Mechanisms of differential desensitization of metabotropic glutamate receptors

Abstract

G protein-coupled receptors (GPCRs) interact with intracellular transducers to control both signal initiation and desensitization, but the distinct mechanisms that control the regulation of different GPCR subtypes are unclear. Here we use fluorescence imaging and electrophysiology to examine the metabotropic glutamate receptor (mGluR) family. We find distinct properties across subtypes in both rapid desensitization and internalization, with striking differences between the group II mGluRs. mGluR3, but not mGluR2, undergoes glutamate-dependent rapid desensitization, internalization, trafficking, and recycling. We map differences between mGluRs to variable Ser/Thr-rich sequences in the C-terminal domain (CTD) that control interaction with both GPCR kinases and β-arrestins. Finally, we identify a cancer-associated mutation, G848E, within the mGluR3 CTD that enhances β-arrestin coupling and internalization, enabling an analysis of mGluR3 β-arrestin-coupling properties and revealing biased variants. Together, this work provides a framework for understanding the distinct regulation and functional roles of mGluR subtypes.

Keywords: G protein-coupled receptor; GRK; desensitization; internalization; melanoma; metabotropic glutamate receptor; meto; trafficking; β-arrestin.

Figure 1.. Glutamate-dependent internalization of mGluRs

(A) Surface fluorescence from HEK293T cells expressing either SNAP-tagged mGluRs or β₂AR with and without 60 min agonist stimulation (1–10 mM Glu and 10 μM Iso) before BG-Alexa 546 labeling. Values were normalized to the fluorescence of a given receptor without an agonist. Unpaired t tests, ***p < 0.0001 for mGluR3, *p = 0.02 for mGluR7, *p = 0.03 for mGluR8, and ***p = 0.0004 for β₂AR. (B) Images of cells expressing SNAP-mGluR2 (left) or SNAP-mGluR3 (right) before and after 30 min 1 mM Glu treatment with intensity line scans (bottom). Dotted lines denote the plasma membrane location. (C) TIRF image (left) and line scan (right) showing colocalization of SNAP-mGluR3 (red) and GFP-2xFYVE (green) following Glu treatment. (D) Quantification of the percentage of GFP-2xFYVE puncta that show colocalization with SNAP-mGluR2 or SNAP-mGluR3 before and after 15 min of Glu application. Lines connect values for individual cells, and bars show average values. Paired t test, **p = 0.004. (E) Confocal images showing colocalization of SNAP-mGluR3 and Cy3-transferrin (Cy3-Tf) following 30 min treatment with 1 mM Glu. (F) Pearson’s correlation coefficient (PCC) comparing the top 10% of pixels between mGluR2 or mGluR3 with Cy3-Tf following incubation in 1 nM Glu for 30 min. Unpaired t test, ***p < 0.0001. (G) Quantification of PCC comparing colocalization of mGluR3 and Cy3-Tf, mCh-TGN38, or LAMP1-YFP following incubation in 1 mM Glu for 30 min. (H) Top, schematic describing a recycling experiment in which receptors are internalized with Glu, remaining surface receptors are labeled with BG-Alexa 488, and subsequent labeling with BG-Alexa 546 is indicative of recycled receptors. For the control (blue dotted line), no Glu treatment was given. Unpaired t tests, ***p = 0.0002 for 10 min, ***p = 0.0002 for 30 min, and ***p = 0.0003 for 60 min. (I) Schematic summarizing major results. mGluR2 remains on the surface following activation, whereas mGluR3 internalizes into early endosomes, traffics to the ERC, and recycles back to the surface. Error bars show SEM. Scale bar, 10 μm (B and E) or 5 μm (C). Number of cells tested (F and G) or fields of cells analyzed (A) is in parentheses.

Figure 2.. GRK and β-arrestin dependence of mGluR internalization

(A and B) DN β-arr1 (S412D) and cmpd101 reduce internalization of mGluR3, whereas PTX enhances internalization, as seen in representative images (A) and quantification of surface fluorescence reduction following Glu application (B). Red arrows denote internalized receptors. Unpaired t tests, **p = 0.001 for PTX, **p = 0.001 for cmpd101, and **p = 0.001 for DN β-arr1. (C and D) Overexpression of GRK2 or β-arr enhances internalization of mGluR3, but not mGluR2. Unpaired t test, ***p < 0.0001 for GRK2. (E) Overexpression of GRK2 enhances internalization of mGluR7 and mGluR8, but not mGluR1 or mGluR5. Unpaired t tests, ***p = 0.0003 for mGluR3, **p = 0.004 for mGluR7, and **p = 0.006 for mGluR8. (F) SNAP-mGluRs were expressed in a CRISPR β-arr DKO HEK293 cell line. Internalization for mGluRs in β-arr DKO cells compared with the parental cell line using quantification of surface fluorescence reduction following Glu application. Unpaired t tests, ***p < 0.0001 for mGluR3, *p = 0.02 for mGluR7, and *p = 0.04 for mGluR8. Number of fields of cells analyzed is in parentheses. Error bars show SEM. Scale bar, 10 μm.

