No abstract

G protein—coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Gα16/zchimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca2+mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 Gs- or Gi-coupled receptors was examined for their functional association with the Gα16/zchimeras. Although most of the GPCRs tested were incapable of inducing Ca2+mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca2+mobilization. In contrast, only 16 of the GPCRs tested were capable of using Gα16to mobilize intracellular Ca2+. Analysis of dose-response curves obtained with the δ-opioid, dopamine D1, and Xenopus melatonin Mel1c receptors revealed that the Gα16/zchimeras possess better sensitivity than Gα16in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Gα16/zchimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca2+mobilization. ( Journal of Biomolecular Screening 2003:39-49)

Opioid receptors are involved in regulating neuronal survival. Here we demonstrate that activation of the µ-opioid receptor in human neuroblastoma SH-SY5Y cells led to the phosphorylations of IĸB kinase (IKK) and p65, denoting the stimulation of the nuclear factor-ĸB (NFĸB) transcription factor. This response was mediated through pertussis toxin-sensitive G proteins. The µ-opioid-induced IKK phosphorylation required extracellular signal-regulated protein kinase, phosphatidylinositol 3-kinase and c-Src. Moreover, c-Jun N-terminal kinase and calmodulin-dependent kinase II also participated in the IKK activation, despite the lack of involvement of phospholipase Cβ and protein kinase C. These data suggest that the µ-opioid receptor is capable of simulating NFĸB signaling via the phosphorylation of IKK and p65 in human neuroblastoma SH-SY5Y cells.