Phospholipase Cβ (PLCβ) isoforms, which are under the control of Gα_q and Gβγ subunits, generate Ca²⁺ signals induced by a broad array of extracellular agonists, whereas PLCδ isoforms depend on a rise in cytosolic Ca²⁺ for their activation. Here we find that PLCβ₂ binds strongly to PLCδ₁ and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gβγ subunits. Such competition has consequences for signaling, because in HEK293 cells PLCβ₂ suppresses elevated basal [Ca²⁺] and inositol phosphates levels and the sustained agonist-induced elevation of Ca²⁺ levels caused by PLCδ₁. Also, expression of both PLCs results in a synergistic release of [Ca²⁺] upon stimulation in A10 cells. These results support a model in which PLCβ₂ suppresses the basal catalytic activity of PLCδ₁, which is relieved by binding of Gβγ subunits to PLCβ₂ allowing for amplified calcium signals.

Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gα_q family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gα_q. In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gα_q for PLCβ1 binding, and excess TRAX reverses Gα_q activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), Förster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gα_q for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments.

Caveolae are protein-dense plasma membrane domains structurally composed of caveolin-1 or -3 along with other proteins. Our previous studies have shown that caveolae enhance calcium signals generated through the Gα_q/phospholipase Cβ signaling pathway and that subjecting cells to hypo-osmotic stress reverses this enhancement. In this study, we have used super-resolution fluorescence microscopy supplemented by fluorescence correlation studies to determine the structural factors that underlie this behavior. We find similar and significant population of Gα_q and one of its receptors, bradykinin type 2 receptor (B2R), as well as a significant population of Gα_i and its coupled β2-adrenergic receptor (βAR), are localized to caveola domains. Although mild osmotic stress deforms caveolae and alters interactions between the caveolae and these proteins, the general structure and the localization of caveola components remain largely unchanged. This deformation eliminates the ability of caveolae to stabilize calcium signals mediated through Gα_q-B2R, but does not affect cAMP signals mediated through Gα_i and βAR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 newtons/m² increases the domain diameter by ∼30% and increases the fluorescence intensity in the center of the domain mouth suggesting a flattening of the invagination. Approximate calculations show that caveolae in muscle tissue have the strength to handle the stress of muscle movement.

Caveolae are membrane domains having caveolin-1 (Cav1) as their main structural component. Here, we determined whether Cav1 affects Ca²⁺ signaling through the Gα_q–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 Gα_q-eYFP are similarly associated in both cell lines by Forster resonance energy transfer (FRET). Upon stimulation, the amount of FRET between Gα_q-eYFP and eCFP-Gβγ remains high in FRTcav^– cells, but decreases almost completely in FRTcav⁺ cells, suggesting that Cav1 is increasing the separation between Gα_q-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 Gα_q and Cav1 that increase upon stimulation. Fluorescence correlation spectroscopy studies show that the mobility of Gα_q-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, Gα_q(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.

Caveolae are membrane domains that may influence cell signaling by sequestering specific proteins such as G-protein-coupled receptors (GPCRs). While previous reports largely show that Gα_q subunits, but not other G-proteins, interact strongly with the caveolae protein, Caveolin-1 (Cav1), the inclusion of GPCRs in caveolae is controversial. Here, we have used fluorescence methods to determine the effect of caveolae on the physical and functional properties of two GPCRs that have been reported to reside in caveolae, bradykinin receptor type 2 (B₂R), which is coupled to Gα_q, and the μ-opioid receptor (μOR), which is coupled to Gα_i. While caveolae do not affect cAMP signals mediated by μOR, they prolong Ca²⁺ signals mediated by B₂R. In A10 cells that endogenously express B₂R and Cav1, downregulation of Cav1 ablates the prolonged recovery seen upon bradykinin stimulation in accord with the idea that the presence of caveolae prolongs Gα_q activation. Immunofluorescence and Förster resonance energy transfer (FRET) studies show that a significant fraction of B₂R resides at or close to caveolae domains while none or very little μOR resides in caveolae domains. The level of FRET between B₂R and caveolae is reduced by downregulation of Gα_q or by addition of a peptide that interferes with Gα_q–Caveolin-1 interactions, suggesting that Gα_q promotes localization of B₂R to caveolae domains. Our results lead to the suggestion that Gα_q can localize its associated receptors to caveolae domains to enhance their signals.