluminescence immunosensor for detection of ochratoxin A in pepper

Rapid, portable, and accurate detection tools for monitoring ochratoxin A (OTA) in food are essential for the guarantee of food safety and human health. Herein, as a proof-of-concept, this study proposed a ratiometric bioluminescence immunosensor (RBL-immunosensor) for homogeneous detection of OTA in pepper. The construct of the RBL-immunosensor consists of three components, including the large fragment of the split nanoluciferase (NanoLuc)-tagged nanobody (NLg), the small fragment of the split NanoLuc-tagged mimotope peptide heptamer (MPSm), and the calibrator luciferase (GeNL). The specific nanobody-mimotope peptide interaction between NLg and MPSm induces the reconstitution of the NanoLuc, which catalyzes the Nano-Glo substrate and produces a blue emission peak at 458 nm. Meanwhile, GeNL can produce a green emission peak at 518 nm upon substrate conversion via bioluminescent resonance energy transfer (BRET). Therefore, the concentration of OTA can be linked to the variation of the bioluminescence signal (λ458/λ518) measured by microplate reader and the variation of the blue/green ratio measured by smartphone via the competitive immunoreaction where OTA competes with MPSm to bind NLg. The immunosensor is ready-to-use and works by simply mixing the components in a one-step incubation of 10 min for readout. It has a limit of detection (LOD) of 0.98 ng/mL by a microplate reader and an LOD of 1.89 ng/mL by a smartphone. Good selectivity and accuracy were confirmed for the immunosensor by cross-reaction analysis and recovery experiments. The contents of OTA in 10 commercial pepper powder samples were tested by the RBL-immunosensor and validated by high-performance liquid chromatography. Hence, the ready-to-use RBL-immunosensor was demonstrated as a highly reliable tool for detection of OTA in food.

Glycoprotein-glycoprotein Receptor Binding Detection Using Bioluminescence Resonance Energy Transfer.

The glycoprotein receptors, members of the large G protein-coupled receptor family, are characterized by a large extracellular domains responsible for binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays, most often using radiolabeling but also by thermal shift assays. Despite their high sensitivity, these assays require appropriate laboratory conditions and, often, purified plasma cell membranes, which do not provide information on receptor localization or activity because the assays typically focus on measuring binding only. Here, we apply bioluminescence resonance energy transfer in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc)-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (human chorionic gonadotropin or LH) fused to the enhanced green fluorescent protein. The Gluc-LHCGR, as well as other Gluc-G protein-coupled receptors such as the somatostatin and the C-X-C motif chemokine receptors, is expressed on the plasma membrane, where luminescence activity is equal to membrane receptor expression, and is fully functional. The chimeric enhanced green fluorescent protein-ligands are properly secreted from cells and able to bind and activate the wild-type LHCGR as well as the Gluc-LHCGR. Finally, bioluminescence resonance energy transfer was used to determine the interactions between clinically relevant mutations of the hormones and the LHCGR that show that this bioassay provides a fast and effective, safe, and cost-efficient tool to assist the molecular characterization of mutations in either the receptor or ligand and that it is compatible with downstream cellular assays to determine receptor activation/function.

Membrane Heteroreceptor Complexes as Second-Order Protein Modulators: A Novel Integrative Mechanism through Allosteric Receptor–Receptor Interactions

Bioluminescence and fluorescence resonance energy transfer (BRET and FRET) together with the proximity ligation method revealed the existence of G-protein-coupled receptors, Ionotropic and Receptor tyrosine kinase heterocomplexes, e.g., A2AR–D2R, GABAA–D5R, and FGFR1–5-HT1AR heterocomplexes. Molecular integration takes place through allosteric receptor–receptor interactions in heteroreceptor complexes of synaptic and extra-synaptic regions. It involves the modulation of receptor protomer recognition, signaling and trafficking, as well as the modulation of behavioral responses. Allosteric receptor–receptor interactions in hetero-complexes give rise to concepts like meta-modulation and protein modulation. The introduction of receptor–receptor interactions was the origin of the concept of meta-modulation provided by Katz and Edwards in 1999, which stood for the fine-tuning or modulation of nerve cell transmission. In 2000–2010, Ribeiro and Sebastiao, based on a series of papers, provided strong support for their view that adenosine can meta-modulate (fine-tune) synaptic transmission through adenosine receptors. However, another term should also be considered: protein modulation, which is the key feature of allosteric receptor–receptor interactions leading to learning and consolidation by novel adapter proteins to memory. Finally, it must be underlined that allosteric receptor–receptor interactions and their involvement both in brain disease and its treatment are of high interest. Their pathophysiological relevance has been obtained, especially for major depressive disorder, cocaine use disorder, and Parkinson’s disease.

Molecular characterization of GPR84 in domestic cats

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83–89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein–protein interaction with FFAR2/GPR43 on the cell membrane.

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor.

The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HT2AR for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and β-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G protein-selective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.

CXCL17 is an allosteric inhibitor of CXCR4 through a mechanism of action involving glycosaminoglycans.

