enkephalin--ala(2)-mephe(4)-gly(5)- and Melanoma

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.

Topics: Actins; Cell Line, Tumor; Contractile Proteins; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Activation; Filamins; Humans; Melanoma; Microfilament Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Receptors, Opioid, mu

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 subclone of the M2 melanoma cells, stably transfected with filamin A cDNA. The results of binding experiments showed that after chronic morphine treatment (24 h) of A7 cells, MOP-binding sites were down-regulated to 63% of control, whereas, unexpectedly, in M2 cells, MOP binding was up-regulated to 188% of control naive 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 up-regulation 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 GTPgammaS binding. In M2 cells DAMGO stimulation of GTPgammaS 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.

Topics: Blotting, Western; Cell Line; Cell Line, Tumor; Contractile Proteins; Data Interpretation, Statistical; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Filamins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Melanoma; Microfilament Proteins; Morphine; Narcotic Antagonists; Narcotics; Pertussis Toxin; Radioligand Assay; Receptors, Opioid, mu; Tubulin; Up-Regulation