Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Abstract

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Figure 1

The use of snap-tag to label surface proteins with TR-FRET compatible fluorophores. (a) Ribbon representations at the same scale of the heterodimeric GABA_B receptor (left, as based on the structure of the dimer of mGlu1 extracellular domains (pdb accession number: 1EWK), and the structure of a possible rhodopsin dimer (pdb: 1N3M)), an IgG (center, pdb: 1IGT) and a snap-tag (right, pdb 1EH6). (b) Covalent labeling of snap-tag fusion protein using 0-benzyl guanine derivatives carrying a fluorophore (F). (c) Cell surface specific binding with increasing concentrations of BG-K (filled circles) or BG-d2 (open circles) on ST-GABA_B1 co-expressed with GABA_B2. (d) Specific BG-K labeling of mock transfected cells, and cells expressing the ST-GABA_B1 alone (ST-GB1) or co-expressed with GABA_B2 (ST-GB1:GB2), ST-GABA_B1 mutated in its intracellular retention motif (ST-GB1_ASA), and the G-protein αi1 subunit fused to snap-tag (Gi1-ST). (e) Confocal imaging of cells over-expressing a ST-GABA_B1-GFP fusion alone (right) or together with GABA_B2 (left) and labeled with BG-d2. (f) Amount of d2 (open circles) and K (closed circles) fluorophores specifically bound to cells expressing various amounts of ST-GABA_B receptors at the cell surface. Cells were transfected with a fixed amount of ST-GABA_B1 and increasing amounts of GAB A_B2 plasmids. The specific number of cell surface receptors (Bmax) was determined by Scatchard analysis using [³H]-CGP54626, a non permeant GABA_B1 specific antagonist. Linear regression revealed a slope (number of fluorophores per GABA_B dimers) of 1.05 and 1.04 for the BG-K and BG-d2 labeling, respectively. Data in c, d and f are means ± sem of triplicate determinations from a representative experiment.

Figure 2

Detection of GABA_B heteromers at the cell surface using snap-tag and TR-FRET. (a) FRET intensity between d2-labeled anti-flag antibodies and BG-K labeled snap-tags in cells expressing increasing amounts of surface ST-GABA_B1 and flag-GABA_B2 receptors. FRET is measured as the specific d2 emission at 665nm after excitation of europium cryptate at 337 nm, the background signal measured in the absence of d2-antibodies being substracted. The FRET intensity is represented according to the total number of receptors expressed at the cell surface, (b) TR-FRET was measured between flag-GABA_B receptors labeled with d2-antibodies, and the indicated HA-snap-tag fusion proteins labeled with BG-K. Data were obtained with the same amount of snap-tag proteins at the cell surface as measured with anti-anti-HA ELISA, and a constant amount of flag-GABA_B receptors. Positive control (left column) was performed with cells expressing ST-GABA_B1 and flag-GABA_B2. (c) FRET intensity was measured in cells expressing ST-GABA_B1 and ST-GABA_B2 with varying concentration of BG-d2 and 5μM BG-K. (d) FRET intensity is directly proportional to the amount of ST-GABA subunits at the cell surface. The number of snap-tags was deduced from the Bmax of [³H]-CGP54626. (e) FRET efficacy as determined by the ratio of the specific d2 emission at 665 nm resulting from FRET, and the fluorescence intensity (at 620 nm) of the specifically bound BG-K, is plotted as a function of the amount of snap-tags at the cell surface deduced from the Bmax of [³H]-CGP54626. Data in (a) and (b) are means ± sem of triplicate determinations from a typical experiments. Data in (d) and (e) are triplicate determinations from 4 independent experiments.

Figure 3

Detection of cell surface protein oligomers using snap-tag fusions and TR-FRET. (a) Both TR-FRET intensity and HA-ELISA were measured for various expression levels of either GABA_B (filled squares) or V2 vasopressin (open squares) HA-ST-fusions. (b) Experiments were conducted as in (a) for various other cell surface proteins, and means TR-FRET intensity over the ELISA signal (representing the slope) are presented. Negative control (right column “mGlu1 dimer”) was performed using a mGlu1 receptor dimer carrying a single ST (see (c)). (c) FRET intensity was plotted as a function of the amount of mGlu1 receptor at the cell surface, when both subunits are fused to snap-tag (filled symbols) or when only one subunit per dimer is labeled (open symbols). To control the subunit composition of the mGlu1 receptor dimer, each subunit carries the C-terminal tail of either GABA_B1 (C1 in blue) with its natural intracellular retention signal (blue ball) or GABA_B2 (C2 in red) in which an intracellular retention signal was added (red ball). Coiled-coil interaction between Cl and C2 prevents intracellular retention of both proteins such that neither subunit reach the surface alone, but do so when co-expressed together (supp Fig. D).

Figure 4

Detection of GABA_B dimers of dimers at the cell surface, (a) FRET intensity (left panel) and FRET efficacy (right panel) measured in cells expressing various amounts of the GABA_B receptor combinations illustrated in the top schemes: when both subunits carry a snap-tag (filled squares), when only GABA_B1 carries a snap-tag (grey circles), or when only GABA_B2 carries a snap-tag (open triangles). FRET intensity is plotted as a function of the amount of snap-tags at the cell surface deduced from the Bmax of [³H]-CGP54626. (b) FRET intensity as a function of the amount of ST-GAB A_B2 expressed alone, (c) FRET between ST-GABA_B2 subunits as a function of the amount of transfected GABA_B1 plasmid. (d) GABA activation of the GABA_B heteromer affects neither the FRET between GABA_B2 subunits (GB1+ST-GB2), nor the FRET between GABA_B1 subunits (ST-GB1+GB2), nor the FRET between GABA_B1 and GABA_B2 subunits (ST-GB1+ST-GB2).

Figure 5

Functional correlate of the association between GABA_B heterodimers (a) top: Scheme representing the various combinations of subunits co-expressed: ST-GABA_B1(blue) with flag-GAB A_B2KKXX (red, the KKXX motif being the intracellular retention sequence added after the coiled coil domain), with (right) or without (left) the HA-GB1-HD, corresponding to the GB1 subunit deleted of both its extracellular and intracellular domains. Bottom: TR-FRET intensities measured between two ST-GABA_B1 labeled with BG-K and BG-d2 (left (1)), between ST-GABA_B1 labeled with BG-K and HA-GB1-HD labeled with d2-conjugated anti-HA antibodies (middle (2)), and between ST-GABA_B1 labeled with BG-K and flag-GAB A_B2KKXX labeled with d2-conjugated anti-flag antibodies (right (3)), in the absence (white columns) and in the presence (grey columns) of HA-GB1-HD. b) Ca²⁺ signals generated in cells expressing ST-GABA_B1 and flag-GABA_B2KKXX together (filled squares), or in the presence of over-expressed HA-GB1-HD (open squares), or in the presence of over-expressed CD4 (filled circles). Data are means ± sem of triplicate determinations from a typical experiment.