(359) GRK2 constitutively downregulates delta opioid receptor activity in the periphery

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Opioids remain the gold standard for prescribed analgesic care, yet opioid treatments systemically produce negative side effects that contraindicate long-term use. Low dose peripheral administration of opioids has been utilized in the clinic and produces sufficient analgesia without debilitating central side effects. However, the analgesic potential of peripheral opioid agonists is significantly limited in the absence of tissue inflammation through an unknown mechanism. Bradykinin, a potent inflammatory mediator, has been demonstrated to increase peripheral opioid efficacy in a protein kinase C (PKC)-dependent manner, thus, “priming” the receptor for agonist activation. Here, we report that G Protein-Coupled Receptor Kinase 2 (GRK2) constitutively associates with delta opioid receptor (DOR) at the plasma membrane in peripheral sensory neurons, which prevents optimal G protein association with the receptor thereby limiting DOR responsiveness. Notably, bradykinin receptor activation induces PKC-dependent phosphorylation of Raf Kinase Inhibitory Protein (RKIP), followed by RKIP self-dimerization and cytosolic sequestration of GRK2. Consequentially, bradykinin-induced scaffolding of GRK2 away from the plasma membrane restores DOR functional competence in peripheral sensory neurons. To determine whether GRK2 governs peripheral DOR analgesic competence, rats were injected intrathecally with antisense oligodeoxynucleotides (AS-ODN) complementary to GRK2 mRNA that nearly ablated GRK2 protein in the dorsal root ganglia. In mismatch control rats, pretreatment with bradykinin was required to elicit peripheral DOR-mediated inhibition of PGE2-induced thermal allodynia. Importantly, GRK2 knockdown eliminated the bradykinin priming requirement for DOR analgesia in the periphery. Collectively, these results characterize a novel role for GRK2 in maintaining DOR analgesically incompetent at the plasma membrane. Furthermore, results provide a more complete mechanism of peripheral opioid priming where bradykinin induces PKC-dependent RKIP phosphorylation, self-dimerization, and scaffolding of GRK2 away from plasma membrane DOR to enhance receptor responsiveness in peripheral sensory neurons. Funding: NINDS/RO1 NS082746 (N.A.J.), NIDCR/T32 DE14318 (A.P.D.), and NIDCR/F31 DE025551 (A.P.D.). Opioids remain the gold standard for prescribed analgesic care, yet opioid treatments systemically produce negative side effects that contraindicate long-term use. Low dose peripheral administration of opioids has been utilized in the clinic and produces sufficient analgesia without debilitating central side effects. However, the analgesic potential of peripheral opioid agonists is significantly limited in the absence of tissue inflammation through an unknown mechanism. Bradykinin, a potent inflammatory mediator, has been demonstrated to increase peripheral opioid efficacy in a protein kinase C (PKC)-dependent manner, thus, “priming” the receptor for agonist activation. Here, we report that G Protein-Coupled Receptor Kinase 2 (GRK2) constitutively associates with delta opioid receptor (DOR) at the plasma membrane in peripheral sensory neurons, which prevents optimal G protein association with the receptor thereby limiting DOR responsiveness. Notably, bradykinin receptor activation induces PKC-dependent phosphorylation of Raf Kinase Inhibitory Protein (RKIP), followed by RKIP self-dimerization and cytosolic sequestration of GRK2. Consequentially, bradykinin-induced scaffolding of GRK2 away from the plasma membrane restores DOR functional competence in peripheral sensory neurons. To determine whether GRK2 governs peripheral DOR analgesic competence, rats were injected intrathecally with antisense oligodeoxynucleotides (AS-ODN) complementary to GRK2 mRNA that nearly ablated GRK2 protein in the dorsal root ganglia. In mismatch control rats, pretreatment with bradykinin was required to elicit peripheral DOR-mediated inhibition of PGE2-induced thermal allodynia. Importantly, GRK2 knockdown eliminated the bradykinin priming requirement for DOR analgesia in the periphery. Collectively, these results characterize a novel role for GRK2 in maintaining DOR analgesically incompetent at the plasma membrane. Furthermore, results provide a more complete mechanism of peripheral opioid priming where bradykinin induces PKC-dependent RKIP phosphorylation, self-dimerization, and scaffolding of GRK2 away from plasma membrane DOR to enhance receptor responsiveness in peripheral sensory neurons. Funding: NINDS/RO1 NS082746 (N.A.J.), NIDCR/T32 DE14318 (A.P.D.), and NIDCR/F31 DE025551 (A.P.D.).