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 ...

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)

A large variety of neurotransmitters, hormones, and chemokines regulate cellular functions via cell surface receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins) belonging to the G i subfamily. All members of the G i subfamily, with the sole exception of G z , are substrates for the pertussis toxin ADP-ribosyl transferase. G z also exhibits unique biochemical and regulatory properties. Initial portrayals of the cellular functions of G z bear high resemblance to those of other G i proteins both in terms of the receptors and eectors linked to G z . However, recent discoveries have begun to insinuate a distinct role for G z in cellular communication. Functional interactions of the a subunit of G z (Ga z ) with the NKR-P1 receptor, Ga z -speci®c regulator of G protein signaling, p21-activated kinase, G protein-regulated inducers of neurite outgrowth, and the Eya2 transcription cofactor have been demonstrated. These ®ndings provide possible links for G z to participate in cellular development, survival, proliferation, dierentiation and even apoptosis. In this review, we have drawn a sketch of a signaling network with G z as the centerpiece. The emerging picture is one that distinguishes G z from other members of the G i subfamily.