Figure 3.. GRK2-mediated rapid desensitization of mGluRs

(A and B) Representative whole-cell patch-clamp recordings from HEK293T cell expressing SNAP-mGluR2 (A) or SNAP-mGluR3 (B) showing an inward GIRK current induced by Glu application. (C) Quantification of the percentage of desensitization of GIRK currents showing larger desensitization of mGluR3 responses. Unpaired t test, ***p < 0.0001. (D and E) Recordings showing sensitivity of GIRK currents induced by mGluR2 (D) or mGluR3 (E) with and without GRK2 overexpression. (F) Summary bar graph showing the percentage increase in desensitization on mGluR2- and mGluR3-mediated GIRK currents with overexpression of GRK2, GRK2 mutants, or β-arr. Unpaired t test versus mGluR3 control, *p = 0.04 for 1xGRK2 and ***p < 0.0001 for 2xGRK2. (G) Schematic showing differential GRK recruitment and rapid desensitization for mGluR2 (left) and mGluR3 (right). (H) Recordings showing modest desensitization of mGluR8-mediated GIRK currents with and without GRK2 overexpression over 30 s Glu application. (I) Summary bar graph showing the percentage increase in desensitization of mGluR4-, mGluR7-, and mGluR8-mediated GIRK currents with overexpression of WT GRK2 or K220R. Unpaired t test of mGluR8 control versus mGluR8 + GRK, *p = 0.03. (J) Average trace representing calcium transients produced by ~50 cells expressing only mGluR1 or with GRK2 overexpression. (K) Summary bar graph showing the full duration at half-maximum of calcium transients mediated by mGluR1 and the first calcium transient mediated by mGluR5 activation, without and with overexpression of WT GRK2 or K220R. ** indicates statistical significance. Unpaired t tests, p = 0.002 for mGluR1 control versus GRK2, p = 0.001 for mGluR1 control versus K220R, p = 0.005 for mGluR5 control versus GRK2, and p = 0.009 for mGluR5 control versus K220R. Number of cells analyzed are as follows: n = 142 for mGluR1 control, 134 for GRK2, and 149 for K220R; n = 287 for mGluR5 control, 126 for GRK2, and 88 for K220R. (L) Summary heatmap showing the relative propensities for acute desensitization and internalization across mGluRs, and their dependence on interactions with β-arr and GRK2. Error bars show SEM. Number of cells recorded from (A) or independent experiments (F, I, and K) is in parentheses.

Figure 4.. mGluR3 recruits β-arrestins via scaffold and catalytic coupling

(A) Left, representative images and intensity line scans showing Glu-dependent surface recruitment of β-arr2 by mGluR3. Right, quantification ofthe percentage of cells that exhibit β-arr surface recruitment. Unpaired t tests for mGluR3, ***p < 0.0001 for β-arr1 and ***p < 0.0001 for β-arr2. (B) Time-lapse images showing Glu-dependent surface recruitment of β-arr2 and subsequent internalization of only mGluR3. Arrows point at β-arr2 accumulation on the cell surface. Arrowheads denote internalized receptors. (C–E) Representative TIRF images showing a lackof β-arr2 puncta for mGluR2 (C) but a high density of β-arr2 puncta generated by activation of mGluR3 (D) and β₂AR (E). Line scans reveal colocalization of receptor (red) and β-arr2 (green) puncta. Black arrows denote overlapping peaks. (F) Summary bar graph of receptor and β-arr2 puncta densities under basal conditions and agonist treatment. Paired t tests, *p = 0.03 for mGluR3 basal and *p = 0.01 for mGluR3 + Glu. (G) Quantification of the percentage of β-arr2 puncta that are colocalized with either mGluR3 (under basal or +Glu conditions) or β₂AR (+Iso) puncta. Unpaired t tests, *p = 0.03 for mGluR3 basal versus +Glu and ***p = 0.0005 for mGluR3 + Glu versus β₂AR + Iso. (H) Left, representative TIRF images showing that mGluR3 activation leads to β-arr2 puncta that colocalize extensively with the CCP marker, CLC. Right, intensity line scan through the white dotted line on the images that shows overlapping β-arr2 (green) and CLC (red) peaks, denoted by the black arrows. (I) Schematic of the working model for β-arr coupling with mGluR3. Error bars show SEM. Scale bar, 10 μm (B–E) or 5 μm (H). Number of fields of cells (A) or cells (F and G) analyzed is in parentheses.

