Dynorphin peptides differentially regulate the human κ opioid receptor

Abstract

Dynorphins, endogenous peptides for the κ opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and α-neoendorphin (α-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [ 35 S]GTPγS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with α-Neo at a concentration of ∼600× EC 50 value (from [ 35 S]GTPγS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations (∼10% vs. ∼65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of α-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced α-Neo-, but not Dyn A-or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of α-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.

The dynorphin/κ-opioid receptor system and its role in psychiatric disorders

“…However, disparate findings were obtained. Heikkila et al [402] reported enhanced CSF DYN A levels in unmedicated patients with schizophrenia relative to healthy and psychiatric controls using an antibody recognizing DYN A (9)(10)(11)(12)(13)(14)(15)(16)(17). CSF DYN A levels significantly correlated with increased psychotic rating scores on the Brief Psychiatric Ratings Scale.…”

“…Other biologically active DYNs include DYN B1-13 (DYN B/rimorphin), DYN B1-29 (leumorphin), DYN A/B1-32 (big DYN) and a-and b-neoendorphin [10][11][12][13][14]. The affinity and efficacy of these peptides differ with DYN A being the most potent and b-neoendorphin the least [15,16]. Importantly, DYNs bind with high affinity to other opioid and non-opioid receptors (e.g., acid-sensing ion channels and NMDA receptors), [17][18][19].…”

“…It seems likely, however, that these result from alternative splicing, post-translational modifications and/or protein-protein interactions [34,35]. KOR reportedly couples to both inhibitory G bc , Ga i , Ga o , Ga z and Ga 16 , and stimulatory, Ga s , G-proteins [36]. Nanomolar ligand concentrations result in coupling of KOR to inhibitory G proteins, decreasing membrane excitability and transmitter release via stimulation of K ?…”

“…DYNs A and B, a-neoendorphin, the naturally occurring KOR agonist, salvinorin A, and several synthetic agonists (e.g., U50,488H and U69,593) induce desensitization, internalization and down-regulation of KOR in heterologous expression systems [16,[44][45][46]. In contrast, other synthetic agonists and antagonists (e.g., etorphine and norbinaltorphimine (BNI), respectively) have opposite effects.…”

The role of κ-opioid receptor activation in mediating antinociception and addiction

“…After repeated or sustained exposure to agonists, KORs are desensitized by receptor phosphorylation and recruitment of β-arrestin and endocytosed via a clathrin-and dynamin-dependent pathway. These internalized receptors either return to the membrane by dephosphorylation and EBP50/NHERF-1-dependent recycling or are degraded via both lysosome and proteasome systems [15,16] . G-protein receptor kinase 3 (GRK3) and β-arrestin 1/2 play important roles in the modulation of KOR trafficking [12,17] .…”