The Xenopus melatonin receptor was expressed in human embryonic kidney 293 cells and assayed for cAMP accumulation. In transfected 293 cells expressing the melatonin receptor, melatonin dose-dependently inhibited the endogenous adenylyi cyclases. In contrast, melatonin stimulated the accumulation of cAMP in cells co-expressing the type II adenylyl cyclase. Both the inhibitory and stimulatory responses to melatonin were mediated via Gi-like proteins as they were blocked by pertussis toxin. Upon co-transfection with the oz subunit of G~, the ability of melatonin to regulate both type H and the endogenous adenylyl cyclases became refractory to pertussis toxin, indicating that the melatonin receptor can also couple to Gz. However, other pertussis toxin-insensitive G proteins such as Gq, G~2 and G13 were unable to interact with the melatonin receptor.

The bovine Gα14 is a member of the Gq subfamily of G proteins that can regulate phospholipase Cβ isoforms but the extent to which Gα14 recognizes different receptor classes is not known. Gα14 was cotransfected with a variety of receptors in COS‐7 cells, and agonist‐induced stimulation of phospholipase C was then measured. Activation of the type 2 but not type 1 somatostatin receptor in cells coexpressing Gα14 stimulated the accumulation of inositol phosphates; functional expression of both subtypes of somatostatin receptors was determined by the ability of somatostatin to inhibit cyclic AMP accumulation. Among the three opioid receptors (μ, δ, and κ), only the δ receptor was capable of stimulating IP formation when coexpressed with Gα14 in COS‐7 cells. A panel of Gi‐ and Gs‐linked receptors was screened for their ability to stimulate IP accumulation via Gα14. The adenosine A1, complement C5a, dopamine D1, D2 and D5, formyl peptide, luteinizing hormone, secretin, and the three subtypes of melatonin (mt1, MT2, and Xenopus) receptors were all incapable of activating Gα14, while the α2‐ and β2‐adrenoceptors were able to do so. Gα14‐mediated stimulation of phospholipase Cβ was agonist dose‐dependent. These data demonstrate that although Gα14 can interact with different classes of receptors, it is much less promiscuous than Gα15 or Gα16. British Journal of Pharmacology (2001) 132, 1431–1440; doi:

The pineal neurohormone melatonin modulates a variety of physiological processes through different receptors. It has recently been reported that the cloned melatonin receptors (MT1, MT2 and Mel1c) exhibit differential abilities to stimulate phospholipase C (PLC) via G 16 . Here we examined the molecular basis of such differences in melatonin receptor signaling. Coexpression of MT1 or MT2 with the a subunit of G 16 (Ga 16 ) allowed COS-7 cells to accumulate inositol phosphates in response to 2-iodomelatonin. In contrast, Mel1c did not activate Ga 16 even though its expression was demonstrated by radioligand binding and agonist-induced inhibition of adenylyl cyclase. As Mel1c possesses an exceptionally large C-terminal tail, we further asked if this structural feature prevented productive coupling to Ga 16 . Eleven chimeric melatonin or mutant receptors were constructed by swapping all or part of the C-terminal tail between MT1, MT2 and Mel1c. The primary roles of melatonin, a hormone synthesized and secreted by the pineal gland, are to regulate behavioral and neuroendocrine processes, as well as circadian rhythms in mammals. High affinity melatonin receptors cloned from various organisms have been found to be homologs of one of three receptor subtypes, MT1, MT2 and Mel1c, which make up the G protein-coupled melatonin receptor family (Shiu et al. 1997). The mammalian MT1 and MT2 receptors are 60% identical to each other and they share 60% sequence homology with Xenopus Mel1c (Reppert et al. , 1995a. The receptor subtypes also have distinct expression patterns. Mammalian MT1 is expressed in the hypophyseal pars tuberalis and hypothalamic suprachiasmatic nuclei, regions of the brain that have been implicated as being important for reproduction and circadian rhythms, respectively . The mammalian MT2, on the other hand, is found in the retina (Reppert et al. 1995a). Furthermore, the three receptor subtypes may not necessarily occur in the same organisms. Initial studies indicate that only the MT1

