Christopher P Bailey

Agonist-selective mechanisms of GPCR desensitization

The widely accepted model of G protein‐coupled receptor (GPCR) regulation describes a system where the agonist‐activated receptors couple to G proteins to induce a cellular response, and are subsequently phosphorylated by a family of kinases called the G protein‐coupled receptor kinases (GRKs). The GRK‐phosphorylated receptor then acts as a substrate for the binding of a family of proteins called arrestins, which uncouple the receptor and G protein so desensitizing the agonist‐induced response. Other kinases, principally the second messenger‐dependent protein kinases, are also known to play a role in the desensitization of many GPCR responses. It is now clear that there are subtle and complex interactions between GRKs and second messenger‐dependent protein kinases in the regulation of GPCR function. Functional selectivity describes the ability of agonists to stabilize different active conformations of the same GPCR. With regard to desensitization, distinct agonist‐activated conformations of a GPCR could undergo different molecular mechanisms of desensitization. An example of this is the μ opioid receptor (MOPr), where the agonists morphine and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) induce desensitization of the MOPr by different mechanisms, largely protein kinase C (PKC)‐ or GRK‐dependent, respectively. This can be best explained by supposing that these two agonists stabilize distinct conformations of the MOPr, which are nevertheless able to couple to the relevant G‐proteins and produce similar responses, yet are sufficiently different to trigger different regulatory processes. There is evidence that other GPCRs also undergo agonist‐selective desensitization, but the full therapeutic consequences of this phenomenon await further detailed study.

Agonist-Selective Mechanisms of μ-Opioid Receptor Desensitization in Human Embryonic Kidney 293 Cells

The ability of two opioid agonists, [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and morphine, to induce μ-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.

μ-opioid receptors: correlation of agonist efficacy for signalling with ability to activate internalization.

We have compared the ability of a number of μ-opioid receptor (MOPr) ligands to activate G proteins with their abilities to induce MOPr phosphorylation, to promote association of arrestin-3 and to cause MOPr internalization. For a model of G protein-coupled receptor (GPCR) activation where all agonists stabilize a single active conformation of the receptor, a close correlation between signaling outputs might be expected. Our results show that overall there is a very good correlation between efficacy for G protein activation and arrestin-3 recruitment, whereas a few agonists, in particular endomorphins 1 and 2, display apparent bias toward arrestin recruitment. The agonist-induced phosphorylation of MOPr at Ser375, considered a key step in MOPr regulation, and agonist-induced internalization of MOPr were each found to correlate well with arrestin-3 recruitment. These data indicate that for the majority of MOPr agonists the ability to induce receptor phosphorylation, arrestin-3 recruitment, and internalization can be predicted from their ability as agonists to activate G proteins. For the prototypic MOPr agonist morphine, its relatively weak ability to induce MOPr internalization can be explained by its low agonist efficacy.

μ-Opioid Receptor Desensitization in Mature Rat Neurons: Lack of Interaction between DAMGO and Morphine

μ-Opioid receptors (MORs) exhibit rapid desensitization and internalization during exposure to various opioid agonists. In some studies, however, morphine has been observed to produce little MOR desensitization or internalization. We examined desensitization in mature rat locus ceruleus (LC) neurons and confirmed that morphine is a very poor desensitizing agent, whereas [d-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), a high-efficacy agonist, and methadone, an agonist we observed to be of equivalent efficacy to morphine, produced profound rapid desensitization. Similarly, by measuring plasma membrane receptor levels in HEK293 cells stably expressing T7-epitope-tagged rat MOR1 at near physiological levels (HEK293-MOR1 cells), DAMGO and methadone but not morphine caused rapid MOR internalization. It has been reported that a low concentration of DAMGO, coapplied with morphine, caused morphine to induce MOR internalization. We examined whether this interaction occurred in mature mammalian neurons at the level of receptor desensitization. Coapplication of low concentrations of DAMGO did not increase morphine-induced desensitization in LC neurons but caused a lesser degree of desensitization than DAMGO alone. We also failed to observe an enhancement by DAMGO of morphine-induced desensitization in the electrically stimulated guinea pig ileum myenteric plexus-longitudinal muscle preparation. In HEK293-MOR1 cells, low concentrations of DAMGO did not convert morphine into a receptor-internalizing agent. The data presented here fail to support the theory that low concentrations of DAMGO can increase morphine-induced MOR desensitization or internalization.

Involvement of PKCα and G-protein-coupled receptor kinase 2 in agonist-selective desensitization of µ-opioid receptors in mature brain neurons

Background and purpose:  The ability of an agonist to induce desensitization of the µ‐opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist‐specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high‐efficacy agonist).

Role of protein kinase C and μ-opioid receptor (MOPr) desensitization in tolerance to morphine in rat locus coeruleus neurons

In morphine tolerance a key question that remains to be answered is whether μ‐opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70–80%) of loss of receptor function. Ongoing PKC activity in the MOPr‐expressing neurons themselves, primarily by PKCα, was required to maintain morphine‐induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30–50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in α2‐adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC‐dependent component and that this desensitization underlies the maintenance of morphine tolerance.

Endomorphin-2: A Biased Agonist at the μ-Opioid Receptor

Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K+ current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K+ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

The effect of protein kinase C and G protein-coupled receptor kinase inhibition on tolerance induced by mu-opioid agonists of different efficacy.

