Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between -opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though -receptors and ␦-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.Opioid alkaloids, as well as endogenous opioid peptides, exert their multiple biological effects on target tissues by interacting with specific cell surface receptors including the ␦-, -, and -opioid receptors (1). These opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs).1 The -opioid receptor (MOR) serves as the principle physiological target for most clinically important opioid analgesics, such as morphine and fentanyl (2, 3). Although many opioid alkaloids exert their pharmacological effects via MOR, their binding affinity for the receptor and potency to activate the receptor do not always correspond to their abilities to induce tolerance (4 -8). This suggests that other cellular processes that modulate MOR responsiveness, such as receptor desensitization and internalization, may contribute to opioid tolerance and dependence. Like many other GPCRs, opioid receptors are regulated by agonist-dependent processes and undergo receptor phosphorylation, desensitization, internalization, and down-regulation (1). Interestingly, in addition to the subtype-specific regulation of opioid receptors (9 -12), individual opioid receptors are differentially regulated by distinct opioid agonists (5-8, 13, 14). In the case of MOR, opioid agonists demonstrating equivalent ability to activate receptor signaling exhibit remarkable differences in their abilities to functionally desensitize (5, 6) and induce internalization of the receptor in both transfected cells and neurons (7,8,(13)(14)(15). However, the detailed molecular events underlying this differential regulation of MOR by distinct agonists remain unclear. Using chimeric, truncated, or mutated opioid receptors, several studies reported the crucial role of the C-tail of opioid receptors in regulating their activities and trafficking (11, 16 -20). Whereas several potential phosphorylation sites were suggested to be involved in regulating the activity and trafficking of opioid receptors, the actual ...

Homo-and heterodimerization of the opioid receptors with functional consequences were reported previously. However, the exact nature of these putative dimers has not been identified. In current studies, the nature of the heterodimers was investigated by producing the phenotypes of the 1:1 heterodimers formed between the constitutively expressed -opioid receptor (MOR) and the ponasterone A-induced expression of ␦-opioid receptor (DOR) in EcR293 cells. By examining the trafficking of the cell surface-located MOR and DOR, we determined that these two receptors endocytosed independently. Using cell surface expression-deficient mutants of MOR and DOR, we observed that the corresponding wild types of these receptors could not rescue the cell surface expression of the mutants, whereas the antagonist naloxone could. Furthermore, studies with constitutive or agonist-induced receptor internalization also indicated that MOR and DOR endocytosed independently and could not "drag in" the corresponding wild types or endocytosis-deficient mutants. Additionally, the heterodimer phenotypes could be eliminated by the pretreatment of the EcR293 cells with pertussis toxin and could be modulated by the deletion of the RRITR sequence in the third intracellular loop that is involved in the receptor-G protein interaction and activation. These data suggest that MOR and DOR heterodimerize only at the cell surface and that the oligomers of opioid receptors and heterotrimeric G protein are the bases for the observed MOR-DOR heterodimer phenotypes.The ability of G protein-coupled receptors (GPCRs) 1 to homoor heterodimerize has implications in the functions of the receptors. Dimerization of the receptors has been reported for the class A GPCRs such as the adenosine (1), adrenergic (2-5), angiotensin (6), dopamine (7,8), muscarinic (9), vasopressin (2, 10), and opioid (11-15) receptors and the class C GPCRs such as the calcium-sensing (16), the metaboropic glutamate receptors (17), and the ␥-amino-n-butyric acid type B (GABA B ) receptors (18 -20). The homo-and heterodimerization of these receptors have been demonstrated by co-immunoprecipitation experiments (11, 21) and subsequently by the fluorescence resonance energy transfer or bioluminescence resonance energy transfer techniques (3,12,23). The heterodimerization of the GPCRs was shown to be selective, with formation of heterodimers with some but not all subtypes of the receptors (13, 24, 25). Most importantly, there are functional differences between the monomers and the homo-and heterodimers of the GPCRs. The classic example is the inability of individual GABA B1 and GABA B2 subunit to form a functional receptor (18 -20). Alteration in the GPCR function or expression was also observed with the heterodimerization of 5HT1B and -1D (26), dopamine D1 and adenosine A1 (27), muscarinic M2 and M3 (28), or dopamine and somatostatin (29) receptors. Heterooligomerization of the GPCRs with other receptor types, such as the ionotropic GABA A receptor, has been observed, resulting in the alteration ...

