GRK2 Constitutively Governs Peripheral Delta Opioid Receptor Activity

Abstract

Summary Opioids remain the standard for analgesic care, however adverse effects of systemic treatments contraindicate long-term administration. While most clinical opioids target mu opioid receptors (MOR), those that target the delta class (DOR) also demonstrate analgesic efficacy. Furthermore, peripherally-restrictive opioids represent an attractive direction for analgesia. However, opioid receptors including DOR are analgesically incompetent in the absence of inflammation. Here, we report that G Protein-Coupled Receptor Kinase 2 (GRK2) naively associates with plasma membrane DOR in peripheral sensory neurons to inhibit analgesic agonist efficacy. This interaction prevents optimal Gβ subunit association with the receptor, thereby reducing DOR activity. Importantly, bradykinin stimulates GRK2 movement away from DOR and onto Raf Kinase Inhibitory Protein (RKIP). Protein kinase C (PKC)-dependent RKIP phosphorylation induces GRK2 sequestration, restoring DOR functionality in sensory neurons. Together, these results expand the known function of GRK2, identifying a non-internalizing role to maintain peripheral DOR in an analgesically incompetent state.

Identification of a signaling cascade that maintains constitutive δ-opioid receptor incompetence in peripheral sensory neurons

“…In the periphery, DOR lies dormant in an analgesically incompetent state unless primed by inflammation (4,(7)(8)(9)(10)(11)30). Such a prerequisite for receptor activation would be expected to limit the therapeutic efficacy of peripherally restricted DOR agonists to inflammatory pain patients only.…”

“…Under non-inflammatory conditions, DOR is chronically bound to GRK2, which renders the receptor dormant or unresponsive to agonist stimulation in peripheral sensory neurons (11). In immortalized cells, phosphorylation by PKA facilitates GRK2 translocation to the plasma membrane to negatively modulateGPCRsignaling (24).GiventhatPKA-dependentphosphorylation dictates plasma membrane translocation of GRK2 ( Fig.…”

“…To build on this, we next carried out a functional Ca 2ϩ imaging assay in peripheral sensory neurons to determine whether PKA-dependent phosphorylation of GRK2 governs DOR incompetence. We measured opioid inhibition of voltage-gated Ca 2ϩ channels (VGCCs) elicited by KCl-evoked Ca 2ϩ transients in cultured sensory neurons, which is a validated method that has been used by multiple groups to quantify DOR activity in a heterogeneous neuronal population (10,11,27). In contrast to patch-clamp electrophysiology, this method circumvents inevitable user bias in determining whether to include a cell that does not respond to a DOR agonist due to lack of receptor expression versus lack of functional receptor competence.…”

“…We previously reported that small to medium capsaicin (CAP; 1 M)-sensitive DRG neurons transfected with FITC-GRK2 siRNA produce significant DOR-mediated inhibition of VGCCs compared with mock-treated cells that have little to no DOR activity in the presence of a DOR agonist (11). To test whether PKA-dependent phosphorylation of GRK2 underlies functional DOR incompetence, we used siRNA-mediated knockdown of GRK2 followed by co-nucleofection of GFP with empty vector, wild-type GRK2, GRK2-S685D, or GRK2-S685G cDNA.…”

“…However, agonist activation of peripheral opioid receptors, including DOR, requires an inflammatory prestimulus or "priming" event (4 -11). Peripheral DOR incompetence is maintained by a constitutive interaction between the receptor and G-protein-coupled receptor kinase 2 (GRK2) (11). Exposure to inflammatory mediator bradykinin induces a "priming" cascade that involves protein kinase C (PKC)-induced phosphorylation of Raf-kinase-inhibitory protein (RKIP), followed by RKIP self-dimerization and GRK2 sequestration to the cytosol.…”