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. 2016 Oct 5;92(1):143-159.
doi: 10.1016/j.neuron.2016.08.036. Epub 2016 Sep 15.

Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors

Joshua Levitz  1 , Chris Habrian  1 , Shashank Bharill  2 , Zhu Fu  2 , Reza Vafabakhsh  2 , Ehud Y Isacoff  3
Affiliations

Mechanism of Assembly and Cooperativity of Homomeric and Heteromeric Metabotropic Glutamate Receptors

Joshua Levitz et al. Neuron. .

Abstract

G protein-coupled receptors (GPCRs) mediate cellular responses to a wide variety of extracellular stimuli. GPCR dimerization may expand signaling diversity and tune functionality, but little is known about the mechanisms of subunit assembly and interaction or the signaling properties of heteromers. Using single-molecule subunit counting on class C metabotropic glutamate receptors (mGluRs), we map dimerization determinants and define a heterodimerization profile. Intersubunit fluorescence resonance energy transfer measurements reveal that interactions between ligand-binding domains control the conformational rearrangements underlying receptor activation. Selective liganding with photoswitchable tethered agonists conjugated to one or both subunits of covalently linked mGluR2 homodimers reveals that receptor activation is highly cooperative. Strikingly, this cooperativity is asymmetric in mGluR2/mGluR3 heterodimers. Our results lead to a model of cooperative activation of mGluRs that provides a framework for understanding how class C GPCRs couple extracellular binding to dimer reorganization and G protein activation.

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Figures

Figure 1
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% 2-step photobleaching is consistent with a strict dimer with ~75% GFP maturation. (D) Left, Schematic showing SimPull technique for pulldown 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-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 S.E.M. calculated from multiple experiments (N>3).
Figure 2
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 3 regions proposed to form the LBD dimer interface. (B-C) Summary of stoichiometry of dimer interface mutants in oocytes (H) and SimPull from HEK293T cell lysate (I). “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 S.E.M. calculated from multiple experiments (N>3).
Figure 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 3 FRET states obtained from Gaussian fits of smFRET histograms. (B) Histogram showing smFRET distributions for mGluR2-3xLB1. Distributions for mGluR2WT in 0 (grey) 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 uM for C121A; 300 uM for WT, 3xLB1). (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 S.E.M. calculated from multiple experiments (N≥3).
Figure 4
Figure 4. mGluR2 heterodimerizes with Group II/III mGluRs and prefers intra- over inter-group assembly
(A-B) Coexpression of excess untagged group II or III mGluRs decreases 2-step photobleaching of mGluR2-GFP in Xenopus oocytes. (C) Co-expression of chimeras between mGluR1 and mGluR2 (left) decreases 2-step photobleaching with a stronger effect when the ECD is from mGluR2 rather than mGluR1 (right). * indicates statistical significance (unpaired t-test, p=.0002). Dotted line shows the level of 2-step bleaching observed for mGluR2-GFP alone. (D) Images (left) showing colocalization between mGluR2-mCherry and mGluR3-GFP and representative trace (right) showing 1-step photobleaching in red and green. (E) Photobleaching step analysis showing primarily 1-step GFP photobleaching for mGluR3-GFP in complex with mGluR2-mCherry. (F) Summary of colocalization analysis for mGluR2-mCherry with either mGluR2-GFP, mGluR3-GFP, or mGluR7-GFP. * indicates statistical significance (unpaired t-test, p=0.0003 between mGluR2 and mGluR7 and p=0.0007). Error bars show S.E.M. calculated from multiple experiments (N≥3).
Figure 5
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 100uM 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-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 S.E.M. calculated from multiple experiments (N≥3).
Figure 6
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-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 2 agonists is >5× as efficient as 1 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-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 S.E.M. calculated from multiple experiments (N.≥3).
Figure 7
Figure 7. mGluR2/mGluR3 heterodimers exhibit trans-activation, intermediate glutamate affinity, and assymmetric 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 1 subunit liganding of mGluR2 in mG2-mG2, in mG2-mG3, or 1 subunit liganding of mGluR3 in mG2-mG3, or 2 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 S.E.M. calculated from multiple experiments (N≥3).
Figure 8
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 S.E.M. calculated from multiple experiments (N≥3).
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