Long-term Kappa Opioid Receptor Inhibition by Norbinaltorphimine and 14-3-3γ

Abstract ID 93671 Poster Board 549 G protein-coupled receptors (GPCRs) are the largest family of eukaryotic membrane receptors, mediate diverse physiological functions, and are targets for ∼30% of marketed drugs, including opioids used to treat pain. Despite considerable resource allocation for more than a decade, the opioid epidemic continues to grow, with >80,000 American lives lost per year due to opioid overdose. The kappa opioid receptor (KOR) is highly expressed in the nervous system and is a promising therapeutic target for pain. Activation of KOR in peripheral sensory neurons produces profound antinociceptive responses, and peripherally-restricted KOR agonists avoid the addictive and deadly adverse effects of centrally-targeted opioids. However, development of peripherally-restricted KOR agonists has been limited by unidentified regulatory pathways that reduce effectiveness and/or induce hyperalgesia. A single injection of norbinaltorphimine (norBNI) can result in weeks-long inhibition of KOR via an unknown mechanism. This long-term antagonism is not through sustained receptor occupancy, but requires norBNI-mediated KOR activation and subsequent c-Jun N-terminal kinase (JNK) signaling. We have shown that this long-term antagonism requires protein synthesis, as this effect is blocked by protein translation inhibitors. Here we identify that the scaffolding protein 14-3-3γ is upregulated in response to norBNI-mediated JNK activation. Knockdown of 14-3-3γ by siRNA eliminated norBNI's long-term antagonism of KOR-mediated antinociception in the rat hind paw and KOR-mediated cAMP signaling in primary cultures of sensory neurons. Thus, 14-3-3γ is a critical mediator necessary for the long-term KOR antagonism by norBNI. Notably, not all KOR-mediated signaling pathways were disrupted by norBNI pretreatment, as KOR-mediated activation of extracellular signal regulated kinase (ERK) was maintained. Further, overexpression of 14-3-3γ in CHO cells also prevented KOR-mediated cAMP signaling, and these effects appeared selective to KOR as other Gi coupled receptors were not affected. Next, we sought to understand the mechanism of 14-3-3γ inhibition of KOR. The family of 14-3-3 proteins are ubiquitously expressed, highly conserved scaffolds that can modulate signaling complexes through protein-protein interactions with a large and diverse set of interacting partners. Thus, we considered that KOR and 14-3-3γ may form a complex in cells that can bias KOR signaling. KOR and 14-3-3γ co-immunoprecipitate in transfected CHO and HEK293T cells, suggesting the formation of a KOR-14-3-3γ complex. This idea is supported by recent literature identifying 14-3-3 proteins as regulators of certain G protein-coupled receptors. Molecular modeling suggests that 14-3-3γ may interact directly with KOR in a similar manner as beta-arrestins, via the KOR phosphorylated C-terminus or intracellular loops. Ongoing studies are investigating the molecular underpinnings of this interaction, which may be widely applicable to the GPCR family as ∼90% of GPCRs have predicted 14-3-3 binding motifs. This work was supported by the National Institutes of Health grant [R01 DA038645] to WC and KB; RJ was supported in part by the Translational Science Training program at UT Health San Antonio (TST128233); And MW was supported by the NIDA T32 Postdoctoral Training Program in Drug Abuse Research: Behavior & Neurobiology [T32DA031115] and a National Research Service Award [F32DA056212].