ORCID Profile
0000-0001-7990-371X
Current Organisations
University of Western Australia
,
The Harry Perkins Institute of Medical Research
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Publisher: Portland Press Ltd.
Date: 26-08-2021
DOI: 10.1042/BST20201110
Abstract: Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the in idual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.
Publisher: Frontiers Media SA
Date: 26-05-0010
DOI: 10.3389/FENDO.2022.848816
Abstract: The angiotensin type 2 (AT 2 ) receptor and the bradykinin type 2 (B 2 ) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT 2 receptor and the B 2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gα q , GRK2 and β-arrestin2 recruitment proximal to AT 2 receptors only occurred upon B 2 receptor coexpression and activation, all of which is indicative of AT 2 -B 2 receptor heteromerization. Additionally, we also observed specific coupling of the B 2 receptor with the Gα z protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gα z , Gα q , GRK2 and β-arrestin2 was inhibited by B 2 receptor but not AT 2 receptor antagonism, indicating the importance of B 2 receptor activation within AT 2 -B 2 heteromers. The close proximity between the AT 2 receptor and B 2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT 2 receptor and B 2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gα q /GRK2/β-arrestin2 recruitment (AT 2 receptor) and Gα z protein coupling (B 2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT 2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the ersity of GPCR pharmacology and signaling.
Publisher: Springer Science and Business Media LLC
Date: 29-09-2015
Publisher: Springer Science and Business Media LLC
Date: 26-03-2021
DOI: 10.1038/S41467-021-22101-7
Abstract: Adipogenesis associated Mth938 domain containing (AAMDC) represents an uncharacterized oncogene lified in aggressive estrogen receptor-positive breast cancers. We uncover that AAMDC regulates the expression of several metabolic enzymes involved in the one-carbon folate and methionine cycles, and lipid metabolism. We show that AAMDC controls PI3K-AKT-mTOR signaling, regulating the translation of ATF4 and MYC and modulating the transcriptional activity of AAMDC -dependent promoters. High AAMDC expression is associated with sensitization to dactolisib and everolimus, and these PI3K-mTOR inhibitors exhibit synergistic interactions with anti-estrogens in IntClust2 models. Ectopic AAMDC expression is sufficient to activate AKT signaling, resulting in estrogen-independent tumor growth. Thus, AAMDC -overexpressing tumors may be sensitive to PI3K-mTORC1 blockers in combination with anti-estrogens. Lastly, we provide evidence that AAMDC can interact with the RabGTPase-activating protein RabGAP1L, and that AAMDC, RabGAP1L, and Rab7a colocalize in endolysosomes. The discovery of the RabGAP1L-AAMDC assembly platform provides insights for the design of selective blockers to target malignancies having the AAMDC lification.
Publisher: Springer Science and Business Media LLC
Date: 09-06-2017
DOI: 10.1038/S41598-017-03486-2
Abstract: Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.
Publisher: Frontiers Media SA
Date: 2012
Publisher: Elsevier BV
Date: 12-2018
Publisher: MDPI AG
Date: 28-12-2020
DOI: 10.3390/IJMS22010209
Abstract: Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, which are known binding sites of allosteric modulators in other GPCRs. Our data demonstrate the allosteric effect of LVV-H7 on AngII binding, which is consistent with the positive modulation of AT1R activity and signaling previously reported. This further supports the pharmacological targeting of AT1R by hemorphins, with implications in vascular and renal physiology.
Publisher: Public Library of Science (PLoS)
Date: 25-03-2019
Publisher: Elsevier BV
Date: 06-2021
Publisher: Frontiers Media SA
Date: 26-03-2019
Publisher: Frontiers Media SA
Date: 30-08-2022
DOI: 10.3389/FENDO.2022.931573
Abstract: G protein-coupled receptors (GPCRs) are capable of interacting to form higher order structures such as homomers and heteromers. Heteromerisation in particular has implications for receptor function, with research showing receptors can attain unique expression, ligand binding, signalling and intracellular trafficking upon heteromerisation. As such, GPCR heteromers represent novel drug targets with extensive therapeutic potential. Changes to ligand affinity, efficacy and G protein coupling have all been described, with alterations to these pharmacological aspects now well accepted as common traits for heteromeric complexes. Changes in internalisation and trafficking kinetics, as well as β-arrestin interactions are also becoming more apparent, however, few studies to date have explicitly looked at the implications these factors have upon the signalling profile of a heteromer. Development of ligands to target GPCR heteromers both experimentally and therapeutically has been mostly concentrated on bivalent ligands due to difficulties in identifying and developing heteromer-specific ligands. Improving our understanding of the pharmacology and physiology of GPCR heteromers will enable further development of heteromer-specific ligands with potential to provide therapeutics with increased efficacy and decreased side effects.
