Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes

Abstract

A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described.

Keywords: Allosteric modulation; BRET competition assays; BRET kinetics and dose–response assays; BRET saturation assays; Bioluminescence resonance energy transfer (BRET); G protein-coupled receptors (GPCRs); GPCR–RTK heteroreceptor complexes; Heterodimerization; Heteroreceptor complexes; Homodimerization; Receptor tyrosine kinases (RTKs); Receptor–receptor interactions.

Figure 1. BRET² saturation assay shows specific A2AR and FGFR1 interaction in HEK293T cells

(A) The existence of a A2AR-FGFR1 heteroreceptor complexes and their agonist regulation by CGS21680 and/or FGF2 have been validated using quantitative BRET² saturation curves assay in HEK293T27 cells co-transfected with a constant amount of FGFR1-Rluc8 plasmid and increasing amount of the A2AR-GFP2 plasmid. In the current analysis the amount of each receptor effectively expressed in transfected cells was monitored for each individual experiment by correlating both total luminescence and total fluorescence to the number of receptor-binding sites (biochemical binding analysis) in permeabilized cells The linear regression equations derived from these data were thus used to convert fluorescence and luminescence values into femtomoles/mg protein of receptor in order to obtain accurate values. Cells were pre-incubated 10 min with vehicle, CGS21680 (100 nM), FGF-2 (50ng/ml), or with both CGS21680 and FGF-2 (100nM and 50ng/ml, respectively). The A2AR/FGFR1 curve fitted better to a saturation curve than to a linear regression, F test (P < 0.001). Data are means ± s.e.m. (n=5). The BRETmax values were significantly enhanced by combined, CGS21680 and FGF-2 treatment alone versus vehicle or CGS21680 treatment alone; and FGF2 treatment alone versus vehicle or CGS21680 treatment alone (P<0.01). (B) The BRET50 values were significantly reduced by combined, CGS21680 and FGF-2 treatment alone versus vehicle (P<0.001).

Figure 1. BRET² saturation assay shows specific A2AR and FGFR1 interaction in HEK293T cells

(A) The existence of a A2AR-FGFR1 heteroreceptor complexes and their agonist regulation by CGS21680 and/or FGF2 have been validated using quantitative BRET² saturation curves assay in HEK293T27 cells co-transfected with a constant amount of FGFR1-Rluc8 plasmid and increasing amount of the A2AR-GFP2 plasmid. In the current analysis the amount of each receptor effectively expressed in transfected cells was monitored for each individual experiment by correlating both total luminescence and total fluorescence to the number of receptor-binding sites (biochemical binding analysis) in permeabilized cells The linear regression equations derived from these data were thus used to convert fluorescence and luminescence values into femtomoles/mg protein of receptor in order to obtain accurate values. Cells were pre-incubated 10 min with vehicle, CGS21680 (100 nM), FGF-2 (50ng/ml), or with both CGS21680 and FGF-2 (100nM and 50ng/ml, respectively). The A2AR/FGFR1 curve fitted better to a saturation curve than to a linear regression, F test (P < 0.001). Data are means ± s.e.m. (n=5). The BRETmax values were significantly enhanced by combined, CGS21680 and FGF-2 treatment alone versus vehicle or CGS21680 treatment alone; and FGF2 treatment alone versus vehicle or CGS21680 treatment alone (P<0.01). (B) The BRET50 values were significantly reduced by combined, CGS21680 and FGF-2 treatment alone versus vehicle (P<0.001).

Figure 2. Effects of combined and single treatment with 8-OH-DPAT and FGF2 on FGFR1 homodimerization in HEK293T27 cells containing FGFR1-5-HT1A heteroreceptor complexes