CXCL17 is a chemokine principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, and its expression is increased during viral infections of the lung. However, the exact role of CXCL17 in health and disease requires further investigation, and there is a need for confirmed molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET)-based assays, here we demonstrated that CXCL17 inhibited CXCR4-mediated signaling and ligand binding. Moreover, CXCL17 interacted with neuropillin-1, a VEGFR2 coreceptor. In addition, we found that CXCL17 only inhibited CXCR4 ligand binding in intact cells and demonstrated that this effect was mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. Disruption of putative GAG binding domains in CXCL17 prevented CXCR4 binding. This indicated that CXCL17 inhibited CXCR4 by a mechanism of action that potentially required the presence of a glycosaminoglycan-containing accessory protein. Together, our results revealed that CXCL17 is an endogenous inhibitor of CXCR4 and represents the next step in our understanding of the function of CXCL17 and regulation of CXCR4 signaling.

Function of mast cell and bile-cholangiocarcinoma interplay in cholangiocarcinoma microenvironment

ObjectiveThe correlation between cholangiocarcinoma (CCA) progression and bile is rarely studied. Here, we aimed to identify differential metabolites in benign and malignant bile ducts and elucidate the generation, function and...

The Expression and Functionality of CB1R-NMDAR Complexes Are Decreased in A Parkinson's Disease Model.

One of the hallmarks of Parkinson's disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB1R). The presence of CB1R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB1R in CB1R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB1R-NMDAR complex in transfected HEK-293T cells. Moreover, α-synuclein treatment induced a decrease in the cAMP and MAP kinase (MAPK) signaling of both CB1R and NMDAR not only in transfected cells but also in neuronal primary cultures. Finally, the interaction between CB1R and NMDAR was studied by Proximity Ligation Assay (PLA) in neuronal primary cultures, where it was observed that the expression of CB1R-NMDAR complexes was decreased upon α-synuclein treatment. These results point to a role of CB1R-NMDAR complexes as a new therapeutic target in Parkinson's disease.

Bioluminescence Resonance Energy Transfer (BRET)-based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells.

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

Attenuation of phytofungal pathogenicity of Ascomycota by autophagy modulators

Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. Here, we describe bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 that is critical for autophagosome formation. We identified ebselen (EB) and its analogs ebselen oxide (EO) and 2-(4-methylphenyl)−1,2-benzisothiazol-3(2H)-one (PT) as inhibitors of fungal pathogens Botrytis cinerea and Magnaporthe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, B. cinerea, M. oryzae, Sclerotinia sclerotiorum and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Our findings provide molecular insights to develop the next generation of antifungal compounds by targeting autophagy in important fungal pathogens.

Mechanistic Insights into the Bacterial Luciferase-based Bioluminescence Resonance Energy Transfer Luminescence: The Role of Protein Complex Dimer.

Bacterial bioluminescence holds significant potential in the realm of optical imaging due to the inherent advantages of bioluminescence and ease of operation. However, its practical utility is hindered by its low light intensity. The fusion of bacterial luciferase with a highly fluorescent protein has been demonstrated to significantly enhance autonomous luminescence. Nevertheless, the underlying mechanism behind this enhancement remains unclear, and there is a dearth of research investigating the mechanistic aspects of bioluminescence resonance energy transfer (BRET) luminescence, whether it occurs naturally or can be achieved through experimental means. In this study, we investigated the phenomenon of bacterial luciferase-based BRET luminescence employing a range of computational techniques, including structural modeling, molecular docking, molecular dynamics simulations, as well as combined quantum mechanics and molecular mechanics calculations. The theoretical findings suggest that the BRET luminescence occurs through resonance energy transfer between the excited bioluminophore and the ground chromophore within the protein complex dimer. The proposed mechanism of the protein complex dimer offers a microscopic understanding of the intriguing BRET phenomenon and has the potential to inspire further practical applications in the field of optical imaging.

Distinct roles of the extracellular surface residues of glucagon-like peptide-1 receptor in β-arrestin 1/2 signaling

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with β-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in β-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated β-arrestin 1/2 recruitment for diverse ligands, and β-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased β-arrestin 1 recruitment but increased β-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected β-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in β-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive β-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in β-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in β-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on β-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without β-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and β-arrestins. Our study offers valuable information about ligand induced β-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

Fine-tuning activation specificity of G-protein-coupled receptors via automated path searching

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.

Activation of Multiple G Protein Pathways to Characterize the Five Dopamine Receptor Subtypes Using Bioluminescence Technology.

