The bradykinin receptor is a G protein-coupled receptor (GPCR) that is coupled to the G␣ q family of heterotrimeric G proteins. In general, a GPCR can exert intracellular signals either by transiently associating with multiple diffusing G protein subunits or by activating a G protein that is stably bound to the receptor, thus generating a signal that is limited by the stoichiometry of the complex. Here we have distinguished between these models by monitoring the association of type 2 bradykinin receptor (B 2 R) and the G␣ q /G␤␥ heterotrimer in living human embryonic kidney 293 cells expressing fluorescent-tagged proteins. Stable B 2 R-G␣ q ⅐G␤␥ complexes are observed in resting cells by fluorescence resonance energy transfer from either G␣ q -eCFP or eCFP-G␤␥ to B 2 R-eYFP. Stimulating the cells with bradykinin causes detachment of B 2 R from the G protein subunits as the receptor internalizes into early endosomes, with a corresponding elimination of B 2 R-G protein fluorescence resonance energy transfer because G␣ q and its associated G␤␥ remain on the plasma membrane. Single point and scanning fluorescence correlation spectroscopy measurements show that a portion of B 2 R molecules diffuses with a mobility corresponding to dimers or small oligomers, whereas a second fraction diffuses in higher order molecular assemblies. Our studies support a model in which receptors are pre-coupled with their corresponding G proteins in the basal state of cells thereby limiting the response to an external signal to a defined stoichiometry that allows for a rapid and directed cellular response.Cell signaling through heterotrimeric G proteins involves the binding of an extracellular agonist to its corresponding 7-transmembrane receptor (GPCR).2 The agonist-bound receptor is then able to catalyze the exchange of GTP for GDP on the G␣ subunit of a heterotrimeric G protein (i.e. G␣␤␥). The G␣(GTP) has a weaker affinity for G␤␥ allowing G␣(GTP) and G␤␥ to interact with diverse effector proteins (for background see Ref. 1). This mechanism allows G protein signaling to be transduced by two ways. In the first, a diffusion model, the agonist-bound receptor activates a large number of G proteins through rapid association and diffusion. This model also indicates that the dynamics of the activated receptor that determines its encounter with G proteins impacts the efficiency of signal propagation (2). In the second, a stoichiometric model, the receptor, its associated G protein subunits, and effectors exist in a pre-formed complex. This model limits the number of G proteins that a single receptor can activate to the number of proteins in the complex, and it restricts the temporal and spatial aspects of the associated signal. Importantly, a pre-formed signaling complex would direct a signal to a particular pathway as opposed to the scenario where G proteins were freely diffusing and could contact multiple receptors and effectors.The organization of GPCRs in their natural cellular environment is unknown. There is increasing evidence that GP...

Phospholipase Cβ1 (PLCβ1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCβ1 binds to the RNA processing protein translin-associated factor X (TRAX) with nanomolar affinity and that this binding competes with G proteins. Here, we show that endogenous PLCβ1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCβ1. In HEK293 cells, TRAX overexpression ablates Ca(2+) signals generated by G protein-PLCβ1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLCβ1 overexpression completely reverses the 2- to 4-fold down-regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by an ∼4-fold recovery in both the transcript and protein levels. Also, down-regulation of endogenous PLCβ1 in HEK293 and HeLa cells allows for an ∼20% increase in siRNA(GAPDH) silencing. While PLCβ1 overexpression results in a 50% reversal of cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g., cyclophilin, Hsp90, translin). PLCβ1 overexpression in HEK293 and HeLa cells causes a 30% reduction in the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis that allows cells to progress through the S phase. We find that PLCβ1 reverses the cell death and completely rescues H2B levels caused by siRNA knockdown of LDH or GAPDH. Taken together, our study shows a novel role of PLCβ1 in gene regulation through TRAX association.

Summary Background Receptors that couple to Gi and Gq often interact synergistically in cells to elicit cytosolic Ca2+ transients that are several-fold higher than the sum of those driven by each receptor alone. Such synergism is commonly assumed to be complex, requiring regulatory interaction between components, multiple pathways, or multiple states of the target protein. Results We show that cellular Gi-Gq synergism derives from direct supra-additive stimulation of phospholipase C-β3 (PLC-β3) by G protein subunits Gβγ and Gαq, the relevant components of the Gi and Gq signaling pathways. No additional pathway or proteins are required. Synergism is quantitatively explained by the classical and simple two-state (inactive↔active) allosteric mechanism. We show generally that synergistic activation of a two-state enzyme reflects enhanced conversion to the active state when both ligands are bound, not merely the enhancement of ligand affinity predicted by positive cooperativity. The two-state mechanism also explains why synergism is unique to PLC-β3 among the four PLC-β isoforms and, in general, why one enzyme may respond synergistically to two activators while another does not. Expression of synergism demands that an enzyme display low basal activity in the absence of ligand and becomes significant only when basal activity is ≤ 0.1% of maximal. Conclusions Synergism can be explained by a simple and general mechanism, and such a mechanism sets parameters for its occurrence. Any two-state enzyme is predicted to respond synergistically to multiple activating ligands if, but only if, its basal activity is strongly suppressed.

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca2+ signaling through the Gαq–phospholipase-Cβ (PLCβ) pathway using Fischer rat thyroid cells that lack Cav1 (FRTcav–) and a sister line that forms caveolae-like domains due to stable transfection with Cav1 (FRTcav+). In the resting state, we found that eCFP-Gβγ and Gαq-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Gαq-eYFP and eCFP-Gβγ remains high in FRTcav– cells, but decreases almost completely in FRTcav+ cells, suggesting that Cav1 is increasing the separation between Gαq-Gβγ subunits. In FRTcav– cells overexpressing PLCβ, a rapid recovery of Ca2+ is observed after stimulation. However, FRTcav+ cells show a sustained level of elevated Ca2+. FRET and colocalization show specific interactions between Gαq and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Gαq-eGFP is unaffected by activation in either cell type. The mobility of eGFP-Gβγ remains slow in FRTcav– cells but increases in FRTcav+ cells. Together, our data suggest that, upon stimulation, Gαq(GTP) switches from having strong interactions with Gβγ to Cav1, thereby releasing Gβγ. This prolongs the recombination time for the heterotrimer, thus causing a sustained Ca2+ signal.