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.