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...

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Currently, most basic and clinical research on depression is focused on either central serotonergic, noradrenergic, or dopaminergic neurotransmission as affected by various etiological and predisposing factors. Recent evidence suggests that there is another system that consists of a subset of brain a 1B -adrenoceptors innervated primarily by brain epinephrine (EPI) that potentially modulates the above three monoamine systems in parallel and plays a critical role in depression. The present review covers the evidence for this system and includes findings that brain a 1 -adrenoceptors are instrumental in behavioral activation, are located near the major monoamine cell groups or target areas, receive EPI as their neurotransmitter, are impaired or inhibited in depressed patients or after stress in animal models, and are restored by a number of antidepressants. This 'EPI-a 1 system' may therefore represent a new target system for this disorder.

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The role of brain epinephrine (EPI) in the regulation of motor activity and movement in mice was examined. Blockade of EPI synthesis with i.p. 2,3-dichloro-alpha-methylbenzylamine (DCMB) or LY134046 was found to produce marked behavioral inactivity which could be significantly reversed by intraventricular injection of EPI and by three other alpha(1)-adrenoceptor agonists, norepinephrine (NE), 6-fluoronorepinephrine (6FNE), and phenylephrine (PE), as well as by serotonin (5HT). EPI had the largest effect of these agonists and also was the only one that reversed nondrug-induced inactivity of mice in their home cages during the light phase. The effects of EPI were blocked by coinfusion of an alpha(1)-adrenoceptor antagonist (terazosin) but not of an alpha(2)-(atipamezole) or beta(1) (betaxolol)-blocker. The rank order of maximal behavioral responses to EPI, 6FNE, and PE in DCMB-treated mice was the same as the rank order of their maximal stimulation of hydrolysis of phosphatidylinositol at cloned alpha(1B)-adrenoceptors in cell culture. On the basis of the above findings and of the central distributions of adrenergic neurons and alpha(1)-adrenoceptors, the existence of a central EPI-innervated alpha(1)-adrenergic receptor system is postulated which serves to coexcite or enhance signaling in several monoaminergic brain regions involved in movement and motor activity.