The modulatory effect of 5-HT1A agonist 8-OH-DPAT was studied on the FGF2 induced FGFR1/FGFR1 homodimer formation by means of BRET² analysis. HEK293T27 cells were transiently co-transfected at a constant ratio (1:1:1) with 5-HT1A, FGFR1-Rluc8 and FGFR1-GFP2. (A) A concentration-response curve with FGF-2 was performed on the development of the BRET² signal from the FGFR1 homodimer in the HEK293T27 cells. The cells were transiently co-transfected at a constant ratio (1:1:1) of 5-HT1A, FGFR1-Rluc8 and FGFR1-GFP2 and treated with the agonist ligands for 5 min before BRET² measurement. Treatment with 8-OH-DPAT (with two different concentrations: 50nM and 250nM) shifted the curves of the BRET² signal to the left which indicate an enhanced potency of combined treatment with FGF2 and the 5-HT1A agonist vs FGF-2 treatment alone to promote FGFR1 homodimer formation. (B) The kinetics of the FGFR1-Rluc/FGFR1-GFP2 interaction after FGF2 treatment and its modulation by 8-OHDPAT was also studied in transiently transfected HEK293T27 cells using the BRET² assay to study the FGFR1 homodimer over a period of 20 minutes. FGF-2 and the combined FGF-2 and 8-OH-DPAT treatments showed no clear-cut changes of the BRET² value over the 8 min period. However, the combined treatment had a weak tendency to increase the BRET² signal over time whereas the FGF2 alone treatment had a markedly tendency to decrease the BRET₂ signal over time.

Figure 2. Effects of combined and single treatment with 8-OH-DPAT and FGF2 on FGFR1 homodimerization in HEK293T27 cells containing FGFR1-5-HT1A heteroreceptor complexes

The modulatory effect of 5-HT1A agonist 8-OH-DPAT was studied on the FGF2 induced FGFR1/FGFR1 homodimer formation by means of BRET² analysis. HEK293T27 cells were transiently co-transfected at a constant ratio (1:1:1) with 5-HT1A, FGFR1-Rluc8 and FGFR1-GFP2. (A) A concentration-response curve with FGF-2 was performed on the development of the BRET² signal from the FGFR1 homodimer in the HEK293T27 cells. The cells were transiently co-transfected at a constant ratio (1:1:1) of 5-HT1A, FGFR1-Rluc8 and FGFR1-GFP2 and treated with the agonist ligands for 5 min before BRET² measurement. Treatment with 8-OH-DPAT (with two different concentrations: 50nM and 250nM) shifted the curves of the BRET² signal to the left which indicate an enhanced potency of combined treatment with FGF2 and the 5-HT1A agonist vs FGF-2 treatment alone to promote FGFR1 homodimer formation. (B) The kinetics of the FGFR1-Rluc/FGFR1-GFP2 interaction after FGF2 treatment and its modulation by 8-OHDPAT was also studied in transiently transfected HEK293T27 cells using the BRET² assay to study the FGFR1 homodimer over a period of 20 minutes. FGF-2 and the combined FGF-2 and 8-OH-DPAT treatments showed no clear-cut changes of the BRET² value over the 8 min period. However, the combined treatment had a weak tendency to increase the BRET² signal over time whereas the FGF2 alone treatment had a markedly tendency to decrease the BRET₂ signal over time.

Figure 3. Different BRET² strategy assays to study GPCR-RTK heteroreceptor complexes dynamic

BRET² assays seems to be particularly suited for the molecular characterization and study of GPCR-RTK heteroreceptor complexes. Using different combination of receptor donor/acceptor fused receptor, it could be possible to unravel the molecular mechanisms underlying GPCR-RTK heteroreceptor complex trans-activation/trans-inhibition processes, internalization and signalling. (centre) For instance, it could be possible to study the existence of the GPCR-RTK heteroreceptor complexes and the pharmacological properties of drugs acting on these important therapeutic targets using as a BRET pairs, the GPCR-fused receptor as a donor and the RTK-fused receptor as acceptor. (left) Furthermore, the effects of combined and single treatment with GPCR and RTK agonist on the RTK homodimerization in cells containing the GPCR-RTK heteroreceptor complexes could be feasible. (right) In addition, the effects of combined treatment with the GPCR and RTK agonist on GPCR receptor homodimerization in cells containing the GPCR-RTK heteroreceptor complexes could be possible. Each of these different BRET approach could bring new light in the conformation changes and dynamic process that can take place upon agonists treatment in the GPRC-RTK heteroreceptor complexes.