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2914-6_13
Abstract: Bioluminescence resonance energy transfer (BRET) is a well-established technique for investigating G protein-coupled receptor (GPCR) pharmacology. BRET enables the monitoring of molecular proximity through the use of heterologously expressed proteins of interest and/or fluorophore-labeled ligands. Fusion to a donor luciferase enzyme or an acceptor fluorophore and subsequent detection of resonance energy transfer indicate the close proximity of the molecules of interest. As BRET is readily applied to the study of numerous GPCR signaling and regulatory paths, it is an ideal technique for investigating the pharmacology of biased ligands and receptors.
Publisher: Frontiers Media SA
Date: 13-04-2022
DOI: 10.3389/FNCEL.2022.812359
Abstract: The orexin system comprises two G protein-coupled receptors, OX 1 and OX 2 receptors (OX 1 R and OX 2 R, respectively), along with two endogenous agonists cleaved from a common precursor (prepro-orexin), orexin-A (OX-A) and orexin-B (OX-B). For the receptors, a complex array of signaling behaviors has been reported. In particular, it becomes obvious that orexin receptor coupling is very erse and can be tissue-, cell- and context-dependent. Here, the early signal transduction interactions of the orexin receptors will be discussed in depth, with particular emphasis on the direct G protein interactions of each receptor. In doing so, it is evident that ligands, additional receptor-protein interactions and cellular environment all play important roles in the G protein coupling profiles of the orexin receptors. This has potential implications for our understanding of the orexin system’s function in vivo in both central and peripheral environments, as well as the development of novel agonists, antagonists and possibly allosteric modulators targeting the orexin system.
Publisher: Elsevier BV
Date: 02-2019
DOI: 10.1016/J.CELLSIG.2018.11.018
Abstract: Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A
Publisher: Springer Science and Business Media LLC
Date: 06-2015
DOI: 10.1038/NMETH.3398
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 11-09-2019
Abstract: Studies have shown that the hormone serelaxin, which has organ-protective actions mediated via relaxin family peptide receptor 1 (RXFP1), its cognate G protein–coupled receptor, requires the angiotensin II type 2 receptor (AT 2 R) to ameliorate renal fibrogenesis in vitro and in vivo . In this study, the authors describe a functional interaction between RXFP1, AT 2 R, and the angiotensin II type 1 receptor (AT 1 R), all of which are expressed on extracellular matrix–producing myofibroblasts, the cellular basis of progressive fibrosis. The crosstalk between these G protein–coupled receptors allows antagonists acting at each receptor to directly or allosterically block the antifibrotic actions of agonists acting at AT 2 R or RXFP1. These findings have significant therapeutic implications for a mechanistic understanding of the concomitant use of drugs acting at each receptor. Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein–coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT 2 R) to ameliorate renal fibrogenesis in vitro and in vivo . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT 1 R), which is expressed on myofibroblasts along with RXFP1 and AT 2 R, is unknown. We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro , and in three models of renal- or cardiomyopathy-induced fibrosis in vivo . The AT 1 R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo . Candesartan also ameliorated serelaxin’s antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan’s inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT 1 Rs but that constitutive AT 1 R–RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT 2 R agonist (compound 21). These findings have significant implications for the concomitant use of RXFP1 or AT 2 R agonists with AT 1 R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression.
Publisher: Springer Science and Business Media LLC
Date: 09-2020
DOI: 10.1038/S41698-020-00129-0
Abstract: Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee ( Apis mellifera ) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the anticancer selectivity of melittin, and outlines treatment strategies to target aggressive breast cancers.
Publisher: MDPI AG
Date: 22-01-2021
DOI: 10.3390/IJMS22031082
Abstract: Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).
Location: Australia
No related grants have been discovered for Elizabeth Johstone.