The~i-opioidreceptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G15 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both 6-and K-opioid receptors are likely to activate G16. Interactions of 6-and K-opioid receptors with G15 were examined by coexpressing the oploid receptors and Ga15 in COS-7 cells. The 6-selective agonist [D-Pen 2,DPen5] enkephalin potently stimulated the formation of mositol phosphates in cells coexpressing the 6-opioid receptorand Ga 16. The 6-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of Ga16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the pi-opioid receptor, whereas the K-opioid receptor produced moderate phospholipase C activity. Ga16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the 1i-opioid receptor was expressed at a lower level than those of the 6-and K-Opioid receptors. To examine if differential coupling to Ga16 is prevalent, a panel of G5-or G1-coupled receptors was coexpressed with Ga16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of a2-and /12-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-l and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1 .5-to almost 17-fold. These findings suggest that the promiscuous Ga16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.

In the complex signal transduction networks involving G protein-coupled receptors there are numerous examples where G i -linked receptors augment G q -dependent signals. The mechanistic basis of such occurrences is thought to entail signal convergence at phospholipase C␤ (PLC␤) via the G protein ␤␥-dimers. Herein, we explored the possibility that augmentation by ␤␥-dimers requires preactivation of PLC␤. COS-7 cells were transiently cotransfected with cDNAs encoding various combinations of receptors and G protein subunits. The G icoupled ␦-and -opioid receptors could not stimulate PLC␤ unless they were coexpressed with G␣ 16 . The opioid-induced response was dose-dependent and partially inhibited by pertussis toxin or coexpression with transducin, indicating the involvement of ␤␥-subunits released from the G i proteins.When PLC␤ was preactivated by constitutively active mutants of G␣ 16 , G␣ q , or G␣ 14 , opioids enhanced the activity by 80 to 300% and such responses were mostly pertussis toxin-sensitive. The opioid-induced enhancement was dose-dependent and could not be blocked by staurosporin, a protein kinase C inhibitor. Other G i -coupled receptors that were ineffective on their own also acquired the ability to stimulate PLC␤ in the presence of a constitutively active mutant of G␣ q . Coactivation of endogenous or exogenous G q -coupled receptors with the ␦-opioid receptor produced strong stimulations of PLC␤ and such responses could be partially blocked by pertussis toxin. These results show that enhancement of G q -dependent signals by G i -coupled receptors requires activated PLC␤ and is mediated via the ␤␥-dimer.In the nervous system, different extracellular signals are often required to coordinate complex neuronal activities such as neurotransmission and cognition. The multitude of extracellular signals is usually detected by a variety of cell surface receptors that use distinct yet overlapping signal transduction mechanisms. The ability to integrate and process incoming signals is an important characteristic of neurons. The superfamily of G protein-coupled receptors (GPCRs) constitutes a large array of cell surface detectors for neurotransmitters, hormones, lipids, pheromones, and photons. Multiple GPCRs are often coexpressed in any particular cell type, where they regulate the levels of intracellular second messengers independently, in synergism, or by antagonism. Of the two most widely studied effectors of GPCRs, adenylyl cyclase and phospholipase C (PLC), intricate regulatory mechanisms for the former have been discerned.The mechanism by which signals generated from different GPCRs become integrated inside the cell is best exemplified by the type 2 adenylyl cyclase. Type 2 adenylyl cyclase can be stimulated by the G protein ␤␥-subunits only when it is already preactivated by either G␣ s or protein kinase C-mediated phosphorylation (Tsu and Wong, 1996). Hence, the ␤␥-subunits released on the activation of G i -linked receptors can enhance the activity of type 2 adenylyl cyclase only if G s ...

The δ‐opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ‐opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ‐selective agonist [d‐Pen2,d‐Pen5]enkephalin. The inhibitory response of [d‐Pen2,d‐Pen5]enkephalin was dependent on the expression of the δ‐opioid receptor and exhibited an EC50 of 1 nM. The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ‐opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ‐opioid receptor with type II adenylyl cyclase and an activated mutant of αs converted the δ‐opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk− fibroblasts, the cloned δ‐opioid receptor was able to stimulate the formation of inositol phosphates (EC50 = 8 nM). This response was sensitive to pertussis toxin. The opioid‐mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ‐opioid receptor to couple to G proteins other than Gi was also examined. Cotransfection studies revealed that the δ‐opioid receptor can utilize Gz to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.