Constitutive activation of the delta opioid receptor by mutations in transmembrane domains III and VII.

We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129 (domain III) [corrected], and Tyr308 (domain VII) in the mouse delta opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPgammaS (where GTPgammaS is guanosine 5'-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [35S]GTPgammaS binding was increased for every mutant receptor under study (1.6-2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents alpha-aminobutyric acid) efficiently reduced basal [35S]GTPgammaS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPgammaS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp128 (III) and Tyr308 (VII) interact with each other and that Tyr129 (III) undergoes H bonding with His278 (VI). Thus, Asp128, Tyr129, and Tyr308 may be involved in a network of interhelical bonds, which contributes to maintain the delta receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.     

In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists

Background

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.

Methods and Findings

In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.

Conclusions

Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.     

Mu and Delta Opioid Receptors Oppositely Regulate Motor Impulsivity in the Signaled Nose Poke Task

Impulsivity is a primary feature of many psychiatric disorders, most notably attention deficit hyperactivity disorder and drug addiction. Impulsivity includes a number of processes such as the inability to delay gratification, the inability to withhold a motor response, or acting before all of the relevant information is available. These processes are mediated by neural systems that include dopamine, serotonin, norepinephrine, glutamate and cannabinoids. We examine, for the first time, the role of opioid systems in impulsivity by testing whether inactivation of the mu- (Oprm1) or delta- (Oprd1) opioid receptor gene alters motor impulsivity in mice. Wild-type and knockout mice were examined on either a pure C57BL6/J (BL6) or a hybrid 50% C57Bl/6J–50% 129Sv/pas (HYB) background. Mice were trained to respond for sucrose in a signaled nose poke task that provides independent measures of associative learning (responses to the reward-paired cue) and motor impulsivity (premature responses). Oprm1 knockout mice displayed a remarkable decrease in motor impulsivity. This was observed on the two genetic backgrounds and did not result from impaired associative learning, as responses to the cue signaling reward did not differ across genotypes. Furthermore, mutant mice were insensitive to the effects of ethanol, which increased disinhibition and decreased conditioned responding in wild-type mice. In sharp contrast, mice lacking the Oprd1 gene were more impulsive than controls. Again, mutant animals showed no deficit in associative learning. Ethanol completely disrupted performance in these animals. Together, our results suggest that mu-opioid receptors enhance, whereas delta-opioid receptors inhibit, motor impulsivity. This reveals an unanticipated contribution of endogenous opioid receptor activity to disinhibition. In a broader context, these data suggest that alterations in mu- or delta-opioid receptor function may contribute to impulse control disorders.     

Differential involvement of the mu and kappa opioid receptors in spatial learning

In order to test the role of mu and kappa opioid receptors (Mu opioid receptor (MOR) and Kappa opioid receptor (KOR)) in hippocampal-dependent spatial learning, we analyzed genetically engineered null mutant mice missing the functional MOR or KOR gene. Compared to wild-type mice, the homozygous MOR null mutants exhibited an impairment in the ultimate level of spatial learning as shown in two distinct tasks, the 8-arm radial-maze and the Morris water-maze. Control behaviors were normal. The learning impairment could be associated with the impairment we found in the maintenance of long-term potentiation in mossy fibers in CA3. In comparison, there was no impairment in spatial learning in our KOR mutants or in mossy fibers (mf) in CA3 region long-term potentiation (LTP). Our work suggests that the MOR may play a positive role in learning and memory by increasing LTP in CA3 neurons.