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

[Image: see text] 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 E(max) 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 Ca(2+) mobilization to produce this response. Moreover, superactivation by Met-ENK, which was the most-effective Ca(2+) mobilizing agonist, required Gαi/o activation, availability of Gβγ subunits at the membrane, and activation of Ca(2+) 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 Gβγ dimer and Ca(2+) mobilization, relying instead on Src and Raf-1 to induce this cellular adaptation.