Caveolin-1 alters Ca2+ signal duration through specific interaction with the Gαq family of G proteins

Abstract

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.

Super-resolution Visualization of Caveola Deformation in Response to Osmotic Stress

“…Once activated by receptors, G␣ q subunits stimulate PLC␤, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate leading to Ca 2ϩ release from intracellular stores (6,7). Based on previous work (8,9), we found that Cav-1 or -3 specifically binds G␣ q , and the affinity between the two proteins increases when G␣ q is activated (4). The stabilization of activated G␣ q by Cav1/3, coupled with release of G␤␥ from caveola domains during the activation cycle, has the net effect of prolonging PLC␤ activation and increasing the extent and duration of Ca 2ϩ responses.…”

“…Our laboratory has shown that the presence of caveolae directly affects Ca 2ϩ signals mediated through the G␣ q /phospholipase C␤ (PLC␤) 2 pathway (4,5). Receptors that activate this pathway bind dopamine, acetylcholine, bradykinin, and angiotensin II as well as other hormones and neurotransmitters.…”

Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation

“…Previously, Escribá et al showed that PE enhances the binding of Gα-subunit to model membranes (45). FRET experiments using X-rhodamine isothiocyanate (XRITC)-Gα q -subunit, XRITC-Gβγ-subunit, and caveolin-1 (Cav1)-EGFP showed that Gα q -subunit has a stronger affinity for Cav1-EGFP than Gβγ-subunit has (46). Our results, together with results of others, suggest that PE provides a platform for PLCβ1 activation by sensing curved membranes, such as caveolae, via the C-terminal domains.…”

β-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release

“…Although PLC1 is concentrated at the cell membrane, it is also present throughout the cell, whereas G q is more-or-less membrane specific, where it stably associates with PLC1 to allow for rapid transmission of intracellular signals via GPCRs such as the M3 muscarinic receptor (Dowal et al, 2006). Recent evidence suggests that caveolin-1 can have a regulatory role, promoting dissociation of G subunits from  subunits to facilitate GPCR signal transduction (Sengupta et al, 2008). Interaction between caveolin-1 and -dystroglycan appears to be necessary for such a functional role, because in human airway smooth muscle cells with stably silenced -dystroglycan, PLC1 and G q were lost from caveolae and accumulated in fractions that are typically devoid of caveolae.…”

“…Collectively, these results support a role for the actin cytoskeleton, through its tethering to a DGC-caveolin-1 complex, in the establishment and maintenance of discrete caveolae microdomains in smooth muscle cells. release in the smooth muscle and other cell systems (Daniel et al, 2009;Darby et al, 2000;El-Yazbi et al, 2008;Gosens et al, 2007b;Prakash et al, 2007;Sengupta et al, 2008;Zhu et al, 2008). We assessed whether this functional role is linked to the unique ordered distribution of caveolae in contractile smooth muscle cells.…”