Recent biochemical, biophysical, and functional studies suggest that G protein-coupled receptors (GPCRs) 1 can assemble as homo-or heterodimeric complexes (1, 2). Heterodimerization has been shown to alter both ligand binding affinity and signaling efficacy of GPCRs (1, 2). ␦-and -opioid receptors form stable heterodimers with ligand binding and signaling properties resembling that of the 2 receptor (3). Formation of heterodimers between the sst 1 and sst 5 somatostatin receptors has been found to modulate the pharmacology and signaling of both receptors (4). The ␥-aminobutyric acid receptor B is unique in that heterodimerization of the nonfunctional ␥-aminobutyric acid receptors B1 and B2 is required for native affinity for ligands and complete functional activity (5-9). Heteromeric assembly of fully functional AT 1 angiotensin II and B 2 bradykinin receptors results in increased efficacy of angiotensin II and decreased efficacy of bradykinin (10). Heterodimerization has also been shown to alter endocytotic trafficking of GPCRs (3, 4, 10, 11). The -␦ heterodimer exhibited a decrease in agonist-mediated receptor endocytosis (3). Oligomerization of ␦-and -opioid receptors with the distantly related ␤ 2 -adrenergic receptor results in increased and decreased receptor endocytosis, respectively (11). AT 1 -B 2 heterodimerization induced a switch to a clathrin-and dynamindependent endocytotic pathway for both receptors (10). Signaling of GPCRs is often terminated by phosphorylation of intracellular serine and threonine residues. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin. Although changes in trafficking have been clearly documented, agonistinduced phosphorylation and desensitization of these GPCR heterodimers has not been examined.We have recently shown that the sst 2A and sst 3 somatostatin receptors exist as constitutive homodimers when expressed alone and as constitutive heterodimers when coexpressed in human embryonic kidney (HEK) 293 cells (12). Whereas the sst 2A -sst 3 heterodimer behaved like the sst 2A homodimer, it did not reproduce the pharmacological characteristics of the sst 3 homodimer, suggesting that physical interaction of sst 3 with sst 2A induced functional inactivation of the sst 3 subtype (12). Here we report that the sst 2A receptor also forms stable heterodimers with the -opioid receptor (MOR1), a member of a closely related GPCR family. Unlike that observed for the sst 2A -sst 3 heterodimer, sst 2A -MOR1 heterodimerization did not significantly affect the ligand binding or coupling properties but promoted cross-modulation of phosphorylation, internalization, and desensitization of these receptors. EXPERIMENTAL PROCEDURES Materials

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human l-opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased b-endorphin binding affinity and a decreased potency of morphine-6-glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist-induced desensitization, internalization and resensitization of the human l-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human l-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or b-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.

The main analgesic effects of the opioid alkaloid morphine are mediated by the -opioid receptor. In contrast to endogenous opioid peptides, morphine activates the -opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse -opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [D-Ala 2 ,Me-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four -receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell-and tissuespecific C-terminal splicing of the -opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.Opioid receptors couple via G proteins to a variety of downstream effectors including adenylate cyclase (1) and mitogenactivated protein kinases (2-5). During repeated or continuous agonist stimulation these responses undergo rapid desensitization. An important mechanism of desensitization of G proteincoupled receptors is the phosphorylation of intracellular receptor domains by G protein-coupled receptor kinases or second messenger-regulated protein kinases such as Ca 2ϩ /calmodulindependent protein kinase II, cAMP-dependent protein kinase, or protein kinase C. After phosphorylation of the receptor, arrestins are frequently recruited to the plasma membrane, at which they facilitate endocytosis by serving as scaffolding proteins that bind to clathrin (6, 7). For some time it has been assumed that the rapid removal of ligand-activated receptors from the cell surface plays a major role in the receptor degradation, thus enhancing functional desensitization. Recent studies have suggested that endocytozed receptors are predominantly recycled to the cell surface in a reactivated state (8, 9).We have shown previously that two C-terminal splice variants (rMOR1 and rMOR1B) 1 of the rat -opioid receptor differ in their DAMGO-mediated internalization and resensitization rates (10, 11). The rapid internalizing variant rMOR1B revealed a faster resensitization and consequently a slower desensitization as compared with rMOR1. These results clearly show that receptor recycling after interna...

