Levitz J , Habrian C , Bharill S , Fu Z , Vafabakhsh R , Isacoff EY .

PN2 EY018241 NEI NIH HHS , S10 RR028971 NCRR NIH HHS , T32 GM008295 NIGMS NIH HHS , R01 GM117051 NIGMS NIH HHS

	Figure 1. Dimerization of mGluR2 Is Mediated Primarily by the Ligand-Binding Domain (A) Left: domain structure of the mGluR2-GFP construct. LBD, ligand-binding domain; CRD, cysteine-rich domain; TMD, transmembrane domain. Right: schematic showing TIRF image of single mGluR2-GFP molecules in the plasma membrane of oocytes. (B) Representative photobleaching trace for a single mGluR2-GFP complex. Arrows show photobleaching steps. (C) Summary of photobleaching step analysis for mGluR2-GFP and truncations. ∼60% two-step photobleaching is consistent with a strict dimer with ∼75% GFP maturation. (D) Left: schematic showing SimPull technique for pull-down with an anti-mGluR2 antibody. Right: representative TIRF image of single mGluR2-GFP molecules isolated from HEK293T cell lysate. (E) Representative photobleaching trace for a single mGluR2-GFP complex. (F) Summary of photobleaching step analysis for mGluR2-GFP and ΔECD-GFP in SimPull. (G and H) TIRF images of single GFP-LBD (G) or TMD-GFP (H) subunits isolated using an anti-HA antibody in the presence or absence of full-length HA-mGluR2. (I) Summary of pull-down efficiency for GFP-LBD and TMD-GFP in the absence or presence of HA-mGluR2. (Unpaired t test, p = 0.00003 between GFP-LBD with and without HA-SNAP; p = 0.0001 between TMD-GFP with and without HA-SNAP; p = 0.004 between GFP-LBD and TMD-GFP.) Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 2. Mutational Analysis of mGluR2 Dimer Interface: Assembly and Functional Effects (A) Schematic (left) and crystal structure of mGluR1 in the “active” state (PDB: 1EWK) (right) showing three regions proposed to form the LBD dimer interface. (B and C) Summary of stoichiometry of dimer interface mutants in oocytes (B) and SimPull from HEK293T cell lysate (C). “3x-LB1” is the construct containing L103A, L154A, and F158A mutations. (D) Representative FRET trace showing glutamate-induced reductions in intersubunit FRET between LBDs for N-terminally SNAP- and CLIP-tagged versions of mGluR2-3xLB1. (E) Summary of glutamate EC50 determinations from activation of GIRK channels (current) versus LBD conformational change (FRET) in mGluR2WT (WT) and dimer interface mutants. Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 3. smFRET Analysis of mGluR2 LBD Interface Mutants (A) smFRET traces of mGluR2-3xLB1 showing spontaneous dynamics in the absence of glutamate. Top: donor (green) and acceptor (red) fluorescence for a single mGluR2-3xLB1 dimer. Bottom: smFRET calculated from donor and acceptor fluorescence values in top traces. Dotted gray lines show the three FRET states obtained from Gaussian fits of smFRET histograms. (B) Histogram showing smFRET distributions for mGluR2-3xLB1. Distributions for mGluR2WT in 0 (gray) or 1 mM glutamate (black) are shown as solid lines for comparison. (C) Representative smFRET traces of mGluR2-C121A in the absence of glutamate (top) and for mGluR2-C121A showing transitions to resting conformation in the presence of saturating 10 mM glutamate (bottom). (D) Histogram showing smFRET distributions for mGluR2-C121A. Note the small increase in high FRET population for C121A in the presence of saturating glutamate compared to WT. (E) Representative smFRET trace for WT, 3xLB1, and C121A showing the level of dynamics in the presence of saturating DCG-IV (100 mM for WT and 3xLB1; 300 mM for C121A). (F) Histogram showing smFRET distributions for WT, 3xLB1, and C121A in the presence of saturating DCG-IV. (G) Cross-correlation plots showing relative dynamics for WT, 3xLB1, and C121A in the presence of saturating DCG-IV. Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 4. mGluR2 Heterodimerizes with Group II/III mGluRs and Prefers Intra- over Inter-group Assembly (A and B) Schematic of co-assembled GFP-tagged mGluR2 and untagged mGluR3 (left) and a representative single-step photobleaching trace (A). A summary of the photobleaching step distribution for mGluR2-GFP co-expressed with different mGluR subtypes (B). (C) Co-expression of chimeras between mGluR1 and mGluR2 (left) decreases two-step photobleaching with a stronger effect when the ECD is from mGluR2 rather than mGluR1 (right). ∗ indicates statistical significance (unpaired t test, p = 0.0002). Dotted line shows the level of two-step bleaching observed for mGluR2-GFP alone. (D) Images (left) showing co-localization between mGluR2-mCherry and mGluR3-GFP and representative trace (right) showing one-step photobleaching in red and green. (E) Photobleaching step analysis showing primarily one-step GFP photobleaching for mGluR3-GFP in complex with mGluR2-mCherry. (F) Summary of co-localization analysis for mGluR2-mCherry with mGluR2-GFP, mGluR3-GFP, or mGluR7-GFP. ∗ indicates statistical significance (unpaired t test, p = 0.0003 between mGluR2 and mGluR7 and p = 0.0007 between mGluR3 and mGluR7). Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 5. Photoactivation of Tethered Ligands Reveals Cooperative Activation in mGluR2 (A) Chemical structure of the D-MAG-0 photoswitchable tethered ligand. Irradiation with 500 nm light (green arrow) induces the trans-configuration and 380 nm light (violet arrow) induces the cis-configuration. (B) Schematic of photoactivation of mGluR2 that is conjugated to D-MAG-0 (“LimGluR2”). (C) Representative HEK293T whole-cell recording where LimGluR2 is co-expressed with GIRK1(F137S) as a reporter. 380 nm light (violet bar) induces an inward current that is turned off by 500 nm light (green bar) compared to current evoked by 100 μM glutamate. (D) Low-affinity LimGluR2(R57A) shows large photocurrents and diminished glutamate response. (E) Partial D-MAG-0 labeling yields weak LimGluR2 photocurrent that is potentiated by a low concentration of glutamate. (F) Summary of photocurrent potentiation (y axis) by 10 μM glutamate as a function of degree of D-MAG-0 labeling (photoswitch efficiency; x axis). Red line shows linear fit. (G) Summary of accelerating effect of 10 μM glutamate on photocurrent kinetics. Individual cells (gray) in two conditions connected by lines with average (red). ∗ indicates statistical significance (paired t test, p = 0.007). (H and I) Concentration dependence of glutamate-mediated photocurrent potentiation for LimGluR2 and LimGluR2(R57A) showing representative trace from cell expressing LimGluR2 (H) and average relation (I). Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 6. Photoactivation Analysis of Receptor Cooperativity in Tandem Dimers and Dimer Interface Mutants (A) Schematic of mGluR2 tandem dimer. Transmembrane linker contains two GFPs (green) and the transmembrane segment of the H+,K+ ATPase (beige). (B and C) Representative traces showing that, compared to saturating glutamate, docking of glutamate of D-MAG-0 in a single subunit within a tandem dimer weakly activates mGluR2 (B), whereas docking in both subunits strongly activates (C). Tethering of D-MAG-0 to subunits is via introduced cysteine in one (300C-WT) or both (300C-300C) subunits. (D) Summary of photoactivation relative to 1 mM glutamate for various conditions. Activation with two agonists is >5× as efficient as one agonist. All constructs are tandem dimers except for “300C,” which is the standard non-tandem LimGluR2 construct. The numbers of cells tested for each condition are shown in parentheses. (E) Model of occupancy-dependent activation of mGluR2, where LBD is either open (O) or closed (C) and the receptor is either resting (R) or activated (A). (F) Schematic of SNAP-mGluR2 photoactivation by BGAG12,460. (G and H) Representative traces showing photoactivation of SNAP-3xLB1 (G) or SNAP-C121A (H). (I) Summary of photoactivation relative to 1 mM glutamate for dimer interface mutants. The numbers of cells tested for each condition are shown in parentheses. Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 7. mGluR2/mGluR3 Heterodimers Exhibit trans-activation, Intermediate Glutamate Affinity, and Asymmetric Cooperativity (A) LimGluR2(F756D) G protein-coupling mutant has no photocurrent (left) unless co-expressed with mGluR2WT (middle) or mGluR3WT (right). (B) Representative trace showing glutamate-induced decreases in ensemble FRET between co-expressed CLIP-mGluR3 (labeled with donor) and SNAP-mGluR2 (labeled with acceptor) in a HEK293T cell. Inset shows cell donor (CLIP-mGluR3) and acceptor (SNAP-mGluR2) fluorescence images. (C) Summary of glutamate EC50 determinations from measurement of GIRK activation (current) versus LBD conformational change (FRET) for mGluR2, mGluR3, and mGluR2/mGluR3 (“mG2/mG3”). FRET for mG2/mG3 obtained from co-expression, as in (B); GIRK current from tandem-linked mGluR2-mGluR3 (“mG2-mG3”). (D) GIRK current traces showing single-subunit photoactivation of linked mG2-mG3 heterodimers via photoactivation of only mGluR2 (top) or only mGluR3 (bottom). (E) Summary of photoactivation (from left to right) with one-subunit liganding of mGluR2 in mG2-mG2 or in mG2-mG3, one-subunit liganding of mGluR3 in mG2-mG3, or two-subunit labeling in unlinked mGluR2, linked mG2-mG3, or unlinked mGluR3. ∗ indicates statistical significance (unpaired t test, p = 0.003 between mG2(300C)-mG2(WT) and mG2(300C)-mG3(WT); p = 0.004 between mG2(300C)-mG3(WT) and mG2(WT)-mG3(306C)). The numbers of cells tested for each condition are shown in parentheses. Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure 8. Basal Conformational Dynamics of mGluR2/3 Heterodimers Provide a Mechanism for Enhanced Single-Subunit Activation (A) Representative ensemble mGluR2/mGluR3 (“mG2/mG3”) FRET trace shows an increase in FRET in response to the orthosteric antagonist LY341495 in the absence of glutamate. (B) Summary of percentage of basal FRET that is LY341495 sensitive in mGluR2, mGluR3, and mG2/mG3 variants. Basal FRET in mGluR3 is reduced by mutation 306C and nearly abolished by mutation S152D. (C) smFRET traces for mG2/mG3 in the absence of glutamate and presence (top) or absence (bottom) of 2 mM Ca2+. (D) smFRET traces for mG2/mG3 in the presence of saturating glutamate (top) or for mGluR2/3(S152D) in the absence of glutamate (bottom). (E) Histogram showing smFRET distribution for mG2/mG3 and mG2/mG3(S152D) in various conditions. (F) Cross-correlation analysis of mGluR2/3 reveals basal dynamics that are diminished by either the removal of Ca2+ or the addition of saturating glutamate. (G) Representative GIRK current traces showing single-subunit photoactivation of mGluR2 in tandem heterodimer of mG2(300C)-mG3(WT) (top) or mG2(300C)-mG3(S152D) (bottom). (H) Summary of effect on photoactivation of introduction of S152D mutation in mG2-mG3 tandem heterodimers (light blue) or mGluR3 homodimers (dark blue). ∗ indicates statistical significance (unpaired t test, p = 0.003 between mGluR2/3 heterodimers and p = 0.008 between mGluR3 homodimers). The numbers of cells tested for each condition are shown in parentheses. (I) Conformational model of ligand occupancy-dependent activation of mGluR2/mGluR3 heterodimers. Error bars show SEM calculated from multiple experiments (n ≥ 3).
