Abstract
The kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor-biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.
Highlights
Ligand-Selective Effects at the Kappa Opioid ReceptorAgonists of the KOR comprise various endogenous and exogenous peptide and non-peptide ligands with diverse chemical scaffolds
Current pharmacological approaches to determine the potency and efficacy of GPCR agonists are based on the measurement of G protein signaling, which can be sampled at multiple levels, e.g. at the level of GTP exchange (GTPγS), at the level of second messenger production, or at the level of gene transcription (Mores et al 2019)
We describe the development and the characteristics of nanobody- and minimal G protein (mini-G) protein-based biosensors, discuss their value in determining ligand effects and ligand bias, and highlight the novel insights into agonist-selective effects at the KOR in living cells
Summary
Agonists of the KOR comprise various endogenous and exogenous peptide and non-peptide ligands with diverse chemical scaffolds. The binding of agonists induces conformational changes in the receptor that allow binding of active state KOR to transducer and regulatory proteins on the cytosolic side, such as G proteins, GPCR kinases (GRKs), and β-arrestins (Bruchas and Chavkin 2010). Their coupling to KOR elicits transmembrane signal transduction. Current pharmacological approaches to determine the potency and efficacy of GPCR agonists are based on the measurement of G protein signaling, which can be sampled at multiple levels, e.g. at the level of GTP exchange (GTPγS), at the level of second messenger production (e.g. cAMP), or at the level of gene transcription (Mores et al 2019). We describe the development and the characteristics of nanobody- and mini-G protein-based biosensors, discuss their value in determining ligand effects and ligand bias, and highlight the novel insights into agonist-selective effects at the KOR in living cells
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