G protein-coupled receptors show preference for G protein subtypes but can recruit multiple G proteins with various downstream signaling cascades. This functional selection can guide drug design. Dopamine receptors are both stimulatory (D1-like) and inhibitory (D2-like) with diffuse expression across the central nervous system. Functional selectivity of G protein subunits may help with dopamine receptor targeting and their downstream effects. Three bioluminescence-based assays were used to characterize G protein coupling and function with the five dopamine receptors. Most proximal to ligand binding was the miniG protein assay with split luciferase technology used to measure recruitment. For endogenous and selective ligands, the G-CASE bioluminescence resonance energy transfer (BRET) assay measured G protein activation and receptor selectivity. Downstream, the BRET-based CAMYEN assay quantified cyclic adenosine monophosphate (cAMP) changes. Several dopamine receptor agonists and antagonists were characterized for their G protein recruitment and cAMP effects. G protein selectivity with dopamine revealed potential Gq coupling at all five receptors, as well as the ability to activate subtypes with the "opposite" effects to canonical signaling. D1-like receptor agonist (+)-SKF-81297 and D2-like receptor agonist pramipexole showed selectivity at all receptors toward Gs or Gi/o/z activation, respectively. The five dopamine receptors show a wide range of potentials for G protein coupling and activation, reflected in their downstream cAMP signaling. Targeting these interactions can be achieved through drug design. This opens the door to pharmacological treatment with more selectivity options for inducing the correct physiological events.

Role of G protein-coupled receptor kinases (GRKs) in β2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the β2 -AR, transfection of βARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the β2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant β2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by β2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and β2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between β2 -AR and β-arrestin2 or between β2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to β2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to β2 -AR agonists occurred in CHO-GLUT4myc cells expressing β2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type β2 -AR. However, β2 -ARs lacking phosphorylation sites failed to recruit β-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the β2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

Evidence for heterodimerization and functional interaction of the urotensin II and the angiotensin II type 1 receptors

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.

System for Self-excited Targeted Photodynamic Therapy Based on the Multimodal Protein DARP-NanoLuc-SOPP3.

Despite the significant potential of photodynamic therapy (PDT) as a minimally invasive treatment modality, the use of this method in oncology has remained limited due to two serious problems: 1) limited penetration of the excitation light in tissues, which makes it impossible to affect deep-seated tumors and 2) use of chemical photosensitizers that slowly degrade in the body and cause photodermatoses and hyperthermia in patients. To solve these problems, we propose a fully biocompatible targeted system for PDT that does not require an external light source. The proposed system is based on bioluminescent resonance energy transfer (BRET) from the oxidized form of the luciferase substrate to the photosensitizing protein SOPP3. The BRET-activated system is composed of the multimodal protein DARP-NanoLuc-SOPP3, which contains a BRET pair NanoLuc-SOPP3 and a targeting module DARPin. The latter provides the interaction of the multimodal protein with tumors overexpressing tumor-associated antigen HER2 (human epidermal growth factor receptor type II). In vitro experiments in a 2D monolayer cell culture and a 3D spheroid model have confirmed HER2-specific photo-induced cytotoxicity of the system without the use of an external light source; in addition, experiments in animals with subcutaneous HER2-positive tumors have shown selective accumulation of DARP-NanoLuc-SOPP3 on the tumor site. The fully biocompatible system for targeted BRET-induced therapy proposed in this work makes it possible to overcome the following limitations: 1) the need to use an external light source and 2) the side phototoxic effect from aberrant accumulation of chemical photosensitizers. The obtained results demonstrate that the fully protein-based self-excited BRET system has a high potential for targeted PDT.

G protein-coupled estrogen receptor (GPER)/GPR30 forms a complex with the β1-adrenergic receptor, a membrane-associated guanylate kinase (MAGUK) scaffold protein, and protein kinase A anchoring protein (AKAP) 5 in MCF7 breast cancer cells

G protein-coupled receptor 30 (GPR30), also named G protein-coupled estrogen receptor (GPER), and the β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCR) that are implicated in breast cancer progression. Both receptors contain PSD-95/Discs-large/ZO-1 homology (PDZ) motifs in their C-terminal tails through which they interact in the plasma membrane with membrane-associated guanylate kinase (MAGUK) scaffold proteins, and in turn protein kinase A anchoring protein (AKAP) 5. GPR30 constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. We hypothesized that this inhibition is a consequence of a plasma membrane complex of these receptors. Using co-immunoprecipitation, confocal immunofluorescence microscopy, and bioluminescence resonance energy transfer (BRET), we show that GPR30 and β1AR reside in close proximity in a plasma membrane complex when transiently expressed in HEK293. Deleting the GPR30 C-terminal PDZ motif (-SSAV) does not interfere with the receptor complex, indicating that the complex is not PDZ-dependent. MCF7 breast cancer cells express GPR30, β1AR, MAGUKs, and AKAP5 in the plasma membrane, and co-immunoprecipitation revealed that these proteins exist in close proximity also under native conditions. Furthermore, expression of GPR30 in MCF7 cells constitutively and PDZ-dependently inhibits β1AR-mediated cAMP production. AKAP5 also inhibits β1AR-mediated cAMP production, which is not additive with GPR30-promoted inhibition. These results argue that GPR30 and β1AR form a PDZ-independent complex in MCF7 cells through which GPR30 constitutively and PDZ-dependently inhibits β1AR signaling via receptor interaction with MAGUKs and AKAP5.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, β2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of β2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.