	Figure S1, Related to Figure 1: Further single molecule analysis of mGluR dimerization. (A-E) mGluR2, mGluR3, mGluR7, mGluR1, and mGluR5 are each dimers. Left, representative TIRF images of each GFP-tagged receptor expressed at low density in the plasma membrane of a Xenopus oocyte. Center, photobleaching step distributions for each condition. Right, representative traces showing functionality of each GFP-tagged constructs: glutamate-evoked GIRK1/2 current (A-C); glutamateevoked calcium-activated chloride current (D-E). Error bars represent the counting uncertainty (see methods). 5 (F-G) Removal of either the LBD (∆LBD) (F) or the entire ECD (∆ECD) (G) eliminates dimerization of mGluR2-GFP in Xenopus oocytes. Error bars show S.E.M. calculated from multiple experiments (N>5 movies). (H-I) Stoichiometry analysis in SimPull from HEK293T cell lysate for mGluR2-GFP (H) and mGluR2(∆ECD)-GFP (I). Left, representative confocal image showing expression of each construct in HEK 293T cells prior to lysis. Center, representative TIRF images of complexes immobilized on a passivated surface at low density. Right, photobleaching step distributions. Error bars represent the counting uncertainty. (J) Summary of photobleaching step distributions from (H-I) for full-length or ∆ECD versions of mGluR2-GFP, mGluR3-GFP, and mGluR1-GFP. Error bars show S.E.M. calculated from multiple experiments (N>5 movies). (K) Photobleaching step distribution for mGluR2(∆ECD)-GFP in SimPull from Xenopus oocyte lysate with high level of expression. Error bars represent the counting uncertainty
	Figure S2, Related to Figure 2: Further analysis of mGluR dimer interfaces (A) Sequence alignment for the upper lobe (LB1) LBD interfaces of mGluRs. Residues mutated to alanine for counting experiments are shown in red. * indicates sequence conservation across all mGluR subfamily members. (B) Crystal structure of mGluR1 in the unliganded “relaxed” (O-O/R) state (PDB: 1EWT) showing the location of residues homologous to those mutated in mGluR2. (C) Sequence alignment for the lower lobe (LB2) LBD interface of mGluRs. Residues mutated to alanine for counting experiments are shown in orange. (D) mGluR2(C121A)-GFP partially reduces dimerization. Left, cartoon and representative TIRF image showing single mGluR2(C121A)-GFP complexes in Xenopus oocytes. Right, photobleaching step distribution. Error bars represent the counting uncertainty. 7 (E) Summary of photobleaching for mGluR2-GFP in the absence or presence of 10 mM DTT. Error bars show S.E.M. calculated from multiple experiments (N>5 movies). (F-H) Analysis of expression of mGluR2 dimer interface mutants in HEK 293T cells. Confocal images show similar expression of either C-terminally GFP-tagged (F) or N-terminally SNAP-tagged and BGAlexa-647 labeled (G) mGluR2 constructs. Quantification of Alexa-647 fluorescence is shown in (H). (I-J) Glutamate dose-response curves for mGluR2(3xLB1) and mGluR2(C121A), compared to mGluR2(WT), with the response as either activation of GIRK current (A) or reduction in ensemble FRET with SNAP and CLIP-tagged receptors labeled with acceptor and donor fluorophores, respectively, (B) in HEK 293T cells. (K-L) Glutamate dose-response curves for LB2 interface mutants, with the response as either activation of GIRK current (C) or reduction in ensemble FRET (D) in HEK293T cells.
