High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence.
Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [(3)H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [(3)H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A(2561-2565) as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A(2561-2565) abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor-Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence.
Wang
Hoau-Yan
HY
Department of Physiology and Pharmacology, City University of New York Medical School, New York, New York, USA. hywang@sci.ccny.cuny.edu
Frankfurt
Maya
M
Burns
Lindsay H
LH
D000818:Animals; D001923:Brain Chemistry; D045744:Cell Line, Tumor; D003285:Contractile Proteins / Q000378:metabolism* / Q000502:physiology*; D003342:Corpus Striatum / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004361:Drug Tolerance; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism* / Q000502:physiology*; D009270:Naloxone / Q000494:pharmacology*; D009292:Narcotic Antagonists; D051381:Rats; D017450:Receptors, Opioid, mu / Q000502:physiology*
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D009292:Narcotic Antagonists; C485764:OPRM1 protein, human; D017450:Receptors, Opioid, mu; D009270:Naloxone