Investigating the ligand agonism and antagonism at the D2long receptor by dynamic mass redistribution

Abstract

The signalling of the D2 receptor (D2R), a G protein-coupled receptor (GPCR), is a complex process consisting of various components. For the screening of D2R ligands, methods quantifying distinct second messengers such as cAMP or the interaction of the receptor with β-arrestin, are commonly employed. In contrast, a label-free biosensor technology like dynamic mass redistribution (DMR), where it is mostly unknown how the individual signalling pathways contribute to the DMR signal, provides a holistic readout of the complex cellular response. In this study, we report the successful application of the DMR technology to CHO-K1 cells stably expressing the human dopamine D2long receptor. In real-time kinetic experiments, studies of D2R reference compounds yielded results for agonists and antagonists that were consistent with those obtained by conventional methods and also allowed a discrimination between partial and full agonists. Furthermore, investigations on the signalling pathway in CHO-K1 hD2longR cells identified the Gαi/o protein as the main proximal trigger of the observed DMR response. The present study has shown that the DMR technology is a valuable method for the characterisation of putative new ligands and, due to its label-free nature, suggests its use for deorphanisation studies of GPCRs.

Shedding Light on the D1‐Like Receptors: A Fluorescence‐Based Toolbox for Visualization of the D1 and D5 Receptors**

“…Label-free techniques such as dynamic mass redistribution (DMR), surface plasmon resonance (SPR) and biolayer interferometry (BLI) are attractive because the isoform of, e. g., the G protein that couples to the receptor of interest doesn't need to be known and genetic modification of the receptor is not required. [35][36][37] This allows studies under more physiological-like conditions, not least because cellular processes are not impaired by the addition of chemical substances that are often required for signal detection in conventional assays. Although not all processes from label-free readouts leading to the observed signal are yet fully understood and sometimes referred to as "black box" readouts, [38] these technique represents a highly interesting alternative to investigate ligand-receptor interactions.…”

“…Although FRET/BRET‐based assays provide valid results and bring several advantages over, e. g., radioassays, modifications must be made to the GPCR and cell protein of interest. Label‐free techniques such as dynamic mass redistribution (DMR), surface plasmon resonance (SPR) and bio‐layer interferometry (BLI) are attractive because the isoform of, e. g., the G protein that couples to the receptor of interest doesn′t need to be known and genetic modification of the receptor is not required [35–37] . This allows studies under more physiological‐like conditions, not least because cellular processes are not impaired by the addition of chemical substances that are often required for signal detection in conventional assays.…”

Fluorescent Tools for the Imaging of Dopamine D2‐Like Receptors**

“…Label-free techniques like dynamic mass redistribution (DMR), surface plasmon resonance (SPR), and bio-layer interferometry (BLI) are particularly interesting because the isoform of, e. g., the G-protein that couples to the receptor of interest does not need to be known and genetic modification of the receptor is not necessary. [24][25][26] This enables investigations under more physiological conditions, not least because the cellular processes are not impaired by the addition of chemical substances, which are often required for signal detection in conventional assays. However, although not all processes leading to the observed signal in label-free readouts are fully understood and are sometimes referred to as "black box" readouts, [27] these techniques offer highly interesting alternatives for studying ligand-receptor interactions.…”

“…Even though FRET/BRET‐based assays deliver reliable results and offer several advantages over e. g. radioassays, modifications have to be made to the GPCR and cell protein of interest. Label‐free techniques like dynamic mass redistribution (DMR), surface plasmon resonance (SPR), and bio‐layer interferometry (BLI) are particularly interesting because the isoform of, e. g., the G‐protein that couples to the receptor of interest does not need to be known and genetic modification of the receptor is not necessary [24–26] . This enables investigations under more physiological conditions, not least because the cellular processes are not impaired by the addition of chemical substances, which are often required for signal detection in conventional assays.…”

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

“…10−12 Dysregulation of the catecholamine dopamine plays diverse roles in pathogenesis and therapy for different diseases such as schizophrenia, Parkinson's disease (PD), drug addiction, and bipolar disorders. 13−15 In the preceding few decades, several test systems have been developed to characterize binding properties or functionality of ligands concerning either the D 1 R or D 2 R. 7,16,17 Unfortunately, the other subtypes have not been as much of a focus.…”