Morphine-like opiates selectively antagonize receptor-arrestin interactions.
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 mu and delta receptors with G protein or beta-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 Gbeta(1). In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was G alpha-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 delta and mu receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (delta) or partial agonists (mu) 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.
Molinari
Paola
P
Department of Pharmacology, Istituto Superiore di Sanità, Rome 00161, Italy.
Vezzi
Vanessa
V
Sbraccia
Maria
M
Grò
Cristina
C
Riitano
Daniela
D
Ambrosio
Caterina
C
Casella
Ida
I
Costa
Tommaso
T
D019390:Arrestins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004745:Enkephalins / Q000037:antagonists & inhibitors; D044385:GTP-Binding Protein alpha Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009294:Narcotics / Q000494:pharmacology*; D010111:Oxymorphone / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000378:metabolism*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D004745:Enkephalins; D044385:GTP-Binding Protein alpha Subunits; D009294:Narcotics; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D010111:Oxymorphone; D037342:Pertussis Toxin