Deletion of a sequence near the fifth transmembrane domain ( 258 RLSKV 262 , i3-1 mutant) and a motif residing at the proximal carboxyl tail ( 344 KFCTR 348 , C-2 mutant) resulted in -opioid receptor mutants that were poorly expressed on the surface of transfected human embryonic kidney 293 cells. Treatment with the opioid antagonist naloxone, the agonist etorphine, and other hydrophobic ligands enhanced cell surface expression of i3-1 and C-2 mutants. The observed enhancement was timeand concentration-dependent, required the ligands to be membrane permeable, and was not the result of the reversal of the constitutive activities of the mutant receptors. The binding of the ligands resulted in the trafficking of the mutant receptors retained in the endoplasmic reticulum to the cell surface. The cell surface-expressed mutant C-2, but not i3-1, fully retained ability to mediate inhibition of adenylyl cyclase activity. Furthermore, the Golgi-disturbing agents brefeldin A and monensin completely blocked naloxone-enhanced expression of i3-1 and C-2 mutants. Results of these studies suggest that intracellular interactions of agonist and antagonist with mutant receptors can serve as chaperones in the trafficking of the mutants to the cell surface.The G protein-coupled receptors (GPCRs), with more than 1000 members, are one of the largest superfamilies of membrane proteins (Wess, 1998). A large body of evidence has revealed that transmembrane and extracellular loop determine selectivity of agonist binding, whereas the intracellular loops are responsible for G-protein coupling (Wess, 1998). Mutation or deletion of the transmembrane domains and intracellular loops resulted in the gain or loss of function in several GPCRs. Gain-of-function receptor mutants are characterized by constitutive activities with agonist-independent activation. The constitutive activities can be suppressed by binding of the negative antagonist (inverse agonist). In addition, inverse agonists are able to increase the expression of the constitutively active mutant (Pei et al., 1994;MacEwan and Milligan, 1996;Gether et al., 1997;McLean et al., 1999;Stevens et al., 2000). Inverse agonist-induced up-regulation and agonist-independent phosphorylation of receptor mutants suggest the existence of constitutive down-regulation of the constitutively active receptor mutants. The down-regulation of the receptor could be the mechanism for the lower expression level of many constitutively active receptor mutants observed when they are expressed in cell lines.In addition to the constitutively active mutants, mutations in any portion of the GPCRs have resulted in the intracellular retention of the mutants at the ER. This retention has no apparent dependence on sequence motif. In particular, the deletion or mutation of the third intracellular (i3) loop or carboxyl tail of many GPCRs has been reported to result in low receptor expression in transfected cells (Cheung et al

In a previous study, we demonstrated that antagonists such as naloxone or naltrexone acted as full agonists at the -opioid receptor (MOR)/␦-opioid receptor (DOR) chimeric receptor (␦2, where the DOR sequence from the first extracellular loop to the carboxyl terminus was spliced to the MOR sequence) when a conserved serine residue in transmembrane 4 (TM4) was mutated to leucine. However, when Ser 196 in the TM4 of MOR was mutated to Leu, antagonists exhibited partial agonistic properties. Since molecular modeling studies suggested transmembrane movement during receptor activation, the observed partial agonistic properties could be due to TM1 and TM7 interaction. Hence, MOR/DOR chimeric mutant receptors with the MOR TM1 and TM7 sequence (␦ 2 7 S196L) or with the MOR TM1 and TM6/7 sequence (␦ 2 67 S196L) were constructed to test such a hypothesis. Using four tests of opioid receptor activation, we found that the opioid antagonists were full agonists in chimeric mutant receptor if the TM1 and TM7 were from different opioid receptors. Additionally, when two of the TM7 amino acid residues of MORS196L receptor mutants were mutated (T327A and C330S), resulting in a mutant receptor with DOR TM7 sequence, opioid antagonist naloxone exhibited full agonistic properties. These data suggest that the efficacy of opioid antagonists in the Ser 196 mutant can be affected by the interaction between TM1 and TM7.