	Figure S3, Related to Figure 3: smFRET analysis of mGluR2 dimer interface mutants. (A) Representative smFRET traces for wild type mGluR2 (SNAP and CLIP-tagged) in the absence (top) or presence of glutamate at a concentration near the EC50 (middle) or at saturation (bottom). (B) Histogram showing smFRET distributions for mGluR2wt. Red dotted lines mark the three FRET peaks. (C) Schematic of 3-state model of LBD activation that involves intra- (open vs. closed) and intersubunit (relaxed vs. active) conformational changes. (D) Representative smFRET traces for mGluR2(3xLB1) shows primarily low FRET state occupancy in a low (top) or saturating (bottom) concentration of glutamate. (E) Cross-correlation analysis for mGluR2(3xLB1) shows enhanced spontaneous dynamics in the absence of glutamate that are eliminated by saturating glutamate. (F) FRET histograms in saturating glutamate show a left shift in low FRET peak location for mGluR2(3xLB1) compared to WT. (G) Representative smFRET trace for mGluR2(C121A) shows rapid dynamics in intermediate glutamate. (H) smFRET histograms for mGluR2(C121) in a range of glutamate concentrations. Note a substantial occupancy of the medium and high FRET states even in saturating (1 or 10 mM) glutamate. (I) Cross-correlation analysis for mGluR2(C121A) shows elevated dynamics even in saturating glutamate. (J-L) smFRET analysis of mGluR2(3xLB1/C121A) shows spontaneous dynamics in the absence of glutamate that are similar to 3xLBI (J), altered high and medium FRET peaks and incomplete low FRET occupancy that are similar to C121A (J, K), and intermediate low FRET occupancy in the presence of DCG-IV compared to 3xLB1 and C121A (L).
	Figure S4, Related to Figure 4: Further single molecule analysis of mGluR2 heterodimerization. (A) Representative TIRF images show that pulldown of mGluR2 efficiently co-immunoprecipiates mGluR3 (left), but not mGluR1 (right), in SimPull from HEK293T cells. 11 (B) Summary of co-pulldown of mGluR3-GFP or mGluR1-GFP by mGluR2. Error bars show S.E.M. calculated from multiple experiments (N>5 movies). (C) Photobleaching step distribution analysis showing that mGluR3-GFP spots immobilized via pulldown of mGluR2 primarily bleach in one step, consistent with the formation of mGluR2/mGluR3 heterodimers.
	Figure S4, Related to Figure 4: Further single molecule analysis of mGluR2 heterodimerization continued (D-E) mGluR1(ECD)-mGluR2(TMD) (“mG1(ECD)-mG2(TMD)-GFP”) (D) and mGluR2(ECD)- mGluR1(TMD) (“mG2(ECD)-mG1(TMD)-GFP”) (E) chimeras form dimers in Xenopus oocytes. Error bars represent the counting uncertainty. (F-H) SimPull analysis shows that, in HEK293T cells, HA-mGluR2 co-assembles with either mG1(ECD)-mG2(TMD)-GFP or mG2(ECD)-mG1(TMD)-GFP, but does so more efficiently with mG2(ECD)-mG1(TMD)-GFP. Error bars show S.E.M. calculated from multiple experiments (N>5 movies).
	Figure S5, Related to Figure 5: D-MAG-0 is a high-efficacy, high-affinity tethered photo-agonist of mGluR2. (A) Glutamate dose-response curves for LimGluR2 (mGluR2(300C) + D-MAG-0) and low affinity mutant LimGluR2(R57A) using GIRK current in HEK293 cells as a readout. 13 (B) Summary of photoactivation (photocurrent amplitude/saturating glutamate current amplitude) for LimGluR2 and LimGluR2(R57A). The numbers of cells tested are shown in parentheses. (C-D) 1 uM of the competitive antagonist LY341495, which binds to the orthosteric glutamate binding site (C), blocks photocurrent in LimGluR2, but not LimGluR2(R57A) (D). (E-F) 1 uM of the negative allosteric modulator RO64-2259, which binds at an allosteric site in the TMD (E), blocks photocurrent in both LimGluR2 and LimGluR2(R57A). (G) Photoactivation in the presence of a range of glutamate concentrations in LimGluR2(R57A) shows photocurrent potentiation at 100 uM glutamate. Error bars show S.E.M. calculated from multiple experiments (N>3 cells).
