Development of NanoBRET-Binding Assays for FKBP-Ligand Profiling in Living Cells.
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ABSTRACT:
SUBMITTER: Gnatzy MT
PROVIDER: S-EPMC8360185 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:IntroductionThe Epidermal Growth Factor Receptor is a member of the Erb receptor tyrosine kinase family. It binds several ligands including EGF, betacellulin (BTC) and TGF-α, controls cellular proliferation and invasion and is overexpressed in various cancer types. Nanobodies (VHHs) are the antigen binding fragments of heavy chain only camelid antibodies. In this paper we used NanoBRET to compare the binding characteristics of fluorescent EGF or two distinct fluorescently labelled EGFR directed nanobodies (Q44c and Q86c) to full length EGFR.MethodsLiving HEK293T cells were stably transfected with N terminal NLuc tagged EGFR. NanoBRET saturation, displacement or kinetics experiments were then performed using fluorescently labelled EGF ligands (EGF-AF488 or EGF-AF647) or fluorescently labelled EGFR targeting nanobodies (Q44c-HL488 and Q86c-HL488).ResultsThese data revealed that the EGFR nanobody Q44c was able to inhibit EGF binding to full length EGFR, while Q86c was able to recognise agonist bound EGFR and act as a conformational sensor. The specific binding of fluorescent Q44c-HL488 and EGF-AF488 was inhibited by a range of EGFR ligands (EGF> BTC>TGF-α).DiscussionEGFR targeting nanobodies are powerful tools for studying the role of the EGFR in health and disease and allow real time quantification of ligand binding and distinct ligand induced conformational changes.
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Project description:Background and purposeInterleukin-23 (IL-23) and its receptor are important drug targets for the treatment of auto-inflammatory diseases. IL-23 binds to a receptor complex composed of two single transmembrane spanning proteins IL23R and IL12Rβ1. In this study, we aimed to gain further understanding of how ligand binding induces signalling of IL-23 receptor complexes using the proximity-based techniques of NanoLuc Binary Technology (NanoBiT) and Bioluminescence Resonance Energy Transfer (BRET).Experimental approachTo monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerisation of receptor subunits can be measured through luminescence. The affinity of NanoBiT complemented complexes for IL-23 was measured using NanoBRET, and cytokine-induced signal transduction was measured using a phospho-STAT3 AlphaLISA assay.Key resultsNanoBiT measurements demonstrated that IL-23 receptor complexes formed to an equal degree in the presence and absence of ligand. NanoBRET measurements confirmed that these complexes bound IL-23 with a picomolar binding affinity. Measurement of STAT3 phosphorylation demonstrated that pre-formed IL-23 receptor complexes induced signalling following ligand binding. It was also demonstrated that synthetic ligand-independent signalling could be induced by high affinity (HiBit) but not low affinity (SmBit) NanoBiT crosslinking of the receptor N-terminal domains.Conclusions and implicationsThese results indicate that receptor complexes form prior to ligand binding and are not sufficient to induce signalling alone. Our findings indicate that IL-23 induces a conformational change in heteromeric receptor complexes, to enable signal transduction. These observations have direct implications for drug discovery efforts to target the IL-23 receptor.
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