Preactivation Permits Subsequent Stimulation of Phospholipase C by G_i-Coupled Receptors

Abstract

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 ...

Constitutively active Gq/11-coupled Receptors Enable Signaling by Co-expressed Gi/o-coupled Receptors

“…Our findings are consistent with the reported necessity for preactivation of various G q/11 -coupled receptors for G i/o -coupled receptor-mediated signaling both in vitro and ex vivo (54). Pretreatment of blood vessels with histamine, for example, is known to yield an enhanced contractile response to 5-HT, which is mediated by previously "silent" 5-HT receptors (55).…”

Gα16 Couples Chemoattractant Receptors to NF-κB Activation

“…In leukocytes and other hemopoietic cells, activation of G␣ 16 may synergize the effect of G␣ i -coupled receptors. This possibility has been suggested by a recent study demonstrating that preactivation of PLC␤ by G␣ 16 permits subsequent stimulation by G␣i-coupled receptors in transiently transfected COS-7 cells (47). Indeed, in macrophages derived from G␣ 15 Ϫ/Ϫ mice, C5a-stimulated phosphoinositide accumulation and Ca 2ϩ release were significantly reduced (19).…”

G16-mediated Activation of Nuclear Factor κB by the Adenosine A1 Receptor Involves c-Src, Protein Kinase C, and ERK Signaling

“…Attenuation by the co-expression of G␣ t (a G␤␥ scavenger) and potentiation by the overexpression of G␤ 1 ␥ 2 confirm the participation of G␤␥ in G 16 -mediated activation of NFB by A1R. Signals arising from G␣ and G␤␥ subunits are often integrated at downstream loci (36,37). One locus for signal integration is the small GTPase Ras (38).…”

“…These extracellular signals activate a key regulatory step in the pathway, the IB kinase (IKK) complex, comprising the catalytic subunits (IKK␣ and IKK␤) and a linker subunit (IKK␥/NEMO). This kinase complex, in turn, phosphorylates IB␣ at Ser 32 and Ser 36 and signals for its ubiquitin-related degradation (2). The released NFB is then translocated into the nucleus and promotes NFBdependent transcription (3).…”