Comprehensive Signaling Profiles Reveal Unsuspected Functional Selectivity of δ-Opioid Receptor Agonists and Allow the Identification of Ligands with the Greatest Potential for Inducing Cyclase Superactivation

Prolonged exposure to opioid receptor agonists triggers adaptations in the adenylyl cyclase (AC) pathway that lead to enhanced production of cyclic adenosine monophosphate (cAMP) upon withdrawal. This cellular phenomenon contributes to withdrawal symptoms, hyperalgesia and analgesic tolerance that interfere with clinical management of chronic pain syndromes. Since δ-opioid receptors (DOPrs) are a promising target for chronic pain management, we were interested in finding out if cell-based signaling profiles as generated for drug discovery purposes could inform us of the ligand potential to induce sensitization of the cyclase path. For this purpose, signaling of DOPr agonists was monitored at multiple effectors. The resulting signaling profiles revealed marked functional selectivity, particularly for Met-enkephalin (Met-ENK) whose signaling bias profile differed from those of synthetic ligands like SNC-80 and ARM390. Signaling diversity among ligands was systematized by clustering agonists according to similarities in Emax and Log(τ) values for the different responses. The classification process revealed that the similarity in Gα/Gβγ, but not in β-arrestin (βarr), responses was correlated with the potential of Met-ENK, deltorphin II, (d-penicillamine2,5)-enkephalin (DPDPE), ARM390, and SNC-80 to enhance cAMP production, all of which required Ca2+ mobilization to produce this response. Moreover, superactivation by Met-ENK, which was the most-effective Ca2+ mobilizing agonist, required Gαi/o activation, availability of Gβγsubunits at the membrane, and activation of Ca2+ effectors such as calmodulin and protein kinase C (PKC). In contrast, superactivation by (N-(l-tyrosyl)-(3S)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-l-phenylalanyl-l-phenylalanine (TIPP), which was set in a distinct category through clustering, required activation of Gαi/o subunits but was independent of the Gβγdimer and Ca2+ mobilization, relying instead on Src and Raf-1 to induce this cellular adaptation

Biased Signaling of the Mu Opioid Receptor Revealed in Native Neurons

Summary: G protein-coupled receptors are key signaling molecules and major targets for pharmaceuticals. The concept of ligand-dependent biased signaling raises the possibility of developing drugs with improved efficacy and safety profiles, yet translating this concept to native tissues remains a major challenge. Whether drug activity profiling in recombinant cell-based assays, traditionally used for drug discovery, has any relevance to physiology is unknown. Here we focused on the mu opioid receptor, the unrivalled target for pain treatment and also the key driver for the current opioid crisis. We selected a set of clinical and novel mu agonists, and profiled their activities in transfected cell assays using advanced biosensors and in native neurons from knock-in mice expressing traceable receptors endogenously. Our data identify Gi-biased agonists, including buprenorphine, and further show highly correlated drug activities in the two otherwise very distinct experimental systems, supporting in vivo translatability of biased signaling for mu opioid drugs. : Biological Sciences; Physiology; Molecular Biology; Neuroscience; Bioengineering; Cell Biology Subject Areas: Biological Sciences, Physiology, Molecular Biology, Neuroscience, Bioengineering, Cell Biolog

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for G(s)). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology