Morphine-like Opiates Selectively Antagonize Receptor-Arrestin Interactions

Abstract

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of and ␦ receptors with G protein or ␤-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent G␤ 1 . In this system, the agonistinduced enhancement of BRET (indicating shortening of distance between the two proteins) was G␣-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at ␦ and receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (␦) or partial agonists () for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.Physiological agonists are usually equally efficient in promoting the interaction of receptors with G protein and arrestin, but manmade analogues can show divergent molecular efficacies for the two transducers (1, 2). This phenomenon, often addressed with a pictorial terminology (3-5), has attracted great interest and if better understood might lead to new types of drugs.The differential efficacy of opioids for G protein and arrestin interactions is also important in the mechanism of opiate addiction. As reported earlier, the addictive opiate morphine cannot induce and actually blocks desensitization and G protein uncoupling of ␦-opioid receptors (DOPR) 2 in neuroblastoma or in transfected cells (6, 7). Subsequent work shows that morphine is a poor inducer of rapid arrestin-dependent endocytosis for both ␦ and (MOPR) receptors (8 -11), although one exception is in striatum neurons with high levels of G protein receptor kinases (12).Two theories predict a relation between lack of endocytosis and the addiction liability of opioids, but the proposed explanations are radically different. One sees rapid endocytosis as a means to quench receptor signaling. Thus, the abnormally sustained signaling pattern produced by a drug that cannot internalize the receptor would promote post-receptor compensatory mechanisms, which may be responsible for the...

6′-Guanidinonaltrindole (6′-GNTI) Is a G Protein-biased κ-Opioid Receptor Agonist That Inhibits Arrestin Recruitment

“…As mentioned above, although not without controversy (17,18), the KOR-mediated arrestin pathway has been suggested to influence the development of antinociceptive tolerance in vivo (20,21). It is noteworthy that arrestin3 KO mice display enhanced and prolonged antinociception after short term treatment with morphine and heroin in the hot plate test, a model of thermal antinociception (35).…”

“…This phenomenon, referred to as functional selectivity, biased agonism, or ligand-directed signaling/receptor trafficking, has been widely observed (15,16). For example, at the level of the DOR and MOR receptors, several compounds have been identified as agonists for G protein coupling but as antagonists (DOR) or partial agonists (MOR) for arrestin (17).…”

“…Although still an area of controversy (see also the "morphine paradox" (17,18)), it has been proposed that the ability of an opioid agonist to promote arrestin-dependent desensitization and internalization in vitro is related to the degree of tolerance that develops after long term treatment in vivo (19,20). Arrestin recruitment to KOR has also been proposed to play a role in antinociceptive tolerance (20,21), making the development of a G protein-biased agonist an important goal for achieving analgesic efficacy without the adverse effects triggered by arrestin (4,11).…”

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

“…Cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 with 10% (v/v) fetal calf serum, 100 g/ml hygromycin B, and 400 g/ml G418 in a humidified atmosphere of 5% CO 2 at 37°C. Enriched membranes from transfected cells were obtained by differential centrifugation (22) and stored in aliquots at Ϫ80°C before use.…”

“…Cells and Membranes-The preparation of retroviral vectors coding for Rluc-tagged human MOP and DOP receptors and RGFP-fused G␤1 or ␤-arrestin 2 and the transduction of SH-5YSY human neuroblastoma cells were described previously (22). Cells were grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and F-12 with 10% (v/v) fetal calf serum, 100 g/ml hygromycin B, and 400 g/ml G418 in a humidified atmosphere of 5% CO 2 at 37°C.…”