	Figure S6, Related to Figure 6: Analysis of tandem mGluR dimers and ligand occupancy-dependent cooperativity. (A) Western blot analysis using an anti-mGluR2 antibody shows that the mGluR2-mGluR2 tandem dimer (tandem) has the expected molecular weight, which is similar in size to the dimer band observed for mGluR2-GFP (~270 kDa). (B) Confocal image of mGluR2-mGluR2 tandem dimers in HEK 293T cells. (C) Glutamate dose-response curves, using GIRK current readout in HEK293T cells, shows similar glutmate sensitivity for mGluR2(WT) and mGluR2-mGluR2 tandem dimers. Error bars show S.E.M. calculated from multiple experiments (N>4 cells). 15 (D-E) Single molecule subunit counting analysis in Xenopus oocytes (D) or SimPull from HEK293T cell lysate (E) shows the expected distribution for mGluR2-mGluR2 tandem dimers. Error bars represent the counting uncertainty. (F) Representative GIRK current trace shows that single subunit photoactivation in mGluR2(300C)- mGluR2(wt) tandem (300C-wt) is blocked by the competitive antagonist, LY341495. (G) Representative trace showing that single subunit activation of 300C-WT tandem dimers is maintained when the WT subunit is mutated to R57A to lower glutamate affinity. This indicates that single subunit activation is not due to binding of ambient glutamate to the WT subunit. (H) Single subunit photoactivation of 300C-WT tandem dimers is potentiated by 1 μM glutamate, a concentration below the EC50 (see Fig. S4A). (I) Summary of photoactivation kinetics, as determined from single component exponential fits, for 300C-WT (“1 agonist”) and 300C-300C (“2 agonists”) tandem dimers. * indicates statistical significance (unpaired t-test, p=0.04). Error bars show S.E.M. calculated from multiple experiments(N>4 cells).
	Figure S7, Related to Figure 7: Characterization of mGluR2-mGluR3 heterodimers. (A-B) Glutamate dose-response curves show an intermediate glutamate affinity for heterodimer of mGluR2 and mGluR3 compared to homodimer of mGluR2 or mGluR3. Activation measured as intersubunit LBD FRET using SNAP-mGluR3 (labeled with BG-Alexa-647 acceptor) co-expressed with CLIP-mGluR2 (labeled with BC-DY-547 donor) (A) or GIRK current using the mGluR2-mGluR3 tandem dimer (B). (C) Representative GIRK current trace shows robust glutamate activation of mGluR2-mGluR3 tandem dimer. (D) Representative trace shows photoactivation of mGluR2(300C)-mGluR3(306C) tandem dimer using D-MAG-0. Error bars show S.E.M. calculated from multiple experiments (N>3).
	Figure S8, Related to Figure 8: Further analysis of conformational dynamics and cooperativity in mGluR2/3 and mGluR3. (A-B) smFRET histograms for mG2/mG3 (A) and mG2/mG3(S152D) (B) heterodimers. SNAP-mGluR3 is labeled with BG-Alexa-647 acceptor and co-expressed CLIP-mGluR2 is labeled with BC-DY-547 donor. Application of competitive antagonist LY341495 or introduction of the S152D mutation (to eliminate the agonist effect of calcium) into the mGluR3 subunit abolishes basal dynamics in mG2/mG3. 18 (C) smFRET histogram reveals enhanced occupancy of the low FRET (activated) state in saturating DCG-IV for mG2/mG3 compared to mGluR2, consistent with stabilization of the activated state. (D) Cross-correlation analysis shows decreased dynamics for mG2/mG3 compared to mGluR2 in the presence of saturating DCG-IV, consistent with stabilization of the activated state. (E-F) Representative traces showing photoactivation of LimGluR3 (mGluR3(306C) + D-MAG-0) (E) and LimGluR3(S152D) (F). Error bars show S.E.M. calculated from multiple experiments (N>3).

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