Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human mu-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 beta-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 mu-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human mu-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild-type receptor. Morphine, morphine-6-glucuronide and beta-endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D-variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d-Ala(2),N-MePhe(4),Glyol(5)]enkephalin (DAMGO) and beta-endorphin but not in response to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or beta-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-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 mu-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 tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.     

Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding.

The mu opioid receptor (MOR) plays a central role in mediating acute and chronic effects of narcotic drugs. Three rare single nucleotide polymorphisms in the hMOR gene have been identified that cause amino acid substitutions in the third intracellular (i3) loop of MOR (R260H, R265H, and S268P). Genotyping 252 individuals of the Coriell collection identified one allele encoding the R265H-MOR variant and a new variant encoding D274N-MOR. Variants R260H-, R265H-, and S268P-MOR were constructed and transfected into HEK293 cells. Morphine stimulated G protein coupling of the three receptor variants to a maximal level approaching that of wild type MOR. In contrast, spontaneous, agonist-independent (basal) MOR signaling, proposed to play a role in opioid tolerance and dependence, was significantly reduced for R260H- and R265H-MOR. Moreover, domains within the i3 loop of MOR have been shown to interact with both G proteins and calmodulin (CaM). CaM binding was deficient for variants R265H- and S268P-MOR, suggesting that domains for G protein coupling and CaM binding overlap partially. Morphine pretreatment significantly enhanced basal G protein coupling of wild type MOR, which is thought to result from release of CaM. In contrast basal G protein coupling activity of the three variants was unaffected by morphine pretreatment consistent with diminished CaM regulation, low basal activity, or both. In conclusion, each of the three single nucleotide polymorphisms mapping to the i3 loop of MOR caused substantial changes in basal G protein coupling, CaM binding, or both. Carriers of the mutant alleles might display altered responses to narcotic analgesics.     

ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis

Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins. Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation. Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles. Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2. Treatment with the mu receptor agonist DAMGO ([d-Ala(2), Me Phe(4), Glyol(5)]enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation. The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent. Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure. Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1. Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor.     

Coupling efficacy and selectivity of the human mu-opioid receptor expressed as receptor-Galpha fusion proteins in Escherichia coli

Two constructs encoding the human micro-opioid receptor (hMOR) fused at its C terminus to either one of two Galpha subunits, Galpha(o1) (hMOR-Galpha(o1)) and Galpha(i2) (hMOR-Galpha(i2)), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4-0.5 pmol/mg). Receptors fused to Galpha(o1) or to Galpha(i2) maintained high-affinity binding of the antagonist diprenorphine. Affinities of the micro-selective agonists morphine, [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding were assessed in the presence of added purified Gbetagamma subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [(35)S]GTPgammaS binding. In the presence of Gbetagamma dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-Galpha(i2) than at hMOR-Galpha(o1), whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [(35)S]GTPgammaS binding at hMOR-Galpha(o1) were similar, whereas at hMOR-Galpha(i2), endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to Galpha(o1) and Galpha(i2) and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled.     

Relevant role of Leu265 in helix VI of the angiotensin AT1 receptor in agonist binding and activity

The finding of critical residues for angiotensin II (AII) binding and receptor signalling in helices V and VI led us to assess if, in this region of the receptor, aliphatic side chains might play a role in the agonist-mediated mechanism. Two mutations of the angiotensin AT1 receptor were designed to explore a possible role of a leucine at two positions, Leu265 and Leu268. Thus two mutants, L265D and L268D, were prepared through single substitutions of Leu265, located in the C-terminal region of transmembrane VI (TM-VI), and Leu268, in the adjoining region of the third extracellular loop (EC-3), for an aspartyl residue, and were stably transfected into Chinese hamster ovary (CHO) cells. Ligand-binding experiments and the functional assays determining inositol phosphate (IP) production were performed in these cells expressing these mutants. No significant changes were found in the binding affinity for the ligands, AII, DuP753, and [Sar1Leu8]AII in the mutant L268D. Moreover, the relative potency and the maximum effect on IP production of this mutant were similar to those of the wild-type receptor. In contrast, L265D mutant AT1 receptor, located within the transmembrane domain, markedly decreased binding affinity and ability to stimulate phosphatidylinositol turnover. Our results suggest that the hydrophobic side chain of Leu265, at the C-terminal portion of the AT1's TM-VI, but not Leu268, which belongs to the EC-3 loop, might interact with the AII molecule. On the other side, the aliphatic side chain of Leu265 may be involved in the formation of the ligand binding sites through allosteric effects, thus helping to stabilize the receptor structure around the agonist binding site for full activity.     

