Abstract
G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, signaling mediated by β-arrestin has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either G protein or β-arrestin-signaling pathways. However, nearly all screening techniques for measuring β-arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure β-arrestin recruitment to the membrane or early endosomes by unmodified receptors. Our strategy uses NanoLuc, an engineered luciferase from Oplophorus gracilirostris (deep-sea shrimp) that is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. We have identified a unique split site within NanoLuc and fused the corresponding N-terminal fragment to either a plasma membrane or early endosome tether and the C-terminal fragment to β-arrestins, which form the basis for the MeNArC and EeNArC assays, respectively. Upon receptor activation, β-arrestin is recruited to the membrane and subsequently internalized in an agonist concentration-dependent manner. This recruitment promotes complementation of the two NanoLuc fragments, thereby reconstituting functional NanoLuc, allowing for quantification of β-arrestin recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification and has promise as a new generic assay for measuring β-arrestin recruitment to diverse GPCR types in heterologous or native cells.
Highlights
An example, the β-adrenergic receptor ligand carvedilol antagonizes G protein activation but recruits β-arrestin and has been shown to increase survival rates in patients suffering from heart failure [1]
We have demonstrated that β-arrestin recruitment to the dopamine D2 receptor (D2R) in indirect pathway neurons in the ventral striatum leads to enhanced locomotion, whereas G protein signaling is necessary for incentive behavior [7], further emphasizing the potential importance of biased signaling in more targeted therapeutics
LinkLight uses a modified luciferase attached to β-arrestin that is cleaved off by a protease fused to the C terminus of the G protein-coupled receptor (GPCR) of interest, thereby activating the luciferase and producing light [12]
Summary
An example, the β-adrenergic receptor ligand carvedilol antagonizes G protein activation but recruits β-arrestin and has been shown to increase survival rates in patients suffering from heart failure [1]. We further validated their use for complementation-based GPCR assays in a direct recruitment assay with β-arrestin, as we have done previously with the D2R receptor in a BRET setup [13], in which the N-terminal Nanoluc fragment was attached to the C-terminal tail of D2R and the C-terminal fragment was fused to the N terminus of βarrestin2 (Fig. 1E).
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