Opioid tolerance and physical dependence in mammals can be rapidly induced by chronic exposure to opioid agonists. Recently, opioid receptors have been shown to interact with the pertussis toxin (PTX)-insensitive G z (a member of the G i subfamily), which inhibits adenylyl cyclase and stimulates mitogen-activated protein kinases (MAPKs). Here, we established stable human embryonic kidney 293 cell lines expressing ␦-opioid receptors with or without G z to examine the role of G z in opioid receptor-regulated signaling systems. Each cell line was acutely or chronically treated with [D-Pen 2 ,DPen 5 ]enkephalin (DPDPE), a ␦-selective agonist, in the absence or presence of PTX. Subsequently, the activities of adenylyl cyclase, cyclic AMP (cAMP)-dependent response element-binding proteins (CREBs), and MAPKs were measured by determining cAMP accumulation and phosphorylation of CREBs and the extracellular signalregulated protein kinases (ERKs) 1 and 2. In cells coexpressing G z , DPDPE inhibited forskolin-stimulated cAMP accumulation in a PTX-insensitive manner, but G z could not replace G i to mediate adenylyl cyclase supersensitization upon chronic opioid treatment. DPDPE-induced adenylyl cyclase supersensitization was not associated with an increase in the phosphorylation of CREBs. Both G i and G z mediated DPDPE-induced activation of ERK1/2, but these responses were abolished by chronic opioid treatment. Collectively, our results show that although G z mediated opioid-induced inhibition of adenylyl cyclase and activation of ERK1/2, G z alone was insufficient to mediate opioid-induced adenylyl cyclase supersensitization.

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.

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptorligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1 § , monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1ˇall inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1ˇwas unable to signal via G 14 -, G 16 -or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and G § 14 , all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G i/o -coupled pathways, but MIP-1ˇ, HCC-1 and MPIF-1 were unable to activate G 14 -mediated stimulation of phospholipase C g activity. In addition, MIP-1ˇwas unable to promote the phosphorylation of extracellular signalregulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and G § 14 or G § 16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

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Many Gi-coupled receptors are known to interact with the pertussis toxin (PTX)-insensitive Gz protein. Given that the alpha subunits of Gi and Gz share only 60% identity in their amino acid sequences, their receptor-interacting domains must be highly similar. By swapping the carboxyl termini of alpha i2 and alpha z with each other or with those of alpha t, alpha12, and alpha13, we examined the relative contributions of the carboxyl-end 36 amino acids of the alpha chains toward receptor recognition. Chimeric alpha chains lacking the site for PTX-catalyzed ADP-ribosylation were coexpressed with the type II adenylyl cyclase (AC II) and one of several Gi-coupled receptors (formyl peptide, dopamine D2, and delta-opioid receptors) in human embryonic kidney 293 cells. The alpha i2/alpha z chimera was able to interact with both aminergic and peptidergic receptors, resulting in betagamma-mediated stimulation of AC II in the presence of agonists and PTX. Functional and mutational analyses of alpha i2/alpha z revealed that this chimera can inhibit the endogenous ACs of 293 cells. Similarly, the alpha z/alpha i2 chimera seemed to retain the abilities to interact with receptors and inhibit cAMP accumulation. Fusion of the carboxyl-terminal 36 amino acids of alpha z to a backbone of alpha t1 produced a chimera, alpha t1/alpha z, that did not couple to any of the Gi-coupled receptors tested. Interestingly, an alpha13/alpha z chimera (with only the last five amino acids switched) displayed differential abilities to interact with receptors. Signals from aminergic, but not peptidergic, receptors were transduced by alpha13/alpha z. A similar construct, alpha12/alpha z, behaved just like alpha13/alpha z. These results indicated that "alpha i-like" or "alpha z-like" sequences at the carboxyl termini of alpha subunits are not always necessary or sufficient for specifying interaction with Gi-coupled receptors.