Threonine 180 is required for G-protein-coupled receptor kinase 3- and beta-arrestin 2-mediated desensitization of the mu-opioid receptor in Xenopus oocytes

To determine the sites in the mu-opioid receptor (MOR) critical for agonist-dependent desensitization, we constructed and coexpressed MORs lacking potential phosphorylation sites along with G-protein activated inwardly rectifying potassium channels composed of K(ir)3.1 and K(ir)3.4 subunits in Xenopus oocytes. Activation of MOR by the stable enkephalin analogue, [d-Ala(2),MePhe(4),Glyol(5)]enkephalin, led to homologous MOR desensitization in oocytes coexpressing both G-protein-coupled receptor kinase 3 (GRK3) and beta-arrestin 2 (arr3). Coexpression with either GRK3 or arr3 individually did not significantly enhance desensitization of responses evoked by wild type MOR activation. Mutation of serine or threonine residues to alanines in the putative third cytoplasmic loop and truncation of the C-terminal tail did not block GRK/arr3-mediated desensitization of MOR. Instead, alanine substitution of a single threonine in the second cytoplasmic loop to produce MOR(T180A) was sufficient to block homologous desensitization. The insensitivity of MOR(T180A) might have resulted either from a block of arrestin activation or arrestin binding to MOR. To distinguish between these alternatives, we expressed a dominant positive arrestin, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-independent manner. arr2(R169E) produced robust desensitization of MOR and MOR(T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3- and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-protein-coupled receptors.     

Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G‐protein gated inwardly rectifying potassium channels (K_ir3) and directly compared the effects of co‐expression of G‐protein coupled receptor kinase (GRK) and arrestin on agonist‐dependent desensitization of the receptor response. We found, as described previously, that co‐expression of a GRK and an arrestin synergistically increased the rate of agonist‐dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin‐dependent GRK‐independent desensitization of D2R‐K_ir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin‐dependent GRK‐independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist‐dependent desensitization even after GRK co‐expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

     

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells

Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type beta-arrestin + GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.     

Distinct residues in the carboxyl tail mediate agonist-induced desensitization and internalization of the human dopamine D1 receptor.

We have shown in a previous study that desensitization and internalization of the human dopamine D(1) receptor following short-term agonist exposure are mediated by temporally and biochemically distinct mechanisms. In the present study, we have used site-directed mutagenesis to remove potential phosphorylation sites in the third intracellular loop and carboxyl tail of the dopamine D(1) receptor to study these processes. Mutant D(1) receptors were stably transfected into Chinese hamster ovary cells, and kinetic parameters were measured. Mutations of Ser/Thr residues to alanine in the carboxyl tail demonstrated that the single substitution of Thr-360 abolished agonist-induced phosphorylation and desensitization of the receptor. Isolated mutation of the adjacent glutamic acid Glu-359 also abolished agonist-induced phosphorylation and desensitization of the receptor. These data suggest that Thr-360 in conjunction with Glu-359 may comprise a motif necessary for GRK2-mediated phosphorylation and desensitization. Agonist-induced internalization was not affected with mutation of either the Thr-360 or the Glu-359 residues. However, receptors with Ser/Thr residues mutated in the distal carboxyl tail (Thr-446, Thr-439, and Ser-431) failed to internalize in response to agonist activation, but were able to desensitize normally. These results indicate that agonist-induced desensitization and internalization are regulated by separate and distinct serine and threonine residues within the carboxyl tail of the human dopamine D(1) receptor.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation

G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.     

Opioid-receptor gene expression and localization in cancer cells

This study examined the presence and cellular localization of three types of opioid receptors (MOR, DOR and KOR) in five human cancer cell lines: MCF-7, MDA-MB-231, HT-29, MGH-U1 and SH-SY5Y. Expression levels of opioid receptors were measured quantitatively using real-time PCR, and the localizations of the receptors in the cells were determined by immunocytochemistry. All three types of opioid receptors were present in each of the five cell lines examined. However, three of the cell lines (MCF-7, HT-29 and SH-SY5Y) showed significantly higher levels of MOR mRNA and protein than the other two types of receptors (DOR and KOR). Immunocytochemistry revealed that MOR, DOR and KOR receptors were predominantly present on the surface of cell membranes, although these receptors were also occasionally present in the cell cytoplasm.     

