Results: 5

1.
Figure 4

Figure 4. From: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Detection of GABAB 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 GABAB receptor combinations illustrated in the top schemes: when both subunits carry a snap-tag (filled squares), when only GABAB1 carries a snap-tag (grey circles), or when only GABAB2 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 AB2 expressed alone, (c) FRET between ST-GABAB2 subunits as a function of the amount of transfected GABAB1 plasmid. (d) GABA activation of the GABAB heteromer affects neither the FRET between GABAB2 subunits (GB1+ST-GB2), nor the FRET between GABAB1 subunits (ST-GB1+GB2), nor the FRET between GABAB1 and GABAB2 subunits (ST-GB1+ST-GB2).

Damien Maurel, et al. Nat Methods. ;5(6):561-567.
2.
Figure 5

Figure 5. From: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Functional correlate of the association between GABAB heterodimers (a) top: Scheme representing the various combinations of subunits co-expressed: ST-GABAB1(blue) with flag-GAB AB2KKXX (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-GABAB1 labeled with BG-K and BG-d2 (left (1)), between ST-GABAB1 labeled with BG-K and HA-GB1-HD labeled with d2-conjugated anti-HA antibodies (middle (2)), and between ST-GABAB1 labeled with BG-K and flag-GAB AB2KKXX 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-GABAB1 and flag-GABAB2KKXX 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.

Damien Maurel, et al. Nat Methods. ;5(6):561-567.
3.
Figure 3

Figure 3. From: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

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 GABAB (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 GABAB1 (C1 in blue) with its natural intracellular retention signal (blue ball) or GABAB2 (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).

Damien Maurel, et al. Nat Methods. ;5(6):561-567.
4.
Figure 2

Figure 2. From: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Detection of GABAB 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-GABAB1 and flag-GABAB2 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-GABAB 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-GABAB receptors. Positive control (left column) was performed with cells expressing ST-GABAB1 and flag-GABAB2. (c) FRET intensity was measured in cells expressing ST-GABAB1 and ST-GABAB2 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.

Damien Maurel, et al. Nat Methods. ;5(6):561-567.
5.
Figure 1

Figure 1. From: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

The use of snap-tag to label surface proteins with TR-FRET compatible fluorophores. (a) Ribbon representations at the same scale of the heterodimeric GABAB 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 06-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-GABAB1 co-expressed with GABAB2. (d) Specific BG-K labeling of mock transfected cells, and cells expressing the ST-GABAB1 alone (ST-GB1) or co-expressed with GABAB2 (ST-GB1:GB2), ST-GABAB1 mutated in its intracellular retention motif (ST-GB1ASA), and the G-protein αi1 subunit fused to snap-tag (Gi1-ST). (e) Confocal imaging of cells over-expressing a ST-GABAB1-GFP fusion alone (right) or together with GABAB2 (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-GABAB receptors at the cell surface. Cells were transfected with a fixed amount of ST-GABAB1 and increasing amounts of GAB AB2 plasmids. The specific number of cell surface receptors (Bmax) was determined by Scatchard analysis using [3H]-CGP54626, a non permeant GABAB1 specific antagonist. Linear regression revealed a slope (number of fluorophores per GABAB 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.

Damien Maurel, et al. Nat Methods. ;5(6):561-567.

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