Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Abstract

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.

Fig. 1.

Tagging of different GPCRs led to expression at the plasma membrane and maintains their functional coupling to GIRK channels. (A) Representative confocal images showing that expression of tagged GPCRs as indicated. (Scale bar, 10 μm.) (B) Functional coupling of M4, D2, and A1 to GIRK channels in HEK293 cells. Transient transfection of the CFP and YFP constructs into a HEK-293 cell line stably expressing Kir3.1/3.2A. Membrane currents are studied under voltage clamp conditions at a holding potential of -60 mV. The application of relevant agonist elicited inward K⁺ currents. (C) Expression of Go-CFP and A1-YFP in HEK293 cells stably expressing Kir3.1 and Kir3.2A rescues coupling between the receptor and channel. Cells were PTx treated (100 ng/ml for 16 h) to inactivate all endogenous Gi/o G proteins; ^**, P < 0.01.

Fig. 2.

Oligomerization of a2A and M4 receptors on the plasma membrane. Representative confocal images were collected of HEK293 cells expressing M4-CFP, M4-YFP, M4-CFP together with M4-YFP, as well as α2A-CFP together with α2A-YFP. These images were obtained with a laser-scanning confocal microscopy as described in Materials and Methods. Three-cube net FRET signal, shown for the cells coexpressing M4-CFP with M4-YFP and α2A-CFP with α2A-YFP, is delimited to the plasma membrane. (Scale bar, 10 μm.)

Fig. 3.

Receptor precoupling with heterotrimeric G protein subunits. (A) Confocal images of HEKs cotransfected with the indicated plasmids. (Right) The three-cube net FRET images. (Scale bar, 10 μm.) (B) Images obtained with a back illuminated CCD camera (see Materials and Methods). (Right) Images showing the three-cube net FRET signal obtained with the three-cube FRET module (metamorph, Universal Imaging). (Scale bar, 10 μm.)

Fig. 4.

Role of Goα in the translocation of G protein subunits and in the precoupling with the GPCR. (A) Confocal images showing the intracellular distribution of Gβ1-YFP and Gγ2-CFP when expressed in HEK293 cells. (Right) The coexpression of Goα together with Gγ2 or Gβ1 led to translocation of the Gβ1-YFP (Upper) and Gγ2-CFP (Lower), respectively, to the plasma membrane. Under other expression conditions, there was significant intracellular retention. (B) Acceptor (i.e., α2A-YFP) photobleaching was carried out by using the 514-nm laser line. The subtracted image of CFP postbleach - CFP prebleach shows an increase of the donor fluorescence (i.e., Go-CFP) after acceptor photobleaching. (Scale bar, 10 μm.)

Fig. 5.

Receptor precoupling: specificity of the interaction between GPCRs and Gα.(A) Confocal images showing the cellular distribution of Go-CFP, Gs-CFP, and α2A-YFP in HEKs cells. (B) The specificity of the interaction between α2A-YFP and Goα-CFP as well as between IP-YFP (prostacyclin receptor) and Gs-CFP. Parts of the image IP-YFP and Gs-CFP are saturating and arrows indicate regions of interest which are not and have membrane delimited FRET. For all images, cells were transfected with the plasmids as indicated. (Scale bar, 10 μm.)

Fig. 6.

Quantitative analysis of FRET. (A) FRET ratio is plotted against donor intensity/pixel for α2A-YFP+Go-CFP (inverted triangles) and α2A-YFP+Gs-CFP (squares). (B and C) The FRET ratio is independent of the donor plus acceptor intensity/pixel (r² = 0.013, not significant, B) but is strongly correlated with donor/acceptor ratio (r² = 0.593, P < 0.0001, C). The lines indicate the best-fit regression lines.