This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged ␦ and receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulsechase experiments using [35 S]methionine metabolic labeling indicated that the turnover rate of ␦ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G i and G o proteins by pertussis toxin-attenuated down-regulation of the opioid receptor, while down-regulation of the ␦ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on and ␦ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced and ␦ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steadystate and ␦ opioid receptor levels. Immunoprecipitation of and ␦ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.The pharmacological effects of opioid drugs and the physiological effects of endogenous opioid peptides are initiated through the binding and activation of opioid receptors (1), which are members of the G protein-coupled receptor (GPCR) 1 family (2). GPCRs comprise a diverse superfamily of integral membrane proteins that mediate signal transduction across the plasma membrane. All GPCRs are postulated to have amino termini located on the extracellular side of the plasma membrane that are linked to seven-transmembrane helices connected by relatively short intracellular and extracellular loops, and contain carboxyl termini that face the interior of the cell. Ligands approach and engage GPCRs from the extracellular space, and receptor activation results in coupling to heterotrimeric G proteins on the intracellular face of membrane. The amino termini of nearly all G protein-coupled receptors contain consensus amino acid sequences for asparagine-linked glycosylation; two sites for N-linked glycosylation are present in ␦ and receptors, while five are found in the receptor (3).Three types of opioid receptor, ␦, , and , have been cloned and characterized extensively (4 -7). Opioid receptors have unique ligand specificities, anatomical distributions, and physiological functions (8 -15). Opioid receptors exhibit ϳ60% identity in their amino acid sequences, however, marked differences in sequence conservation are evident within receptor subdomains. The amino acid sequences of putative transmembrane spanning segments and the three intracellular loops are highly conserved among opioid receptor types, whereas sequences in the extracellular amino termini, second and third extracellular...

The neurotransmitter dopamine is known to inhibit prolactin secretion and the proliferation of lactotropes in the pituitary gland. In this study, we determined whether dopamine and TGFbeta1 interact to regulate lactotropic cell proliferation. We found that dopamine and the dopamine agonist bromocriptine stimulated TGFbeta1 secretion and TGFbeta1 mRNA expression but inhibited lactotropic cell proliferation both in vivo and in vitro. The dopamine's inhibitory action on lactotropic cell proliferation was blocked by a TGFbeta1-neutralizing antibody. We also found that PR1 cells, which express low amounts of the dopamine D2 receptor, demonstrated reduced expression of TGFbeta1 type II receptor and TGFbeta1 mRNA levels and had undetectable levels of TGFbeta1 protein. These cells showed a reduced TGFbeta1 growth-inhibitory response. Constitutive expression of the D2 receptor short isoform, but not the D2 receptor long isoform, induced TGFbeta1 and TGFbeta1 type II receptor gene expression and recovered dopamine- and TGFbeta1-induced growth inhibition in PR1 cells. The constitutive expression of D2 receptor short isoform also reduced the tumor cell growth rate. These data suggest that a TGFbeta1 system may mediate, in part, the growth-inhibitory action of dopamine on lactotropes.

Significant advances have been made in understanding the structure, function, and regulation of opioid receptors and endogenous opioid peptides since their discovery approximately 25 years ago. This review summarizes recent studies aimed at identifying key amino acids that confer ligand selectivity to the opioid receptors and that are critical constituents of the ligand binding sites. A molecular model of the δ receptor based on the crystal structure of rhodopsin is presented. Agonist‐induced down regulation of opioid receptors is discussed, highlighting recent evidence for the involvement of the ubiquitin/proteasome system in this process. © 2001 John Wiley & Sons, Inc. Biopolymers (Pept Sci) 55: 334–346, 2000

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The mechanism by which ethanol induces ␤-endorphin (␤-EP) neuronal death during the developmental period was determined using fetal rat hypothalamic cells in primary cultures. The addition of ethanol to hypothalamic cell cultures stimulated apoptotic cell death of ␤-EP neurons by increasing caspase-3 activity. Ethanol lowered the levels of adenylyl cyclase (AC)7 mRNA, AC8 mRNA, and/or cAMP in hypothalamic cells, whereas a cAMP analog blocked the apoptotic action of ethanol on ␤-EP neurons. The AC inhibitor dideoxyadenosine (DDA) increased cell apoptosis and reduced the number of ␤-EP neurons, and it potentiated the apoptotic action of ethanol on these neurons. ␤-EP neurons in hypothalamic cultures showed immunoreactivity to transforming growth factor-␤1 (TGF-␤1) protein. Ethanol and DDA increased TGF-␤1 production and/or release from hypothalamic cells. A cAMP analog blocked the activation by ethanol of TGF-␤1 in these cells. TGF-␤1 increased apoptosis of ␤-EP neurons, but it did not potentiate the action of ethanol or DDA actions on these neurons. TGF-␤1 neutralizing antibody blocked the apoptotic action of ethanol on ␤-EP neurons. Determination of TGF-␤1-controlled cell apoptosis regulatory gene levels in hypothalamic cell cultures and in isolated ␤-EP neurons indicated that ethanol, TGF-␤1, and DDA similarly alter the expression of these genes in these cells. These data suggest that ethanol increases ␤-EP neuronal death during the developmental period by cellular mechanisms involving, at least partly, the suppression of cAMP production and activation of TGF-␤1-linked apoptotic signaling.