Nanoluciferase-based complementation assay for systematic profiling of GPCR-GRK interactions.

G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7-GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically ß-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

Atypical opioid receptors: unconventional biology and therapeutic opportunities.

Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating four opioid receptors, namely µ (mu, MOP), δ (delta, DOP), κ (kappa, KOP) and the nociceptin/orphanin FQ receptor (NOP). Interestingly, several other receptors are also activated by endogenous opioid peptides and influence opioid-driven signaling and biology. However, they do not meet the criteria to be recognized as classical opioid receptors, as they are phylogenetically distant from them and are insensitive to classical non-selective opioid receptor antagonists (e.g. naloxone). Nevertheless, accumulating reports suggest that these receptors may be interesting alternative targets, especially for the development of safer analgesics. Five of these opioid peptide-binding receptors belong to the family of G protein-coupled receptors (GPCRs)-two are members of the Mas-related G protein-coupled receptor X family (MrgX1, MrgX2), two of the bradykinin receptor family (B1, B2), and one is an atypical chemokine receptor (ACKR3). Additionally, the ion channel N-methyl-d-aspartate receptors (NMDARs) are also activated by opioid peptides. In this review, we recapitulate the implication of these alternative receptors in opioid-related disorders and discuss their unconventional biology, with members displaying signaling to scavenging properties. We provide an overview of their established and emerging roles and pharmacology in the context of pain management, as well as their clinical relevance as alternative targets to overcome the hurdles of chronic opioid use. Given the involvement of these receptors in a wide variety of functions, including inflammation, chemotaxis, anaphylaxis or synaptic transmission and plasticity, we also discuss the challenges associated with the modulation of both their canonical and opioid-driven signaling.

Nanoluciferase-based methods to monitor activation, modulation and trafficking of atypical chemokine receptors.

Chemokines regulate directed cell migration, proliferation and survival and are key components in various physiological and pathological processes. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). Atypical chemokine receptors (ACKRs) play important roles in the chemokine-receptor network by regulating chemokine bioavailability for the classical receptors through chemokine sequestration, scavenging or transport. Currently, this subfamily of receptors comprises four members: ACKR1, ACKR2, ACKR3 and ACKR4. They differ notably from the classical chemokine receptors by their inability to elicit G protein-mediated signaling, which precludes the use of classical assays relying on the activation of G proteins and related downstream secondary messengers to investigate ACKRs. There is therefore a need for alternative approaches to monitor ACKR activation, modulation and trafficking. This chapter details sensitive and versatile methods based on Nanoluciferase Binary Technology (NanoBiT) and Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) to monitor ACKR2 and ACKR3 activity through the measurement of ß-arrestin and GRK recruitment, and receptor trafficking, including internalization and delivery to early endosomes.

Proadrenomedullin N-Terminal 20 Peptides (PAMPs) Are Agonists of the Chemokine Scavenger Receptor ACKR3/CXCR7.

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two peptides with vasodilative, bronchodilative, and angiogenic properties, originating from a common precursor, proADM. Previous studies proposed that the atypical chemokine receptor ACKR3 might act as a low-affinity scavenger for ADM, regulating its availability for its cognate receptor calcitonin receptor-like receptor (CLR) in complex with a receptor activity modifying protein (RAMP). In this study, we compared the activation of ACKR3 by ADM and PAMP, as well as other related members of the calcitonin gene-related peptide (CGRP) family. Irrespective of the presence of RAMPs, ADM was the only member of the CGRP family to show moderate activity toward ACKR3. Remarkably, PAMP, and especially further processed PAMP-12, had a stronger potency toward ACKR3 than ADM. Importantly, PAMP-12 induced ß-arrestin recruitment and was efficiently internalized by ACKR3 without inducing G protein or ERK signaling in vitro. Our results further extend the panel of endogenous ACKR3 ligands and broaden ACKR3 functions to a regulator of PAMP-12 availability for its primary receptor Mas-related G-protein-coupled receptor member X2 (MrgX2).