International Journal of Molecular Sciences Article Investigation of Receptor Heteromers Using NanoBRET Ligand Binding Elizabeth K. M. Johnstone 1,2,3,* , Heng B. See 1,2,3, Rekhati S. Abhayawardana 1,2,3, Angela Song 4, K. Johan Rosengren 4 , Stephen J. Hill 1,2,5,6 and Kevin D. G. Pfleger 1,2,3,7,* 1 Molecular Endocrinology and Pharmacology Group, Harry Perkins Institute of Medical Research, Nedlands, WA 6009, Australia; [email protected] (H.B.S.); [email protected] (R.S.A.); [email protected] (S.J.H.) 2 Centre for Medical Research, The University of Western Australia, Crawley, WA 6009, Australia 3 Australian Research Council Centre for Personalised Therapeutics Technologies, Canberra, NSW 2609, Australia 4 School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, QLD 4072, Australia; [email protected] (A.S.); [email protected] (K.J.R.) 5 Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham Medical School, Nottingham NG7 2UH, UK 6 Centre of Membrane Proteins and Receptors, University of Nottingham, Midlands NG7 2UH, UK 7 Dimerix Limited, Nedlands, WA 6009, Australia * Correspondence: [email protected] (E.K.M.J.); kevin.pfl[email protected] (K.D.G.P.); Tel.: +61-8-6151-0734 (K.D.G.P.) 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- Citation: Johnstone, E.K.M.; See, dependent modulation of interactions between receptors and specific biomolecules for the detection H.B.; Abhayawardana, R.S.; Song, A.; and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor- Rosengren, K.J.; Hill, S.J.; Pfleger, HIT assays have been intracellular proteins, however in this study we have for the first time used K.D.G. Investigation of Receptor Heteromers Using NanoBRET Ligand bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate Binding. Int. J. Mol. Sci. 2021, 22, heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with 1082. https://doi.org/10.3390/ NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) ijms22031082 receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer). Academic Editor: Fabio Altieri Received: 4 January 2021 Keywords: NanoBRET; Nluc; GPCR; heteromer; ligand binding; Receptor-HIT; angiotensin receptor; Accepted: 19 January 2021 β2 adrenergic receptor Published: 22 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Receptor heteromerization is the formation of a complex composed of two (or more) iations. functional receptor units. Although the constituent receptors are functional in their own right as monomers/homomers, the formation of a heteromer results in a complex that may have distinct biochemical properties [1]. This attainment of novel heteromer pharmacology expands the complexity of receptor signaling networks and adds selectivity and specificity Copyright: © 2021 by the authors. to receptor signaling. Although this makes receptor heteromers exciting potential drug Licensee MDPI, Basel, Switzerland. targets, to date only modest progress has been made towards therapeutically targeting these This article is an open access article complexes. One reason for this is the difficulty in detecting heteromers and monitoring distributed under the terms and their pharmacology. In particular, differentiating heteromer-specific pharmacology from conditions of the Creative Commons Attribution (CC BY) license (https:// monomer/homomer-specific pharmacology can be a major challenge. In order to address creativecommons.org/licenses/by/ this issue, we have developed the Receptor-Heteromer Investigation Technology (Receptor- 4.0/). HIT) [2,3], which has been used to investigate several G protein-coupled receptor (GPCR) Int. J. Mol. Sci. 2021, 22, 1082. https://doi.org/10.3390/ijms22031082 https://www.mdpi.com/journal/ijms Int. J.Int. Mol. J. Sci. Mol. 2021 Sci., 202122, x, 22 , 1082 2 of 182 of 16 receptorheteromers (GPCR) [ 2heteromers,4–11]. In addition, [2,4–11]. In the addition, Receptor-HIT the Receptor assay has-HIT been assay used has tobeen characterize used to characterizereceptor tyrosine receptor kinase tyrosine (RTK) kinase heteromers (RTK) heteromers [12], as well [12], as as heteromers well as heteromers composed com- of both posedGPCRs of both and GPCRs RTKs and [13 –RTKs15] or [1 a3– GPCR15] or a and GPCR the and Receptor the Receptor for Advanced for Advanced Glycation Gly- End- cationproducts End-products [16]. [16]. The TheReceptor Receptor-HIT-HIT assay assay enables enables rapid rapid