Differences in the mechanisms underlying tolerance and μ-opioid receptor desensitization resulting from exposure to opioid agonists of different efficacy have been suggested previously. The objective of this study was to determine the effects of protein kinase C (PKC) and G protein-coupled receptor kinase (GRK) inhibition on antinociceptive tolerance in vivo to opioid agonists of different efficacy. A rapid (8-h) tolerance-induction model was used where each opioid was repeatedly administered to naive mice. Animals were then challenged with the opioid after injection of a kinase inhibitor to determine its effects on the level of tolerance. Tolerance to meperidine, morphine, or fentanyl was fully reversed by the PKC inhibitor 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)carbazole (Gö6976). However, in vivo tolerance to [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) was not reversed by PKC inhibition. The novel small-molecule GRK inhibitors β-adrenergic receptor kinase 1 inhibitor and 2-(8-[(dimethylamino) methyl]-6,7,8,9-tetrahydropyridol[1,2-a]indol-3-yl)-3-(1-methylindol-3-yl)maleimide (Ro 32-0432) did not reverse the tolerance to meperidine, fentanyl, or morphine but did reverse the tolerance to DAMGO. To correlate GRK-dependent DAMGO-induced tolerance with μ-opioid receptor desensitization, we used in vitro whole-cell patch-clamp recording from mouse locus coeruleus neurons and observed that the GRK inhibitors reduced DAMGO-induced desensitization of μ-opioid receptors, whereas the PKC inhibitor had no effect. These results suggest that tolerance induced by low- and moderate-efficacy μ-opioid receptor agonists is dependent on PKC, whereas tolerance induced by the high-efficacy agonist DAMGO is dependent on GRK.

Neurokinin-1 receptors in the rat nucleus tractus solitarius: pre- and postsynaptic modulation of glutamate and GABA release

Neurokinins such as substance P and neurokinin A have long been thought to act as neurotransmitters or modulators in the nucleus tractus solitarius. However, the role and location of the receptors for these peptides have remained unclear. We examined the consequences of activation of the neurokinin-1 (NK1) receptor subtype in the rat nucleus tractus solitarius using whole-cell patch clamp recordings in brain slices. Application of delta-Ala-Phe-Phe-Pro-MeLeu-D-Pro[spiro-gamma-lactam]-Leu-Trp-NH2 (a specific NK1 agonist) or neurokinin A resulted in depolarization, evident as a slow inward current, mediated by direct postsynaptic NK1 receptor activation. The effect was conserved in the presence of tetrodotoxin, and protein kinase C-dependent since it was blocked by 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide, a specific protein kinase C inhibitor. In addition, an increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents was observed, reflecting increased glutamate release induced by NK1 receptor activation. This effect was abolished by tetrodotoxin, suggesting that it resulted from increased firing in afferent neurons, subsequent to somatodendritic excitation via NK1 receptors. Furthermore, spontaneous inhibitory postsynaptic currents were increased in frequency and amplitude showing that GABA release was promoted by NK1 receptor activation. However, amplitude of miniature inhibitory postsynaptic currents was unaltered by NK1 receptor activation, but the increase in frequency persisted. These findings suggest that NK1 receptors are located on presynaptic terminals as well as at somatodendritic sites of GABAergic neurons. The increase in GABA release was also shown to be protein kinase C-dependent. The data presented here show NK1 receptors in the rat nucleus tractus solitarius are present both excitatory and inhibitory neurons. Activation of these receptors can result in increases in release of both GABA and glutamate, suggesting a crucial modulatory role for NK1 receptors in the rat nucleus tractus solitarius.

Pre-treatment with a PKC or PKA inhibitor prevents the development of morphine tolerance but not physical dependence in mice

We previously demonstrated that intracerebroventricular (i.c.v.) administration of protein kinase C (PKC) or protein kinase A (PKA) inhibitors reversed morphine antinociceptive tolerance in 3-day morphine-pelleted mice. The present study aimed at evaluating whether pre-treating mice with a PKC or PKA inhibitor prior to pellet implantation would prevent the development of morphine tolerance and physical dependence. Antinociception was assessed using the warm-water tail immersion test and physical dependence was evaluated by quantifying/scoring naloxone-precipitated withdrawal signs. While drug-naïve mice pelleted with a 75 mg morphine pellet for 3 days developed a 5.8-fold tolerance to morphine antinociception, mice pre-treated i.c.v. with the PKC inhibitors bisindolylmaleimide I, Go-7874 or Go-6976, or with the myristoylated PKA inhibitor, PKI-(14-22)-amide failed to develop any tolerance to morphine antinociception. Experiments were also conducted to determine whether morphine-pelleted mice were physically dependent when pre-treated with PKC or PKA inhibitors. The same inhibitor doses that prevented morphine tolerance were evaluated in other mice injected s.c. with naloxone and tested for precipitated withdrawal. The pre-treatment with PKC or PKA inhibitors failed to attenuate or block the signs of morphine withdrawal including jumping, wet-dog shakes, rearing, forepaw tremor, increased locomotion, grooming, diarrhea, tachypnea and ptosis. These data suggest that elevations in the activity of PKC and PKA in the brain are critical to the development of morphine tolerance. However, it appears that tolerance can be dissociated from physical dependence, indicating a role for PKC and PKA to affect antinociception but not those signs mediated through the complex physiological processes of withdrawal.