Kappa opioid receptor activation of p38 MAPK is GRK3- and arrestin-dependent in neurons and astrocytes

AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (gamma-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.     

Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons

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.     

Co-expressions of different opioid receptor types differentially modulate their signaling via G(16)

Combinations of two different types of opioid receptors - delta-, kappa-, mu-opioid receptors (DOR, KOR, and MOR) and opioid receptor-like receptor 1 (ORL(1)) - were co-expressed with the alpha subunit of G(16) in COS-7 cells, and the ability of various selective agonists to induce activation of phospholipase Cbeta was examined. Nociceptin/orphanin FQ-induced response was enhanced when ORL(1) was co-expressed with MOR or KOR but not DOR. The kappa-agonist U50,488H induced a modest inositol phosphate formation when KOR was expressed alone or with MOR, but the response was attenuated when co-expressing with either DOR or ORL(1). It is suggested that the co-expressions of two different opioid receptor types indeed modify their downstream signaling events.     

6'-Guanidinonaltrindole (6'-GNTI) Is a G Protein-biased κ-Opioid Receptor Agonist That Inhibits Arrestin Recruitment

κ-Opioid receptor (KOR) agonists do not activate the reward pathway stimulated by morphine-like μ-opioid receptor (MOR) agonists and thus have been considered to be promising nonaddictive analgesics. However, KOR agonists produce other adverse effects, including dysphoria, diuresis, and constipation. The therapeutic promise of KOR agonists has nonetheless recently been revived by studies showing that their dysphoric effects require arrestin recruitment, whereas their analgesic effects do not. Moreover, KOR agonist-induced antinociceptive tolerance observed in vivo has also been proposed to be correlated to the ability to induce arrestin-dependent phosphorylation, desensitization, and internalization of the receptor. The discovery of functionally selective drugs that are therapeutically effective without the adverse effects triggered by the arrestin pathway is thus an important goal. We have identified such an extreme G protein-biased KOR compound, 6'-guanidinonaltrindole (6'-GNTI), a potent partial agonist at the KOR receptor for the G protein activation pathway that does not recruit arrestin. Indeed, 6'-GNTI functions as an antagonist to block the arrestin recruitment and KOR internalization induced by other nonbiased agonists. As an extremely G protein-biased KOR agonist, 6'-GNTI represents a promising lead compound in the search for nonaddictive opioid analgesic as its signaling profile suggests that it will be without the dysphoria and other adverse effects promoted by arrestin recruitment and its downstream signaling.     

Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.     

Role of Opioid Receptors Signaling in Remote Electrostimulation - Induced Protection against Ischemia/Reperfusion Injury in Rat Hearts

Aims

Our previous studies demonstrated that remote electro-stimulation (RES) increased myocardial GSK3 phosphorylation and attenuated ischemia/ reperfusion (I/R) injury in rat hearts. However, the role of various opioid receptors (OR) subtypes in preconditioned RES-induced myocardial protection remains unknown. We investigated the role of OR subtype signaling in RES-induced cardioprotection against I/R injury of the rat heart.

Methods & Results

Male Spraque-Dawley rats were used. RES was performed on median nerves area with/without pretreatment with various receptors antagonists such as opioid receptor (OR) subtype receptors (KOR, DOR, and MOR). The expressions of Akt, GSK3, and PKCε expression were analyzed by Western blotting. When RES was preconditioned before the I/R model, the rat''s hemodynamic index, infarction size, mortality and serum CK-MB were evaluated. Our results showed that Akt, GSK3 and PKCε expression levels were significantly increased in the RES group compared to the sham group, which were blocked by pretreatment with specific antagonists targeting KOR and DOR, but not MOR subtype. Using the I/R model, the duration of arrhythmia and infarct size were both significantly attenuated in RES group. The mortality rates of the sham RES group, the RES group, RES group + KOR antagonist, RES group + DOR/MOR antagonists (KOR left), RES group + DOR antagonist, and RES group + KOR/MOR antagonists (DOR left) were 50%, 20%, 67%, 13%, 50% and 55%, respectively.

Conclusion

The mechanism of RES-induced myocardial protection against I/R injury seems to involve multiple target pathways such as Akt, KOR and/or DOR signaling.     

