Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies

Abstract

the δ-opioid receptor (Dop) is an attractive pharmacological target due to its potent analgesic, anxiolytic and anti-depressant activity in chronic pain models. However, some but not all selective DOP agonists also produce severe adverse effects such as seizures. Thus, the development of novel agonists requires a profound understanding of their effects on DOP phosphorylation, post-activation signaling and dephosphorylation. Here we show that agonist-induced Dop phosphorylation at threonine 361 (T361) and serine 363 (S363) proceeds with a temporal hierarchy, with S363 as primary site of phosphorylation. This phosphorylation is mediated by G protein-coupled receptor kinases 2 and 3 (GRK2/3) followed by DOP endocytosis and desensitization. DOP dephosphorylation occurs within minutes and is predominantly mediated by protein phosphatases (PP) 1α and 1β. A comparison of structurally diverse Dop agonists and clinically used opioids demonstrated high correlation between G protein-dependent signaling efficacies and receptor internalization. In vivo, Dop agonists induce receptor phosphorylation in a dose-dependent and agonist-selective manner that could be blocked by naltrexone in Dop-eGfp mice. together, our studies provide novel tools and insights for ligandactivated Dop signaling in vitro and in vivo and suggest that DOP agonist efficacies may determine receptor post-activation signaling. The δ-opioid (DOP) receptor, as member of the opioid receptor family, was first discovered in 1975, based on the preference of [Leu]-enkephalin binding to receptors in mouse vas deferens, significantly later followed by the cloning of the single-copy gene OPRD for DOP receptor 1-3. The endogenous enkephalins ([Met]-enkephalin and [Leu]-enkephalin), and the frog skin peptides dermenkephalin and deltorphins I and II were identified as naturally-occurring ligands 4-6. Deltorphins have high DOP receptor selectivity, whereas enkephalins are moderately DOP receptor-selective 4. Through coupling to Gα i /Gα 0 proteins, DOP receptor activation leads to inhibition of cAMP production and voltage-gated calcium channels (N-and P/Q-type), as well as induction of β-arrestin signaling and activation of G protein-coupled inwardly rectifying potassium (GIRK) channels 7-10. In addition, signaling kinases such as ERK, c-Jun N-terminal kinase (JNK), src, Akt, p38 mitogen-activated protein kinase (p38 MAPK) or phospholipase C (PLC) and phospholipase A 2 (PLA2) are also activated by DOP receptors 11-17. DOP receptor mRNA and protein are widely expressed throughout the brain, spinal cord and dorsal root ganglia (DRG) 18-21. The DOP receptor is involved in the regulation of important physiological processes such as thermal and mechanical hyperalgesia, chronic inflammatory pain, anxiety and depression, migraine, locomotion, seizures, emotions, learning and memory, as well as addiction and tolerance development 22-26. DOP receptor is also involved in wound healing, neuronal, retinal and cardiovascular cytoprotection during hypoxia, as well as cardioprotection d...

The role of structural dynamics in GPCR‐mediated signaling

“…Additionally, ligand-dependent changes in these regions have been shown to lead to bias sampling of distinct arrestin conformations with different impacts on downstream signaling events. In particular, ligand-specific phosphorylation patterns of GPCRs, so-called 'phosphorylation barcodes', caused by selective recruitment of various GRKs that target different sets of phosphorylation sites, have been reported to favor certain arrestin conformations [164][165][166][167][168][169][170][171][172][173][174][175]. These phosphorylation-dependent structural alterations were initially demonstrated by using intramolecular bioluminescence resonance energy transfer (BRET) (reviewed in Refs [137,176]) and FRET biosensors of arrestins in whole cells [157,164,177].…”

GRKs as Modulators of Neurotransmitter Receptors

“…Out of these, Thr358 and Ser363 are targeted by GRK2, with Ser363 being the primary site [ 123 , 124 , 125 ]. Phosphorylation of Thr361 after Ser363 by GRK2/3 has also been observed [ 126 ]. Strong DOR agonists such as DPDPE and DADLE (enkephalin analogues) induced robust DOR phosphorylation at both Ser363 and Thr361 and receptor internalization, whereas agonists that only caused Ser363 to be phosphorylated induced weak internalization [ 126 ].…”

“…Phosphorylation of Thr361 after Ser363 by GRK2/3 has also been observed [ 126 ]. Strong DOR agonists such as DPDPE and DADLE (enkephalin analogues) induced robust DOR phosphorylation at both Ser363 and Thr361 and receptor internalization, whereas agonists that only caused Ser363 to be phosphorylated induced weak internalization [ 126 ]. KOR has only four potential C-terminal phosphorylation sites, and phosphorylation of one, Ser369 (in mouse KOR), by GRK3 is associated with arrestin recruitment and receptor internalization [ 127 , 128 ].…”

GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation

“…The ubiquitous expression of GRK2, 3, 5, and 6 obscures the elucidation of the roles of individual GRKs on receptor phosphorylation. Until now, siRNA/shRNA [16][17][18] , or CRISPR/Cas 9 approaches targeting only a certain subset of relevant GRKs 19 , and the utilisation of GRK inhibitors were the only strategies used to study their impact on living cell function. Yet, in combination with phosphosite-specific antibodies 20,21 or mass-spectrometry 22 , contributions of individual GRKs to the phosphorylation of certain receptors were elucidated to some degree 7 .…”