After stimulation by morphine or not, membranes were solubilized and immunoprecipitated first with immobilized anti-G
After stimulation by morphine or not, membranes were solubilized and immunoprecipitated first with immobilized anti-G. 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 Rabbit Polyclonal to PKA-R2beta 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 receptorCGs 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. Introduction 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]C[4]. Ultra-low-dose opioid antagonists can also reverse hyperalgesia caused by acute, low-dose opioids to produce analgesia [5]. Additionally, ultra-low-dose naltrexone has recently been shown to attenuate opioid reward or addictive properties in conditioned place preference [6] and self-administration and reinstatement paradigms [7]. In a recent clarification of the Salidroside (Rhodioloside) mechanism of action of ultra-low-dose opioid antagonists, we showed that co-treatment with 10 ng/kg naloxone (NLX) prevented a Salidroside (Rhodioloside) 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 G 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 MORCGs coupling [4], [9]. Although Chakrabarti et al (2005) also demonstrated some MORCGs coupling in the opioid na?ve state while we did not, we believe this difference may be due to their use of non-immobilized anti-G antibodies producing some background binding by Fc receptors. First postulated as the sole mediator of excitatory effects of opiates [10], the G interacting with adenylyl cyclases originates from the Gs protein coupling to MOR and not from MOR’s native G proteins [11]. Ultra-low-dose opioid antagonists were initially thought to preferentially bind a subset of MORs [1], and a Gs-coupling MOR subpopulation was again recently proposed [9]. While it is difficult to estimate the relative proportion of MORs signaling via Gs versus Gi/o during tolerance, it seemed unlikely that the ultra-low doses of NLX or naltrexone influencing opioid agonist effects would be sufficient to selectively antagonize such a subpopulation. Based on saturation binding studies [12], which incorporate NLX’s affinity to MOR, Salidroside (Rhodioloside) we estimate receptor occupancy of 10 ng/kg NLX as no more than 1%. More importantly, our co-immunoprecipitation data showed that ultra-low-dose NLX co-treatment reduces MORCGs coupling while levels of coupling to MOR’s native Gi/o proteins; further, in spinal cord of co-treated rats, MORCGi/o coupling levels greatly surpassed those of opioid-na?ve rats [4]. If NLX were selectively antagonizing a subpopulation of Gs-coupled MORs, coupling to native G proteins would likely be unaffected. Since NLX prevents MORCGs coupling at concentrations well below its affinity for MOR and by influencing the coupling behavior of MORs, we considered proteins that interact with MOR and MOR-associated G proteins as the most likely targets, particularly those able to interact with multiple MORs. We first examined proteins that co-immunoprecipitated with MOR during activation. We identified a 300-kDa protein co-immunoprecipitating with MOR as FLNA and then demonstrated specific, high-affinity binding by NLX to FLNA. Best known for cross-linking cytoplasmic actin into dynamic scaffolds to control cell motility, filamins are large cytoplasmic proteins increasingly found to regulate cell signaling by interacting with over 30 different receptors and signaling molecules [13], [14], including MOR [15]. We deduced the precise binding site on FLNA by using overlapping peptides within the c-terminal, since c-terminal FLNA was shown to interact with MOR using a yeast-two hybrid [15]. To assess the functional significance of this high-affinity interaction, we used peptide fragments containing the binding site to prevent NLX from binding full-length FLNA in organotypic striatal slice cultures. Our findings suggest that FLNA interacts with ultra-low-dose NLX and naltrexone to prevent chronic morphine-induced MORCGs coupling, possibly by preventing a critical MORCFLNA interaction. This high-affinity.