Morphine-induced mu-opioid receptor rapid desensitization is independent of receptor phosphorylation and beta-arrestins

Receptor desensitization involving receptor phosphorylation and subsequent betaArrestin (betaArr) recruitment has been implicated in the tolerance development mediated by mu-opioid receptor (OPRM1). However, the roles of receptor phosphorylation and betaArr on morphine-induced OPRM1 desensitization remain to be demonstrated. Using OPRM1-induced intracellular Ca(2+) ([Ca(2+)](i))release to monitor receptor activation, as predicted, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO), induced OPRM1 desensitization in a receptor phosphorylation- and betaArr-dependent manner. The DAMGO-induced OPRM1 desensitization was attenuated significantly when phosphorylation deficient OPRM1 mutants or Mouse Embryonic Fibroblast (MEF) cells from betaArr1 and 2 knockout mice were used in the studies. Specifically, DAMGO-induced desensitization was blunted in HEK293 cells expressing the OPRM1S375A mutant and was eliminated in MEF cells isolated from betaArr2 knockout mice expressing the wild type OPRM1. However, although morphine also could induce a rapid desensitization on [Ca(2+)](i) release to a greater extent than that of DAMGO and could induce the phosphorylation of Ser(375) residue, morphine-induced desensitization was not influenced by mutating the phosphorylation sites or in MEF cells lacking betaArr1 and 2. Hence, morphine could induce OPRM1 desensitization via pathway independent of betaArr, thus suggesting the in vivo tolerance development to morphine can occur in the absence of betaArr.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

Interacting residues in an activated state of a G protein-coupled receptor

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.     

Role of phospholipase D2 in the agonist-induced and constitutive endocytosis of G-protein coupled receptors

We have recently shown that the mu-opioid receptor [MOR1, also termed mu-opioid peptide (MOP) receptor] is associated with the phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane. We further demonstrated that, in human embryonic kidney (HEK) 293 cells co-expressing MOR1 and PLD2, treatment with (D-Ala2, Me Phe4, Glyol5)enkephalin (DAMGO) led to an increase in PLD2 activity and an induction of receptor endocytosis, whereas morphine, which does not induce opioid receptor endocytosis, failed to activate PLD2. In contrast, a C-terminal splice variant of the mu-opioid receptor (MOR1D, also termed MOP(1D)) exhibited robust endocytosis in response to both DAMGO and morphine treatment. We report here that MOR1D also mediates an agonist-independent (constitutive) PLD2-activation facilitating agonist-induced and constitutive receptor endocytosis. Inhibition of PLD2 activity by over-expression of a dominant negative PLD2 (nPLD2) blocked the constitutive PLD2 activation and impaired the endocytosis of MOR1D receptors. Moreover, we provide evidence that the endocytotic trafficking of the delta-opioid receptor [DOR, also termed delta-opioid peptide (DOP) receptor] and cannabinoid receptor isoform 1 (CB1) is also mediated by a PLD2-dependent pathway. These data indicate the generally important role for PLD2 in the regulation of agonist-dependent and agonist-independent G protein-coupled receptor (GPCR) endocytosis.     

Crystal structure of a superstable mutant of human p53 core domain. Insights into the mechanism of rescuing oncogenic mutations

Most of the cancer-associated mutations in the tumor suppressor p53 map to its DNA-binding core domain. Many of them inactivate p53 by decreasing its thermodynamic stability. We have previously designed the superstable quadruple mutant M133L/V203A/N239Y/N268D containing the second-site suppressor mutations N239Y and N268D, which specifically restore activity and stability in several oncogenic mutants. Here we present the x-ray structure of this quadruple mutant at 1.9 A resolution, which was solved in a new crystal form in the absence of DNA. This structure reveals that the four point mutations cause only small local structural changes, whereas the overall structure of the central beta-sandwich and the DNA-binding surface is conserved. The suppressor mutation N268D results in an altered hydrogen bond pattern connecting strands S1 and S10, thus bridging the two sheets of the beta-sandwich scaffold in an energetically more favorable way. The second suppressor mutation N239Y, which is located in close proximity to the DNA-binding surface in loop L3, seems to reduce the plasticity of the structure in large parts of loop L3 as indicated by decreased crystallographic temperature factors. The same is observed for residues in the vicinity of the N268D substitution. This increase in rigidity provides the structural basis for the increase in thermostability and an understanding how N268D and N239Y rescue some of the common cancer mutants.     

Morphine-induced desensitization and down-regulation at mu-receptors in 7315C pituitary tumor cells

Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 microM, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of [3H]diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for [3H]diprenorphine. However, after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 microM or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5 hr.     

Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) and D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 13 amino acids. Except for the D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, and pretreatment with naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [(3)H]diprenorphine as the wild-type. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.49(164)E and D3.32(147) mutants, exhibited enhanced basal [(35)S]GTPgammaS binding which was comparable to the maximally activated level of the wild-type and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [(35)S]GTPgammaS binding of the D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [(35)S]GTPgammaS binding, demonstrating constitutive activation of Galpha(i)/Galpha(o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTPgammaS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in receptor assuming activated conformations. In contrast, the D3.49(164)E mutant displayed significantly lower basal [(35)S]GTPgammaS binding and reduced affinity for DAMGO. Upon incubation of membranes at 37 degrees C, the constitutively active D3.49(164)Y mutant was structurally less stable, whereas the inactivated D3.49(164)E mutant was more stable, than the wild-type. Computational simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the receptor. Taken together, these results indicate that D3.49 plays an important role in constraining the receptor in inactive conformations.     

Inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) on gastrointestinal transit

The inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) (TAPA), a highly selective mu(1)-opioid receptor agonist, on mouse gastrointestinal transit was compared with that of morphine and [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]-enkephalin (DAMGO). When administered intracerebroventricularly 5 min before the oral injection of charcoal meal, TAPA (10-100 pmol), morphine (0.25-4 nmol), and DAMGO (20-80 pmol) dose-dependently inhibited gastrointestinal transit of charcoal. The inhibitory effect of each mu-opioid receptor agonist was completely antagonized by naloxone, a nonselective opioid receptor antagonist. The inhibitory effects of morphine and DAMGO were significantly antagonized by both beta-funaltrexamine, a selective mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist. In contrast, the inhibitory effect of TAPA was not affected at all by beta-funaltrexamine, naloxonazine, nor-binaltorphimine (a selective kappa-opioid receptor antagonist), or naltrindole (a selective delta-opioid receptor antagonist). These results suggest that the inhibitory effect of TAPA on gastrointestinal transit may be mediated through an opioid receptor mechanism different from that of morphine and DAMGO.     

Identification of a mutation cluster in mevalonate kinase deficiency, including a new mutation in a patient of Mennonite ancestry.

Mevalonate kinase (MKase) deficiency (MKD) is a rare autosomal recessive disorder in the pathway of cholesterol and nonsterol isoprenoid biosynthesis. Thus far, two disease-causing missense alleles have been identified, N301T and A334T. We report four additional mutations associated with MKD: L264F, T243I, L265P, and I268T, the last found in a patient of Mennonite ancestry. Electrophoretic analysis of bacterially expressed wild-type and mutant MKase indicated that I268T and T243I mutants produced normal or somewhat reduced amounts of MKase protein; conversely, L264F and L265P mutations resulted in considerably decreased, or absent, MKase protein. Immunoblot analysis of MKase from all patients suggested that the MKase polypeptide was grossly intact and produced in amounts comparable to control levels. Three mutations resulted in significantly diminished MKase enzyme activity (<2%), whereas the I268T allele yielded approximately 20% residual enzyme activity. Our results should allow more-accurate identification of carriers and indicate a mutation "cluster" within amino acids 240-270 of the mature MKase polypeptide.     

Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp.

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.     

Modulating micro-opioid receptor phosphorylation switches agonist-dependent signaling as reflected in PKCepsilon activation and dendritic spine stability

A new role of G protein-coupled receptor (GPCR) phosphorylation was demonstrated in the current studies by using the μ-opioid receptor (OPRM1) as a model. Morphine induces a low level of receptor phosphorylation and uses the PKCε pathway to induce ERK phosphorylation and receptor desensitization, whereas etorphine, fentanyl, and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) induce extensive receptor phosphorylation and use the β-arrestin2 pathway. Blocking OPRM1 phosphorylation (by mutating Ser363, Thr370 and Ser375 to Ala) enabled etorphine, fentanyl, and DAMGO to use the PKCε pathway. This was not due to the decreased recruitment of β-arrestin2 to the receptor signaling complex, because these agonists were unable to use the PKCε pathway when β-arrestin2 was absent. In addition, overexpressing G protein-coupled receptor kinase 2 (GRK2) decreased the ability of morphine to activate PKCε, whereas overexpressing dominant-negative GRK2 enabled etorphine, fentanyl, and DAMGO to activate PKCε. Furthermore, by overexpressing wild-type OPRM1 and a phosphorylation-deficient mutant in primary cultures of hippocampal neurons, we demonstrated that receptor phosphorylation contributes to the differential effects of agonists on dendritic spine stability. Phosphorylation blockage made etorphine, fentanyl, and DAMGO function as morphine in the primary cultures. Therefore, agonist-dependent phosphorylation of GPCR regulates the activation of the PKC pathway and the subsequent responses.