Dynamics of receptor/G protein coupling in living cells

Abstract

The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)-based assay, we measure directly and in living cells the interaction of YFP-labeled alpha(2A)-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Galpha. Simultaneously recorded G protein-regulated inwardly rectifying K(+) channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Galpha mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that alpha(2A)-adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.

Figure 1

Analysis of receptor/G protein interaction by FRET. (A) FRET was measured between YFP-tagged receptors and CFP-tagged Gβγ subunits. When these constructs are excited at 436 nm, emission is shifted from 480 to 535 nm when both fluorophores are close enough to each other to permit FRET. (B) In HEK293T cells transiently expressing α_2A-YFP and CFP-γ₂ together with Gαi₁β₂ (μg transfected DNA: α_2A-YFP 0.4, Gαi₁ 2, Gβ₁ 0.5, Gγ₂ 0.25), α_2A-YFP (left) and CFP-γ₂ (right) colocalize at the cell membrane (scale bar: 5 μM). (C) Upon stimulation with 100 μM NE (bar), a decrease in CFP fluorescence (F_CFP) and an increase in corrected YFP fluorescence (F_YFP) were observed. This resulted in an increase in FRET, assessed as the ratio of F_YFP over F_CFP. The FRET increase is readily reversible upon agonist washout. (D) The average increase in FRET ratio was ∼0.022 (n=8). (E) Fluorescence and FRET changes in a cell (as in panel C) in response to different NE concentrations. The FRET signal is stable over more than 400 s, and the amplitude of the FRET change depends on agonist concentration (concentrations indicated in μM; representative experiment out of eight shown). The agonist-independent increase in the YFP and CFP traces at ∼270 s is due to removal of solution from the coverslip holder; note that no increase is seen in the ratiometric FRET trace.

Figure 2

α_2A-YFP interaction with wild-type Gαi₁ and Gαi₁ND. (A) FRET signals of individual cells in response to 100 μM NE (bar) with wild-type (black curve) and ND mutant (gray curve) Gαi₁ coexpressed with α_2A-YFP and Gβ₁ CFP-γ₂ (n=5, representative recording shown). (B) Concentration–response curves of receptor/G protein interaction for wild-type (black) and ND mutant (gray) (n=5–8) Gαi₁ were determined by measuring amplitudes of FRET changes as in panel A after stimulation with different concentrations of NE. FRET responses following stimulation with 1 mM NE were set to 100%. (C) In membranes prepared from cells expressing α_2A-YFP, Gβ₁ CFP-γ₂ and wild-type Gαi₁ or Gαi₁ND, high-affinity agonist binding sites were determined by competing for [³H]RX821002 binding with NE (n=3 each). Competition binding data for wild-type Gαi₁ (black) were fitted best by a monophasic curve (K_i=21±1.8 μM), while a biphasic fit was significantly better (F-test) for Gαi₁ND (gray; K_i,high=95±70 nM, K_i,low=17±0.8 μM, 16% high-affinity sites). (D) Western blot analysis of expression levels of HEK293T cells transfected with wild-type Gαi₁ (wt) or Gαi₁ND (ND); β-actin was determined in the same samples as control (n=3, representative experiment shown). Cells not transfected with Gαi₁ were also analyzed (mock).

Figure 3

Analysis of absolute FRET levels. (A) Confocal images of cells transfected with α_2A-YFP (left) and Gβ₁ CFP-γ₂ (right; scale bar: 5 μM); here, Gαi₁ was not cotransfected. (B) FRET signal mediated only by endogenous Gα subunits. In cells transfected with α_2A-YFP and Gβ₁ CFP-γ₂, a small increase in FRET in response to agonist stimulation could be detected (representative example of 17 shown). (C) By means of measuring donor recovery after acceptor photobleaching, FRET between the α_2A-YFP and CFP-γ₂ was quantified. CFP fluorescence of cells expressing the indicated constructs was measured before and after acceptor photobleaching for 5 min. In the absence of agonist, the increase in F_CFP was not significantly different when comparing mYFP and the α_2A-YFP as acceptor, regardless of whether Gαi₁ was coexpressed (n=14 and 10 for α_2A-YFP and mYFP, respectively) or not (n=17 and 33 for α_2A-YFP and mYFP, respectively). For cells expressing Gαi₁, F_CFP was also measured in the presence and absence of 100 μM NE in the same cell; here, a significant increase was observed (n=11). ^*P<0.0001 (paired t-test), ^#P<0.001 in a one-way ANOVA followed by Tukey's post test. (D) Increase in F_CFP after acceptor photobleaching of cells transfected with mYFP or α_2A-YFP and Gαi₁-CFP β₁γ₂ (left; n=10 and 9) or Gαi₁ Cerulean-β₁γ₂ (right; n=3 and 7).

Figure 4

Kinetics of the α_2A-adrenergic receptor/Gi interaction. (A) Cells transfected with α_2A-YFP and Gαi₁β₁ CFP-γ₂ were superfused with different NE concentrations. Activation traces of the same cell after stimulation with different concentrations of NE are shown (representative experiment out of eight). (B) Times required to achieve a half-maximal FRET response (T_1/2) are plotted as a function of agonist concentration; P<0.0001 (one-way ANOVA). (C) Kinetics (left) of receptor activation (dark gray; determined with cells expressing the α_2A-CFP/YFP; Vilardaga et al, 2003) and receptor/G protein interaction (light gray) after stimulation with 1 mM NE (representative experiments of 8 and 10, respectively). The maximal amplitude of both traces was set to 100%. Averaged time constants (right) were 44±4.0 ms for receptor activation; for receptor/G protein interaction, the time constants depended on the amount of Gαi₁ and were 86±10, 74±2.3, 58±7.0, 54±3.1 and 44±5.1 ms for 0, 0.2, 0.5, 1 and 2 μg transfected Gαi₁ cDNA, respectively; P=0.0022 for different Gα amounts (one-way ANOVA). (D) Western blot of lysates from cells transfected with α_2A-YFP, Gβ₁ CFP-γ₂ (for amounts, see Materials and methods) and indicated amounts of Gαi₁ (left). In the same samples, β-actin was determined as a control.

Figure 5

Analysis of the simultaneously recorded receptor/G protein interaction and GIRK currents. (A) In cells expressing α_2A-YFP, Gαi₁β₁ CFP-γ₂ and GIRK1+4, FRET responses (thick curves) and GIRK currents (thin curves) were measured simultaneously after stimulation with 100 μM NE (bar; complete reversal of GIRK currents not shown). (B) FRET responses (thick curves) and GIRK currents (thin curves) were recorded during two subsequent stimulations with 1 and 100 μM NE (gray and black curves, respectively) in the same cell. The amplitudes obtained with the 100 μM NE were set to 100%, and the FRET and GIRK current traces following 1 μM NE stimulation were normalized accordingly (n=6, representative recording shown).