Signaling through a G Protein-coupled Receptor and Its Corresponding G Protein Follows a Stoichiometrically Limited Model

Abstract

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...

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

“…Image analysis that monitored the amount of plasma membrane and cytosolic fluorescence from Gα q -eYFP before and 10 minutes after stimulation showed that it remains bound to the plasma membrane after activation, in accord with previous studies using the same methodology (Dowal et al, 2006a;Philip et al, 2007). Similar results were obtained for eCFP-Gβγ (data not shown).…”

“…Importantly, we also found that the extent and timing of Ca 2+ release and recovery were the same in both cell lines, suggesting that the expression of Cav1 does not significantly affect the level of expression or activity of proteins involved in Ca 2+ release. It is also noteworthy that the fluorescent-tagged G-protein subunits were overexpressed by a low amount and were not expected to affect the complexation (see Dowal et al, 2006a;Philip et al, 2007), although overexpression would stabilize G-protein heterotrimers, which was not observed here for the FRTcav + cells. The Ca 2+ measurements were performed on suspension cells, whereas the microscopy studies imaged attached cells.…”

“…These data indicate that CPM-Gα q (GDP) binds threefold more strongly to Cav1-containing membranes. In a second series of membrane-binding studies, we monitored the association between purified G-protein subunits labeled with a FRET acceptor (XRITC) and Cav1-eGFP, in which the eGFP tag on Cav1 acts as a FRET donor (see Philip et al, 2007). These studies were carried out by preparing membranes from FRTcav -cells expressing Cav1-eGFP and monitoring the loss in eGFP fluorescence as the protein with labeled acceptor was incrementally added (see Materials and Methods).…”

“…FRET measurements allow us to monitor protein-protein association on membrane surfaces of living cells in real time (e.g. Philip et al, 2007) and FCS measures the average mobility of cell surface proteins (e.g. Sengupta et al, 2002).…”

WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex

“…However, WDR36 did not regulate TPb-mediated cAMP generation. Gas is known to internalize whereas Gaq stays at the plasma membrane following GPCR activation (Dave et al, 2009;Philip et al, 2007). By contrast, both Gas and Gaq were reported to localize with their receptors on tubular recycling endosomes (Scarselli and Donaldson, 2009).…”