Abstract
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 [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]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 A2561-2565 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 A2561-2565 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.
Highlights
Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia and attenuate tolerance and dependence, with a mechanism long hypothesized as a blockade of excitatory signaling opioid receptors [1,2,3,4]
The exclusive presence of FLNA in the mu opioid receptor (MOR)/Go complexes led us to hypothesize that FLNA is the target through which ultra-low-dose NLX blocks the chronic morphine-induced switch from normal MOR–Gi/o coupling to Gs coupling
We have identified a high-affinity binding site for NLX in the carboxyl-terminal region of the scaffolding protein FLNA that appears to mediate ultra-low-dose NLX’s prevention of the chronic opioid-induced G protein coupling switch by MOR
Summary
Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia and attenuate tolerance and dependence, with a mechanism long hypothesized as a blockade of excitatory signaling opioid receptors [1,2,3,4]. In a recent clarification of the mechanism of action of ultra-low-dose opioid antagonists, we showed that co-treatment with 10 ng/kg naloxone (NLX) prevented a chronic morphine-induced, Gi/o-to-Gs switch in G protein coupling by the mu opioid receptor (MOR) as well as a coincident interaction of the Gbc dimer with adenylyl cyclase II and IV [4]. While opioid receptors preferentially bind Gi and Go proteins to inhibit adenylyl cyclase [8], chronic morphine induces MOR–Gs coupling [4,9]. First postulated as the sole mediator of excitatory effects of opiates [10], the Gbc interacting with adenylyl cyclases originates from the Gs protein coupling to MOR and not from MOR’s native G proteins [11]
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