The intracellular trafficking of opioid receptors directed by carboxyl tail and a di-leucine motif in Neuro2A cells

The mu- and delta-opioid receptors (MOR and DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. By exchanging the carboxyl tail domains of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A cells, it could be demonstrated that the carboxyl tail domain is not the sole determinant in directing the intracellular trafficking in these Neuro2A cells. Deletion of the dileucine motif (Leu245-Leu246) within the third intracellular loop of DOR or the mutation of Leu245 to Met slowed the lysosomal targeting of these delta-opioid receptors. Meanwhile the mutation of Met264 to Leu increased the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the delta-opioid receptor carboxyl tail chimera of the mu-opioid receptor. These studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor.     

Heterodimerization of mu- and delta-opioid receptors occurs at the cell surface only and requires receptor-G protein interactions

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 mu-opioid receptor (MOR) and the ponasterone A-induced expression of delta-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.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

A phosphorylation-regulated brake mechanism controls the initial endocytosis of opioid receptors but is not required for post-endocytic sorting to lysosomes.

The delta-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor down-regulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent protein-tagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of beta-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.     

Prolonged kappa opioid receptor phosphorylation mediated by G-protein receptor kinase underlies sustained analgesic tolerance

Kappa opioid receptor (KOR) desensitization was previously shown to follow agonist-dependent phosphorylation of serine 369 by G-protein receptor kinase (GRK) and beta-arrestin binding in transfected cells. To study the in vivo effects induced by phosphorylation of KOR(S369), C57Bl/6 mice were administered single or repeated doses of the KOR agonist, U50,488, and isolated brain glycoprotein was probed with an antibody, KOR-P, that specifically recognized phosphoserine 369 KOR. Western blot analysis using KOR-P antibody showed that labeling intensity increased after either single or repeated treatment of mice with U50,488 by 59 +/- 22% and 101 +/- 29%, respectively. In contrast, there was no change in labeling intensity by nonphosphoselective KOR antibodies following acute or chronic in vivo treatment with kappa agonist. Moreover, mice lacking GRK3 showed no increase in KOR-P labeling and developed significantly less analgesic tolerance following treatment with kappa agonist. The result suggests that tolerance to kappa agonists includes phosphorylation of serine 369 within KOR by GRK3. Recovery of analgesic potency and reduction of elevated KOR-P labeling in wild-type mice both required 2 weeks to return to base line. Consistent with these results, in vitro phosphorylation by GRK3 of KOR isolated from tolerant mice resulted in 46 +/- 7% less (32)P incorporation than in KOR isolated from untreated mice. In addition, in vitro (32)P incorporation returned to base line levels only in KOR isolated from tolerant mice allowed to recover for 2 weeks. The coincident reversal of analgesic tolerance and slow return to a basal phosphorylation state matched the regeneration rate of functional kappa receptors following irreversible antagonism and suggested that receptor replacement rather than dephosphorylation was required to restore sensitivity.     

RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

Opioid agonists display different capacities to stimulate mu-opioid receptor (MOR) endocytosis, which is related to their ability to provoke the phosphorylation of specific cytosolic residues in the MORs. Generally, opioids that efficiently promote MOR endocytosis and recycling produce little tolerance, as is the case for [d-Ala², N-MePhe⁴,Gly-ol⁵] encephalin (DAMGO). However, morphine produces rapid and profound antinociceptive desensitization in the adult mouse brain associated with little MOR internalization. The regulator of G-protein signaling, the RGS14 protein, associates with MORs in periaqueductal gray matter (PAG) neurons, and when RGS14 is silenced morphine increased the serine 375 phosphorylation in the C terminus of the MOR, a GRK substrate. Subsequently, these receptors were internalized and recycled back to the membrane where they accumulated on cessation of antinociception. These mice now exhibited a resensitized response to morphine and little tolerance developed. Thus, in morphine-activated MORs the RGS14 prevents GRKs from phosphorylating those residues required for β-arresting-mediated endocytosis. Moreover morphine but not DAMGO triggered a process involving calcium/calmodulin-dependent kinase II (CaMKII) in naïve mice, which contributes to MOR desensitization in the plasma membrane. In RGS14 knockdown mice morphine failed to activate this kinase. It therefore appears that phosphorylation and internalization of MORs disrupts the CaMKII-mediated negative regulation of these opioid receptors.