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. 2019 Jun 7;8:e45116. doi: 10.7554/eLife.45116

Figure 3. An inter-TMD FRET assay reveals LBD-independent reorientation in response to positive allosteric modulators.

(A) Top, schematic showing SNAP- and CLIP-tagged mGluR2-TMD constructs labeled with donor and acceptor fluorophores. Bottom, images showing donor and acceptor channels following donor excitation with a 561 nm laser. Scale bars = 40 μm. (B) Representative time course showing donor and acceptor fluorescence intensity (top) during LY48 application. Baseline-normalized FRET is shown in the bottom trace, revealing a large, reversible increase in response to LY48 application. (C–E) Representative traces showing that LY48-induced inter-TMD FRET increase is repeatable (C), blocked by the NAM MNI 137 (D), and dose-dependent (E). The inset to (D) shows the extent of block of a 10 μM LY48 response by different concentrations of MNI 137. (F) Dose–response curve for LY48-induced FRET increase for SNAP-mGluR2-TMD (EC50 = 1.2 ± 0.1 µM), WT-SNAP-mGluR2 + dominant negative G protein (EC50 = 0.6 ± 0.04 µM). The dose–response curves were significantly different (two-way ANOVA, p=0.002). Values are normalized to saturating LY48 and come from at least three separate experiments per conditions. Error bars show s.e.m.

Figure 3.

Figure 3—figure supplement 1. Further characterization of an inter-TMD mGluR FRET Assay.

Figure 3—figure supplement 1.

(A) Configuration of inverted microscope used for ensemble FRET imaging. Briefly, 560 nm laser illumination is used for excitation and donor and acceptor emission is separated with a dichroic in front of two separate, aligned cameras. See Materials and methods for more details. (B) Left, representative donor and acceptor images of HEK 293T cells before and after acceptor channel bleaching for SNAP-mGluR2-TMD. Right, summary bar graph showing a significantly higher donor recovery for SNAP-mGluR2-TMD compared to the prototypical class A GPCR, SNAP-ß2AR (unpaired t test, p=0.003). Error bars show s.e.m. (C) Left, representative images of HEK 293T cells expressing SNAP-mGluR2-TMD before and after application of 10 μM LY48. Right, summary bar graph showing an increase in acceptor (paired t test, p=0.00004) and decrease (paired t test, p=0.00005) in donor fluorescence following LY48 application. Error bars show s.e.m. (D) Representative inter-TMD FRET trace showing that the response to LY48 is sustained and non-desensitizing during a 5-min period. (E) Summary of results showing the SNAP-mGluR2-TMD expression and labeling with donor (BG-LD-555) and acceptor (BG-LD-655) shows identical FRET responses to co-expression of SNAP-mGluR2-TMD and CLIP-mGluR2-TMD. Left, representative images showing donor- and acceptor-labeled SNAP-mGluR2-TMD-expressing HEK 293T cells. Top right, representative FRET trace showing an increase in inter-TMD FRET in response to LY48 application. The inset shows the donor (green) and acceptor (red) channels separately. Bottom right, dose–response curve showing identical curves for SNAP-mGluR2-TMD labeled with donor and acceptor (EC50 = 1.2 ± 0.1 µM) versus co-expression of SNAP- and CLIP-mGluR2-TMD (EC50 = 0.9 ± 0.1 µM). Values are normalized to saturating LY48 and come from at least three separate experiments per conditions. Error bars show s.e.m. (F) Representative images (top) and inter-TMD FRET trace (bottom) for SNAP-mGluR5-TMD in response to the mGluR5 PAM VU 0360172 showing that the LBD-independent rearrangement of TMDs is observed across the mGluR family. All scale bars = 20 µm.
Figure 3—figure supplement 2. Further analysis of G protein effects on inter-TMD rearrangement.

Figure 3—figure supplement 2.

(A) Representative images of HEK 293T cells expression GFP-TM-GαoA-G203T (blue) and SNAP-mGluR2-TMD labeled with donor (green) and acceptor (red). Scale bars = 20 µm. (B) Representative inter-TMD FRET trace showing dose-dependent LY48-induced increases in FRET.
Figure 3—figure supplement 3. A role for a TMD ‘Trigger Switch’ in allosteric agonism and inter-TMD re-arrangement.

Figure 3—figure supplement 3.

(A) mGluR1-TMD crystal structure showing the location of amino acids in TM3, 5 and 6 proposed to form the PAM ‘trigger switch’ including N735 in mGluR2 (red). (B) Representative HEK 293T cell patch recording showing a weak LY48-induced GIRK response relative to saturating glutamate in cells expressing full-length mGluR2. (C) Dose–response curve showing a large decrease in GIRK activation by LY48 for mGluR2-N735D. Values are normalized to saturating glutamate and come from at least three separate experiments per conditions. Error bars show s.e.m. (D) Representative inter-TMD FRET trace showing the response to LY48 for SNAP-mGluR2-TMD-N735D. (E) LY48 dose–response curves showing a large right-shift for SNAP-mGluR2-TMD-N735D (EC50 = 20.5 ± 1.4 µM) relative to wildtype (EC50 = 1.2 ± 0.1 µM). Values are normalized to saturating LY48 and come from at least three separate experiments per conditions. Error bars show s.e.m.