Functional expression of mammalian opioid receptors in insect cells and high-throughput screening platforms for receptor ligand mimetics.

Lepidopteran cell lines have been engineered to constitutively express high levels of mouse delta opioid receptors either alone or in combination with human Galpha16 protein. Biochemical and pharmacological studies demonstrate that these lines contain all the mediator G proteins and downstream effectors required for opioid receptor function, including phospholipase C, and that expression of exogenous Galpha16 does not contribute significantly to increased receptor responses upon activation. The activation of the phospholipase C pathway in the transformed cells upon stimulation with known receptor ligands results in easily and quantitatively measurable increases in free intracellular calcium, which can be monitored by automated fluorescent methods, while the addition of specific antagonists blocks the agonist-induced responses. Therefore, the transformed lepidopteran cell lines can be used as sensitive high-throughput screening platforms for fast detection of delta opioid receptor ligand mimetics (agonists and antagonists) in collections of natural products and synthetic compounds.

Functional domains of delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.

Opioid receptors (delta, mu) belong to the large superfamily of G protein coupled receptors that inhibit adenylyl cyclase. We have studied the effects of seven synthetic peptides representing selected cytoplasmic regions of the murine delta-opioid receptor on forskolin-mediated adenylyl cyclase activity in membranes of D2 and Neuro(2A) cells stably expressing the delta- and mu-opioid receptors respectively. The entire third intracellular loop (i3), its amino-terminal portion (i3.1) and the carboxyl-terminal region of the second cytoplasmic loop (i2.2) enhanced dose-dependently the agonist-mediated inhibition of cAMP accumulation. The peptide-mediated effects are blocked by pertussis toxin treatment and are not observed in parental cells that lack these receptors. The inhibitory effects of the peptides on adenylyl cyclase were markedly attenuated when membranes from D2 and Neuro(2A) cells were preincubated with antisera against Gi(2) alpha and G beta subunits of G proteins. Our results provide evidence on domains of the delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.

Regulation of spontaneous activity of the delta-opioid receptor: studies of inverse agonism in intact cells.

Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine delta-opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin D-Ala2,D-Leu5 enkephalin (DADLE) produced strong inhibition of forskolin-amplified [3H]cyclic AMP production, whereas the delta-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 +/- 0.6 x 10(-8) M) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.

Selective interactions of mu-opioid receptors with pertussis toxin-sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling.

A cDNA encoding the rat mu-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the mu-opioid selective ligand [3H][D-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([3H]DAMGO). In membranes of clone mu11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed [32P]ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2alpha and Gi3alpha were both able to immunoprecipitate a [32P]-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone mu11, indicating that the mu-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the delta-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [3H] agonist binding to the receptor. In membranes from the clone mu11, specific binding of [3H]DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed [32P]ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTP-gammaS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.

Identification of the critical domains of the delta-opioid receptor involved in G protein coupling using site-specific synthetic peptides.

A large body of evidence implicates the second and third intracellular loops and the carboxyl-terminal portion of many G protein-coupled receptors as sites responsible for the interaction to G proteins. We synthesized a number of peptides from selected sites of the murine delta-opioid receptor and measured their ability to modify ligand-stimulated G protein activation and 3H agonist binding to the receptor. In membranes from Rat-1 fibroblasts transfected to express the murine delta-opioid receptor stably (clone D2 cells), the delta-opioid agonist [D-Ser2-Leu5-Thr6]enkephalin (DSLET) stimulated high affinity GTPase activity, which was inhibited by peptides that are derived from the proximal (i3.1) and the distal portions (i3.3) of the third intracellular loop with IC50 values of 15 +/- 5 and 50 +/- 4 microM, respectively. Peptides i3.1 and i3.3 inhibited DSLET-stimulated [35S]guanosine 5'-O-thiotriphosphate binding in the same membranes. However, a peptide designated i4, which was derived from a juxtamembranous region of the carboxyl-terminal tail of the delta-opioid receptor, failed to alter agonist-mediated high affinity GTPase activity or agonist-driven [35S]guanosine 5'-O-thiotriphosphate binding. Specific binding of [3H]DSLET to membrane preparations from clone D2 was reduced by peptides i3.1 and i4. Combinations of these peptides abolished detectable [3H]DSLET binding in the same membranes. Peptides i3.1 and i3.3 also destabilized the high affinity state of the receptor as assessed in 3H agonist binding on membranes from neuroblastoma X glioma (NG108-15) hybrid cells, which express the delta-opioid receptor endogenously; furthermore, delta-opioid receptor-stimulated GTPase activity in the same membranes was inhibited by peptides i3.1 and i3.3 but i4 was inactive. In contrast, peptides derived from the second intracellular loop (i2.1 and i2.2), an intermediate portion of the third intracellular loop (i3.2), and the extreme amino-terminal region of the receptor were without effect in these assays. These observations indicate that although peptides i3.1, i3.3, and i4 act via different mechanisms, they provide evidence that at least two sites of the third intracellular loop and part of the carboxyl-terminal tail of the delta-opioid receptor are important in the interaction between this receptor and cellular G proteins. Collectively, these results provide novel information about regions of the delta-opioid receptor that are involved in G protein coupling and high affinity agonist binding.

Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera.

