The carboxyl tail of the human opioid receptor was shown to bind the carboxyl terminal region of human filamin A, a protein known to couple membrane proteins to actin. Results from yeast two-hybrid screening were confirmed by direct proteinprotein binding and by coimmunoprecipitation of filamin and opioid receptor from cell lysates. To investigate the role of filamin A in opioid receptor function and regulation, we used the melanoma cell line M2, which does not express filamin A, and its subclone A7, transfected with human filamin A cDNA. Both cell lines were stably transfected with cDNA encoding myctagged human opioid receptor. Fluorescent studies, using confocal microscopy, provided evidence that filamin and opioid receptors were extensively colocalized on the membranes of filamin-expressing melanoma cells. The immunostaining of opioid receptors indicated that the lack of filamin had no detectable effect on membrane localization of the receptors. Moreover, opioid receptors function normally in the absence of filamin A, as evidenced by studies of opioid binding and DAMGO inhibition of forskolin-stimulated adenylyl cyclase. However, agonist-induced receptor down-regulation and functional desensitization were virtually abolished in cells lacking filamin A. The level of internalized -opioid receptors, after 30-min exposure to agonist, was greatly reduced, suggesting a role for filamin in opioid receptor trafficking. During these studies, we observed that forskolin activation of adenylyl cyclase was greatly reduced in filamin-lacking cells. An even more unexpected finding was the ability of long-term treatment with [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin of M2 cells, containing opioid receptors, to restore normal forskolin activation. The mechanism of this effect is currently unknown. It is postulated that the observed effects on opioid receptor regulation by filamin A and, by implication, of the actin cytoskeleton may be the result of its role in opioid receptor trafficking.Opioid receptors belong to the superfamily of G protein coupled receptors (GPCRs). They are involved in numerous physiological functions, including analgesia, respiration, and cardiovascular functions. These receptors are also involved in the development of the physical and psychological dependence that are important aspects of drug abuse.Current work in many laboratories, including our own, is directed toward understanding opioid receptor signaling, regulation, and trafficking. Agonist binding induces a change in the conformation of GPCRs because of interaction of the receptors with different kinases, including second messenger-dependent protein kinases and G-protein coupled receptor kinases (GRK). Phosphorylation of the receptors promotes binding of ␤-arrestins and uncouples the activated GPCRs from G-proteins. Uncoupling of the receptor from heterotrimeric G-proteins after short-term agonist exposure leads to receptor desensitization, which results in a decrease in agonist affinity and receptor function. In addition to receptor d...

We investigated the effects of morphine and other agonists on the human mu opioid receptor (MOP) expressed in M2 melanoma cells, lacking the actin cytoskeleton protein filamin A and in A7, a sub clone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed, that after chronic morphine treatment (24 hr) of A7 cells, MOP binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was upregulated to 188% of control naïve cells. Similar up-regulation was observed with the agonists methadone and levorphanol. The presence of antagonists (naloxone or CTAP) during chronic morphine treatment inhibited MOP down-regulation in A7 cells. In contrast, morphine-induced upregulation of MOP in M2 cells was further increased by these antagonists. Chronic morphine desensitized MOP in A7 cells, i.e. it decreased DAMGO-induced stimulation of GTPγS binding. In M2 cells DAMGO stimulation of GTPγS binding was significantly greater than in A7 cells and was not desensitized by chronic morphine. Pertussis toxin treatment abolished morphine-induced receptor up-regulation in M2 cells, whereas it had no effect on morphine-induced down-regulation in A7 cells. These results indicate that, in the absence of filamin A, chronic treatment with morphine, methadone or levorphanol leads to up-regulation of MOP, to our knowledge, the first instance of opioid receptor up-regulation by agonists in cell culture.

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We have previously reported that the protein filamin A (FLA) binds to the carboxyl tail of the mu opioid receptor (MOPr). Using human melanoma cells, which do not express filamin A, we showed that receptor down-regulation, functional desensitization and trafficking are deficient in the absence of FLA (Onoprishvili et al. Mol Pharmacol 64:1092-1100, 2003). Since FLA has a binding domain for actin and is a member of the family of actin cytoskeleton proteins, it is usually assumed that FLA functions via the actin cytoskeleton. We decided to test this hypothesis by preparing cDNA coding for mutant FLA lacking the actin binding domain (FLA-ABD) and expressing FLA-ABD in the human melanoma cell line M2 (M2-ABD cell line). We report here that this mutant is capable of restoring almost as well as full length FLA the down-regulation of the human MOPr. It is similarly very effective in restoring functional desensitization of MOPr, as assessed by the decrease in G-protein activation after chronic exposure of M2-ABD cells to the mu agonist DAMGO. We also found that A7 cells, expressing wild type FLA, exhibit rapid activation of the MAP kinases, ERK 1 and 2, by DAMGO, as shown by a rise in the level of phospho-ERK 1 and 2. This is followed by rapid dephosphorylation (inactivation), which reaches basal level between 30 and 60 min after DAMGO treatment. M2 cells show normal activation of ERK 1 and 2 in the presence of DAMGO, but very slow inactivation. The rapid rate of MAPK inactivation is partially restored by FLA-ABD. We conclude that some functions of FLA do not act via the actin cytoskeleton. It is likely that other functions, not studied here, may require functional binding of the MOPr-FLA complex to actin.

Our laboratory embarked on research to discover proteins the interaction of which with the mu opioid receptor (MOPr) is required for its function and regulation. We performed yeast two-hybrid screens, using the carboxy tail of the human MOPr as bait and a human brain library. This yielded a number of proteins that seemed to bind to the MOPr C-tail. The one we chose to study in detail was filamin A (FLNA). Evidence was obtained that there was indeed protein-protein binding between the C-tail of MOPr and FLNA. A human melanoma cell line (M2) lacking the gene for FLNA and a control cell line (A7) which differed from M2 only in having been transfected with the gene for FLNA and expressing the FLNA protein were made available to us. We transfected these cell lines with the gene for MOPr and used them in our studies. The absence of FLNA strongly reduced MOPr downregulation as well as desensitization of adenylyl cyclase inhibition and G protein activation. A recent finding, published here for the first time, is that FLNA is required for the activation by mu opioid agonists of the MAP kinase p38. Deletion studies indicated that the MOPr binding site on FLNA is in the 24th repeat, close to its C-terminal. It was further found that FLNA lacking the N-terminal actin binding domain is as capable as full length FLNA to restore cells to control status, suggesting that actin binding is not required. A surprising finding was that upregulation of MOPr by morphine and some agonist analogs occurs in M2 cells lacking FLNA, whereas normal receptor downregulation takes place in A7 cells.