The rat opioid receptor is alternatively spliced into two isoforms (MOR1 and MOR1B) which differ in length and amino acid composition at the carboxyl terminus. ]enkephalin (DAMGO) with similar affinity and exhibit functional coupling to adenylyl cyclase with similar efficiency. However, the shorter isoform, MOR1B, desensitized at a slower rate during prolonged DAMGO exposure (4 h) but resensitized at a faster rate than MOR1 during agonist withdrawal (20 min). Immunocytochemical analysis revealed that DAMGO-induced internalization of MOR1B proceeded much faster than that of MOR1 followed by rapid recycling of the receptor to the cell surface. In addition, the greater resistance of MOR1B to homologous desensitization compared with MOR1 as well as MOR1B resensitization was abolished when receptor reactivation/recycling was blocked with monensin, an inhibitor of endosomal acidification. It is concluded that the sequence at the cytoplasmic tail of MOR1B facilitates clathrin-coated vesicle-mediated endocytosis which, in turn, promotes accelerated receptor reactivation. Taken together, our findings suggest that carboxyl-terminal splicing of the rat opioid receptor modulates agonist-induced internalization and receptor resensitization.Prolonged exposure of G protein-coupled receptors to agonists results in a rapid decrease of receptor responsiveness. It is now generally accepted that agonist-induced desensitization involves phosphorylation of intracellular receptor domains. Several kinases have been implicated in opioid receptor desensitization, including cAMP-dependent protein kinase (PKA), 1 protein kinase C (PKC), and calcium/calmodulin-dependent protein kinase II (CaM kinase II) (1-5). Specific phosphorylation sites have been localized in the third intracellular domain and at the carboxyl terminus, which play a critical role in homologous desensitization of the opioid receptor (6 -10). Following phosphorylation, the receptor is being targeted to the endocytotic machinery. A large body of evidence suggests that the main route of internalization of G protein-coupled receptors is via clathrin-coated pits and vesicles into early endosomes. Within the acidic environment of the endosomes, the ligand is effectively separated from the receptor which becomes dephosphorylated and thus resensitized. As a final step, the receptor recycles back to the cell surface (11).For the opioid receptor, desensitization seems to be regulated by CaM kinase II-mediated phosphorylation of two serine residues (Ser 261 /Ser 266 ) in the third intracellular loop (3, 9). Another important phosphorylation site of the opioid receptor is the threonine at position 394 in the carboxyl terminus. Indeed, we and others have recently observed that site-directed mutagenesis of Thr 394 to alanine profoundly delays DAMGOinduced desensitization, suggesting that this site may be a primary target for phosphorylation by GRKs upon agonist binding to the MOR1 (9, 10).We have previously shown, that the cytoplasmic tail of the rat opioid receptor undergoes alternati...

Several recent studies suggest that G protein-coupled receptors can assemble as heterodimers or hetero-oligomers with enhanced functional activity. However, inactivation of a fully functional receptor by heterodimerization has not been documented. Here we show that the somatostatin receptor (sst) subtypes sst 2A and sst 3 exist as homodimers at the plasma membrane when expressed in human embryonic kidney 293 cells. Moreover, in coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst 2A Although G protein-coupled receptors (GPCRs) 1 generally were believed to act as monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. The existence of homodimers has been shown for several GPCRs including the ␤ 2 -adrenergic receptor (1), ␦-and -opioid receptors (2, 3), the metabotrobic glutamate receptor 5 (4), the calcium-sensing receptor (5), the m3 muscarinic receptor (6), and dopamine receptors (7). GPCRs seem to dimerize via different mechanisms. Whereas dimerization of the ␤ 2 -adrenergic receptor (1) and D2 dopamine receptor (8) occurs via their transmembrane regions, dimerization of the ␦-opioid receptor involves the carboxyl terminus (2). In contrast, the metabotrobic glutamate receptor 5 (4) and the calcium-sensing receptor (5, 9, 10) appear to be disulfide-linked dimers, and dimerization occurs via their large amino termini. The question of to what extent agonist binding affects dimerization remains controversial. Recent evidence obtained in living cells using bioluminescence resonance energy transfer suggests that the ␤ 2 -adrenergic receptor exists at the cell surface as a constitutive dimer that is stabilized by agonist binding (11). In contrast, agonist stimulation reduced the level of ␦-opioid receptor dimers suggesting that monomerization precedes agonist-induced internalization of this receptor (2). Biochemical and functional studies suggest that GPCRs can also assemble as heterodimers with enhanced functional activity (3,(12)(13)(14)(15)(16)(17)(18)(19)(20). Formation of heterodimers between two nonfunctional ␥-aminobutyric acid (GABA) receptors, GABA B R1 and GABA B R2, was necessary for a fully functional GABA B receptor (14 -19). ␦-and -Opioid receptors form heterodimers with ligand binding and signaling properties resembling that of the 2 receptor (3). Finally, the somatostatin receptor (sst) sst 5 and the D 2 dopamine receptor heterodimerize to form a new receptor with en-