Solubilization of opioid receptors from rat cortical membranes that retained high-affinity guanine nucleotide-sensitive agonist binding was achieved using 10 mM CHAPS. We report the nature of the interactions of mu and delta opioid receptors with the guanine nucleotide-binding protein G(o) by immunoprecipitation of CHAPS extracts with selective G(o)alpha-subunit protein antisera. Antiserum IM1 raised against amino acids 22-35 of G(o)alpha selectively co-immunoprecipitated G(o)alpha-mu and G(o)alpha-delta opioid receptor complexes detected in the immunoprecipitates by specific [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and [3H][D-Ser2,Leu5,Thr6]enkephalin binding respectively. By contrast, antisera directed against the C-terminal decapeptide (OC2) and the N-terminal hexadecapeptide (ON1) of isoforms of G(o)alpha were unable to immunoprecipitate solubilized opioid receptor-G(o) complexes, although both were able to immunoprecipitate solubilized G(o)alpha and have been shown to reduce the affinity of [D-Ala2,D-Leu5]enkephalin for opioid receptors in rat cortical membranes [Georgoussi, Carr and Milligan (1993) Mol. Pharmacol. 44, 62-69]. These findings demonstrate that CHAPS-solubilized mu and delta opioid receptors from rat cortical membranes form stable complexes with one or more variants of G(o).

Direct measurements of in situ interactions of rat brain opioid receptors with the guanine nucleotide-binding protein Go.

The interactions of rat brain cortical opioid receptors with the guanine nucleotide-binding protein (G protein) Go were probed in membranes by examining the ability of selective antipeptide anti-G protein antisera to disrupt receptor-G protein interactions. This was measured both by antibody-induced alterations in the characteristics of agonist binding to mu and delta receptor binding sites and by antibody attenuation of opioid stimulation of high affinity GTPase activity. Antisera to the amino-terminal 16 amino acids (ON1), amino acids 22-35 (IM1), and the carboxyl-terminal decapeptide (OC2) of forms of Go alpha were able to selectively immunoprecipitate Go from rat cortical membranes. Both antisera OC2 and ON1 were able to immunoprecipitate Go alpha quantitatively. Preincubation of rat cortical membranes with an IgG fraction isolated from antiserum OC2 was able to produce a marked reduction in the ability of the synthetic enkephalin [D-Ala2,D-Leu5] enkephalin (DADLE) (which interacts with delta and mu but not significantly with kappa receptors) to displace specific binding of [3H] diprenorphine (which binds to all of these sites), demonstrating a clear interaction of the mu and delta receptors with one or more variants of Go. An IgG fraction from antiserum ON1 was able to mimic this effect, suggesting that the amino-terminal region of G protein alpha subunits also plays a role in receptor-G protein interactions. In contrast, an IgG fraction from antiserum IM1 was unable to alter the characteristics of DADLE displacement of [3H] diprenorphine binding. Similarly, an antiserum (SG1) directed against the carboxyl-terminal decapeptide common to the alpha subunits of Gi1 and Gi2 was unable to reduce the affinity of DADLE binding to opioid receptors. Use of antiserum OC2 in experiments that allowed pharmacological examination of only the mu-opioid receptor provided independent evidence for the interaction of this receptor site with Go.

Effect of delta-opioid antagonists on the functional coupling between opioid receptors and G-proteins in rat brain membranes.

It is currently accepted that occupancy of opioid receptors by agonists, but not antagonists, promotes the association of the receptors to guanine nucleotide binding proteins (G-proteins) and stimulates a high affinity GTPase as part of the mechanism that links the receptor-ligand complex to adenylate cyclase inhibition. In this work we report that in rat brain membranes selective delta-opioid antagonists, the peptides N,N-Diallyl-Tyr-D-Leu-Gly-Tyr-Leu-OH (Diallyl-G) and N-N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174,864), inhibit the low Km GTPase activity in a concentration dependent way. On the other hand the delta-opioid agonists D-Ala2-D-Leu5-enkephalin (DADLE) and D-Ser2-Leu5-Thr6-enkephalin stimulate dose-dependently the low Km GTPase activity in rat brain membranes. This stimulation was blocked in the presence of Diallyl-G, and reciprocally the inhibition induced by Diallyl-G was reversed by DADLE. The inhibitory effect of Diallyl-G as well as the stimulation induced by DADLE were abolished when membranes were exposed to low concentrations of N-ethylmaleimide or by ADP ribosylation with pertussis toxin which interferes with the ability of the receptor to couple to G-proteins. These observations indicate that the inhibitory effect of Diallyl-G on GTPase requires a functional G-protein and suggest that certain delta-opioid antagonists exhibit negative intrinsic activity and may have the ability to inhibit the receptor-mediated activation of G-proteins.

Purification of the hepatic vasopressin receptor using a novel affinity column.

A vasopressin receptor was purified, using a novel affinity column, from rat liver plasma membranes treated with guanosine 5'-(3-O-thio)triphosphate and solubilized with 0.8% cholate. Incubation of the membranes with the GTP analogue resulted in a dissociation of the receptor-guanine nucleotide regulatory protein complex. This manipulation, although resulting in a low-affinity state of the receptor, facilitated purification. The solubilized receptor was assayed using a new reconstitution procedure in which the soluble extracts were inserted into lipid vesicles composed of phosphatidylcholine and phosphatidylinositol. The receptor was purified by sequential chromatography on Q-Sepharose and hydroxyapatite. The use of a novel affinity column, a V1-vasopressin antagonist-agarose, resulted in a near-homogeneous preparation of a protein which exhibited an Mr = 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of purified receptor, as well as crude membrane preparations cross-linked to [125I]arginine vasopressin, also revealed a protein band with an approximate Mr = 58,000. These findings indicate that V1-antagonist affinity chromatography should be useful for purifying adequate amounts of the receptor for studies of structure and function.

Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity.

Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin-treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE-cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase.

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by 2-mercaptoethanol.

The Ca2+-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0 degree C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22 degrees C was considerably lower than that at 0 degree C. Ca2+-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.