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 ...

Nociceptin/OFQ is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL1) receptor. To elucidate the cellular functions of the ORL1 receptor, we examined its ability to interact with Gãnd G16, two pertussis toxin (PTX) -insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL1 and dopamine D1 receptors, nociceptin/OFQ dose-dependently inhibited dopamine-stimulated cyclic AMP (cAMP) accumulation in a PTX-sensitive manner. However, PTX failed to block the nociceptin/OFQ-induced inhibition of dopamine-stimulated cAMP accumulation in HEK 293 cells co-expressing the £6-subunit of G~. This result indicates functional interaction between the ORL1 receptor and G0. A similar result was obtained with retinoic acid-differentiated SH-SY5Y cells, which endogenously express both the ORL1 receptor and G~. When the ORL1 receptor was transiently co-expressed in COS-7 cells with the a-subunit of G16, nociceptin/OFQ dose-dependently stimulated the formation of inositol phosphates. Nociceptininduced stimulation of phospholipase C was absolutely dependent on the co-expression of £616 and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX-insensitive G proteins, the ORL1 receptor behaves very much like the opioid receptors. Key Words: Adenylyl cyclase-G16-G0-Phospholipase C-ORL1 receptor-Signal transduction.