Figure 5.. The C-terminal domains mediate differential internalization and rapid desensitization of mGluRs

(A) Top, illustrations of CTD deletions and chimeras for mGluR2 and mGluR3. Bottom, representative images of HEK293T cells expressing SNAP-tagged receptors and incubated for 30 min in Glu. Red arrowheads show internalized receptors. Scale bar, 10 μm. (B) Quantification of the percentage of cells that exhibit internalization of the various CTD variants. *** indicates statistical significance. Unpaired t test, p < 0.0001 for mGluR3 versus mGluR3-ΔCTD, p < 0.0001 for mGluR3 versus mGluR3-mGluR2CTD, p < 0.0001 for mGluR2 versus mGluR2-ΔCTD, p < 0.0001 for mGluR2 versus mGluR2-mGluR3CTD, and p = 0.0005 for mGluR3-ΔCTD versus mGluR2. (C) Quantification of surface fluorescence reduction following Glu application, where the control was no Glu. Unpaired t tests, *p = 0.03 mGluR3 and ***p < 0.0001 for mGluR2-mGluR3CTD. (D and E) Representative GIRK current traces for mGluR3-mGluR2CTD (D) and mGluR2-mGluR3CTD (E) with and without GRK2 overexpression. (F) Summary plot showing the percentage increase in desensitization of GIRK currents in the presence of GRK2 overexpression. Unpaired t test versus no GRK2 overexpression condition, **p = 0.005. Error bars show SEM. Number of fields of cells analyzed (B and C) or independent experiments (F) is in parentheses.

Figure 6.. A Ser/Thr-rich stretch of the mGluR3CTD controls GRK- and β-arrestin-dependent internalization and rapid desensitization

(A) Sequence alignment of the CTDs of mGluR2 and mGluR3 displaying the Ser/Thr (ST)-rich sequence and phospho-codes. (B) Top, illustrations of ST-rich region deletion and chimeric constructs. Bottom, representative images of HEK293T cells expressing SNAP-tagged receptor constructs and incubated in 1 mM Glu for 30 min. (C) Quantification of the percentage of cells that exhibit internalization of the various ST sequence variants. Unpaired t tests, ***p < 0.0001 for all comparisons made. (D) Quantification ofthe percentage of the internalized surface receptor population as determined bythe surface labeling assay, in which the control was no Glu. Unpaired t tests, **p = 0.003 for mGluR3 and **p = 0.001 for mGluR2-mGluR3ST. (E) Recordings showing the GRK2 sensitivity of mGluR2-mGluR3ST-mediated GIRK currents. (F) Summary plot showing the percentage increase in desensitization of GIRK currents with GRK2 overexpression. Unpaired t test versus no GRK2 overexpression, **p = 0.006. (G) Top left, sequence alignments of the ST-rich region of mGluR3, showing the residues that were mutated to Ala. Bottom left, images of cells expressing either WT mGluR3 or mGluR3-10xA following 30 min of 1 mM Glu treatment. Red arrows point to internalized receptors. Right, summary bar graph showing the percentage decrease in surface fluorescence following treatment with 1 mM Glu across mutants. Unpaired t tests, *p = 0.02 for WT versus 4xA, ***p < 0.0001 for WT versus 6xA_v1, **p = 0.003 for WT versus 6xA_v2, and ***p < 0.0001 for WT versus 10xA. (H) Working model of GRK2- and β-arr-mediated rapid and long-term desensitization of mGluR3. Error bars show SEM. Scale bars, 10 μm. Number of fields of cells analyzed (B, D, and G) or independent experiments (F) is shown in parentheses.

Figure 7.. Cancer-associated G848E mutation in the mGluR3CTD enables the identification of biased mGluR3 variants.

(A) Top, mutation G848E (green) within the ST-rich region ofthe mGluR3CTD is associated with melanoma. Bottom, mGluR3-G848E exhibits internalization and surface recruitment of β-arr2. (B) Surface labeling assay summary graph showing enhanced internalization for G848E compared with WT. Unpaired t tests, *p = 0.03 for 10 min, *p = 0.04 for 30 min, and **p = 0.003 for 60 min. (C and D) mGluR3-F765D is unable to produce Glu-induced GIRK channel activation (C) or β-arr2-YFP recruitment (D). (E and F) mGluR3-F765D/G848E is unable to produce Glu-induced GIRK channel activation (E) but exhibits β-arr2 recruitment (F). (G and H) Summary bar graphs showing the efficiency of G protein activation as detected via GIRK currents (G) and the efficiency of β-arr recruitment as measured by percentage cell analysis (H). Unpaired t tests, ***p < 0.0001 for WT versus F765D, *p = 0.02 for WT versus G848E, ***p = 0.0001 for G848E versus F765D/G848E, and ***p < 0.0001 for F765D versus F765D/G848E. (I and J) Differential receptor and β-arr2 puncta formation for G848E (I) and F765D/G848E (J) mutants. (K) Summary of mGluR3 and β-arr2 puncta density produced across receptor variants. # indicates statistical significance. Paired t tests, ###p = 0.0008 for WT, ##p = 0.008 for G848E, and ###p = 0.0008 for F765D. * indicates statistical significance. Unpaired t tests, *p = 0.04 for WT versus G848E receptor bars, **p = 0.002 for WT versus G848E β-arr2 bars, and **p = 0.008 for mGluR2ST versus 10xA β-arr2 bars. n = 15 cells for WT, 13 for G848E, 13 for F765D, 10 for F765D/G848E, 13 for ΔCTD, 7 for mGluR2ST, 7 for 10xA). (L) Schematic showing the relative G protein- and β-arr-coupling propensities of group II mGluR variants. Scale bar, 10 μm (A, C, and E) or 5 μm (I and J). Number of fields of cells analyzed (B and H), cells recorded from (G), or cells analyzed (K) is in parentheses.