C-terminal Splice Variants of the Mouse µ-Opioid Receptor Differ in Morphine-induced Internalization and Receptor Resensitization

It is generally accepted that the internalization and desensitization of -opioid receptor (MOR) involves receptor phosphorylation and ␤-arrestin recruitment. However, a mutant MOR, which is truncated after the amino acid residue Ser 363 (MOR363D), was found to undergo phosphorylation-independent internalization and desensitization. As expected, MOR363D, missing the putative agonist-induced phosphorylation sites, did not exhibit detectable agonist-induced phosphorylation. MOR363D underwent slower internalization as reflected in the attenuation of membrane translocation of ␤-arrestin 2 when compared with wild type MOR, but the level of receptor being internalized was similar to that of wild type MOR after 4 h of etorphine treatment. Furthermore, MOR363D was observed to desensitize faster than that of wild type MOR upon agonist activation. Surface biotinylation assay demonstrated that the wild type receptors recycled back to membrane after agonist-induced internalization, which contributed to the receptor resensitization and thus partially reversed the receptor desensitization. On the contrary, MOR363D did not recycle after internalization. Hence, MOR desensitization is controlled by the receptor internalization and the recycling of internalized receptor to cell surface in an active state. Taken together, our data indicated that receptor phosphorylation is not absolutely required in the internalization, but receptor phosphorylation and subsequent ␤-arrestin recruitment play important roles in the resensitization of internalized receptors.The regulation of G protein-coupled receptors (GPCRs) 1 signaling highlights the complex relationship between multiple mechanisms acting at different levels of signal propagation. Following the agonist activation of the receptor, G proteincoupled receptor kinases (GRKs) are recruited to the membrane and phosphorylate the agonist-activated GPCRs. GRKmediated receptor phosphorylation enhances the interaction between GPCRs and cytoplasmic proteins, arrestins, which uncouple the GPCRs from G proteins and also target the GPCRs to clathrin-coated pits for internalization (endocytosis). The internalized receptors can subsequently be dephosphorylated and recycled back to the plasma membrane, which contributes to resensitization. Alternatively, the internalized receptors can be targeted to lysosome for degradation (1-4). Receptor desensitization, the progressive loss of receptor function under continued exposure to an agonist, occurs in many GPCRs. Desensitization can be a consequence of multiple processes of receptor uncoupling, internalization, degradation, and recycling. Therefore, phosphorylation of agonist-activated receptors and subsequent arrestin recruitment are important processes in the modulation of GPCRs responsiveness.However, multiple studies challenge the critical role phosphorylation played in desensitization and internalization of many GPCRs. Desensitization and internalization of human parathyroid hormone receptor (5), AT 1A angiotensin receptor (6), and metabotropic gluta...

Section: Resultsmentioning

confidence: 99%

μ-Opioid Receptor Desensitization

Qiu

Law

Loh

2003

“…More specifically, segments of the carboxyl tail and specific residues contained therein have been identified for both (20,23,24) and ␦ (25) receptors as important for agonist-induced internalization. We hypothesized therefore, that the inability of the ␦ receptor to internalize when co-expressed with the receptor indicated that the relevant region of the carboxyl tail that mediated ␦ opioid receptor internalization was somehow occluded.…”

Section: Discussionmentioning

confidence: 99%

A Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of μ and δ Opioid Receptor Hetero-oligomers

Fan

Varghese

Nguyen

et al. 2005

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for and ␦ receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the -␦ heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the -␦ receptor heteromer resulted in preferential activation of G␣ z , illustrated by incorporation of GTP␥ 35 S, whereas activation of the individually expressed and ␦ receptors preferentially activated G␣ i . The unique pharmacology of the -␦ heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal receptor carboxyl tail modified the ␦-selective ligand-binding pocket, and truncation of the ␦ receptor distal carboxyl tail modified the -selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between and ␦ receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.The endogenous opioid systems mediate important physiological functions related to pain perception, locomotion, motivation, reward, autonomic function, immune modulation, and hormone secretion. Opioid receptors, mediating the actions of the opioid peptides, belong to the family of G protein-coupled receptors (GPCRs) 2 and have distinct pharmacological profiles with discrete but overlapping distributions in brain (reviewed Refs. 1 and 2). The pharmacology of the opioid receptors obtained in brain and other tissues has consistently predicted a greater number of receptor subtypes such as 1, 2, and ␦1, ␦2, which are not explained by the individual pharmacological profiles obtained for the three cloned opioid receptors, , ␦, and , when expressed individually in heterologous systems. One possible mechanism for these findings, gaining increasing credence, has been that of direct receptorreceptor interactions creating novel signaling units and generating distinct post-receptor functional effects.The ability of GPCRs to form homo-oligomers and hetero-oligomers has been described by us and others (reviewed in Refs. 3-5). We have provided evidence for the direct interaction of and ␦ opioid receptors to form hetero-oligomers, with generation of novel pharmacology and G protein coupling properti...

“…Consistent with this, numerous studies have suggested that morphine does not efficiently promote MOR regulation by GRK and arrestin. Morphine has been shown either to be unable to induce MOR phosphorylation (29,30) or to induce less phosphorylation of MOR compared with higher efficacy agonists such as DAMGO (31,32). Additionally, morphine was unable to promote internalization of MOR in heterologous cell systems (7), although more recently morphine was shown to produce internalization in striatal neurons when MOR-and GFP-tagged arrestin were overexpressed (18).…”

Section: Discussionmentioning

confidence: 99%

Mu Opioid Receptor Activation of ERK1/2 Is GRK3 and Arrestin Dependent in Striatal Neurons

Macey

Lowe

Chavkin

2006

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (؊/؊) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3 ؊/؊ mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3 ؊/؊ neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR ؊/؊ , or GRK3 ؊/؊ mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.Opioid receptor activation results in both acute and longlasting changes in neuronal physiology. The mechanisms responsible for the acute changes include G␣ i/o and G␤␥ protein activation that increases potassium conductance, decreases calcium conductance, and results in presynaptic inhibition (1-3). The mechanisms responsible for the long lasting effects of opioids are less clear but may include changes in adenylyl cyclase activity and activation of mitogen activating protein kinase (MAPK) 2 pathways (4, 5). In this study we used primary cultured neurons isolated from mouse striata to address the mechanisms linking mu opioid receptor (MOR) activation to the phosphorylation and activation of the extracellular signal-related kinases 1 and 2 (ERK1/2) members of the MAPK family. Phosphorylation of ERK1/2 by GPCRs involves growth factor receptor transactivation (4). As demonstrated for the ␤-adrenergic receptor, participation of arrestin is an integral part of GPCR signaling through the ERK1/2 signaling pathway in transfected HEK293 cells (6). Following agonist activation of MOR, the receptor becomes phosphorylated by G-protein receptor kinase (GRK), which initiates an arrestin-dependent desensitization process that involves clathrin-mediated endocytosis (7). The opioid activation of ERK1/2 by MOR agonist (D-Ala 2 ,Me-Phe 4 ,Gly-ol5) (DAMGO) was demonstrated in MOR-transfected c...