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

μ-Opioid receptor (MOR) agonists are often used to treat severe pain but can result in adverse side effects. To circumvent systemic side effects, targeting peripheral opioid receptors is an attractive alternative treatment for severe pain. Activation of the δ-opioid receptor (DOR) produces similar analgesia with reduced side effects. However, until primed by inflammation, peripheral DOR is analgesically incompetent, raising interest in the mechanism. We recently identified a novel role for G-protein- coupled receptor kinase 2 (GRK2) that renders DOR analgesically incompetent at the plasma membrane. However, the mechanism that maintains constitutive GRK2 association with DOR is unknown. Protein kinase A (PKA) phosphorylation of GRK2 at Ser-685 targets it to the plasma membrane. Protein kinase A-anchoring protein 79/150 (AKAP), residing at the plasma membrane in neurons, scaffolds PKA to target proteins to mediate downstream signal. Therefore, we sought to determine whether GRK2-mediated DOR desensitization is directed by PKA via AKAP scaffolding. Membrane fractions from cultured rat sensory neurons following AKAP siRNA transfection and from AKAP-knock-out mice had less PKA activity, GRK2 Ser-685 phosphorylation, and GRK2 plasma membrane targeting than controls. Site-directed mutagenesis revealed that GRK2 Ser-685 phosphorylation drives the association of GRK2 with plasma membrane-associated DOR. Moreover, overexpression studies with AKAP mutants indicated that impaired AKAP-mediated PKA scaffolding significantly reduces DORGRK2 association at the plasma membrane and consequently increases DOR activity in sensory neurons without a priming event. These findings suggest that AKAP scaffolds PKA to increase plasma membrane targeting and phosphorylation of GRK2 to maintain DOR analgesic incompetence in peripheral sensory neurons.