identification, identification, screening screening and andprofiling profiling of re- of re- ceptorceptor heteromers heteromers through through ligand ligand-dependent-dependent modulation modulation of interactions of interactions between between the re- the ceptorsreceptors and specific and specific biomolecules. biomolecules. The assay The use assays a proximity uses a proximity-based-based reporter system, reporter such system, as bioluminescencesuch as bioluminescence resonance resonance energy transfer energy (BRET), transfer which (BRET), allows which the allows proximity the proximity be- tweenbetween the receptors the receptors and the and interacting the interacting biomolecules biomolecules to be monitored. to be monitored. Previously, Previously, the in- the teractinginteracting biomolecules biomolecules used in used Receptor in Receptor-HIT-HIT studies have studies been have intracellular been intracellular proteins (Fig- proteins ure 1a),(Figure enabling1a), enabling functional functional interactions interactions to be identified. to be identified. However, However, as we have as previously we have pre- discussedviously [17], discussed Receptor [17-HIT], Receptor-HIT can also be configured can also be to configured investigate to heteromers investigate at heteromers their ex- at tracellulartheir extracellular surface, with surface, the use withof a fluorescent the use of a ligand fluorescent as the ligandlabeled as interacting the labeled biomole- interacting cule biomolecule(Figure 1b). (Figure1b). FigureFigure 1. Intracellular 1. Intracellular and extracellular and extracellular Receptor Receptor-Heteromer-Heteromer Investigation Investigation Technology Technology (Receptor (Receptor-- HIT)HIT) assay assay. Receptor. Receptor-HIT-HIT allows allows for investigation for investigation of receptor of receptor heteromers heteromers through through ligand ligand-dependent-depend- ent modulationmodulation of of interactions interactions between between receptors receptors and and specific specific biomolecules. biomolecules. The The Receptor Receptor-HIT-HIT as- assay say usesuses a a proximity proximity-based-based reporter reporter system system such such as as bioluminescence bioluminescence or or fluoresc fluorescenceence resonance resonance en- energy ergy transfer (BRET or FRET) or protein complementation. The assay has most commonly been used transfer (BRET or FRET) or protein complementation. The assay has most commonly been used to investigate GPCR heteromers (GPCR-HIT) [2] however it can be used to investigate any type of to investigate GPCR heteromers (GPCR-HIT) [2] however it can be used to investigate any type of heteromers, such as RTK heteromers (RTK-HIT) [12]. The assay works by coexpressing each recep- heteromers, such as RTK heteromers (RTK-HIT) [12]. The assay works by coexpressing each receptor tor in cells, one receptor tagged with the first reporter component of the proximity assay (RC1), such in cells, one receptor tagged with the first reporter component of the proximity assay (RC1), such as a as a BRET donor, and the other receptor untagged with respect to the proximity assay system. In- stead,BRET an int donor,eracting and biomolecule the other receptor is tagged untagged with the with second respect reporter to the component proximity assay (RC2), system. such as Instead, a BRETan acceptor. interacting Usually, biomolecule this interacting is tagged biomolecule with the second is a coexpressed reporter component intracellular (RC2), protein such (a) ashow- a BRET ever acceptor.it can also Usually, be a labeled this interacting ligand (b). biomoleculeThe assay works is a coexpressed by adding aintracellular ligand that is protein selective (a) for however the it untaggedcan also receptor. be a labeled If this ligand results (b in). Themodulation assay works of the by proximity adding a ligandsignal thatbetween is selective the tag for on the the untagged re- ceptorreceptor. and the If tag this on results the interacting in modulation biomolecule, of the proximity this indicates signal heteromerization between the tag on of the the receptor two recep- and the tors. tag on the interacting biomolecule, this indicates heteromerization of the two receptors. Int. J. Mol. Sci. 2021, 22, 1082 3 of 16 Recently, we have developed the NanoBRET ligand binding assay [18–23], in which we have successfully