A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers

Abstract

There is growing interest in developing biologics due to their high target selectivity. The G protein-coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

Keywords: G protein–coupled receptor; activation mechanism; agonist; single-domain antibody.

Fig. 1.

DN45 is selective for the human mGlu4 receptor and preferentially binds to the active receptor. (A) Cartoon representing a TR-FRET–based binding assay. SNAP-mGlu receptors were labeled with 100 nM BG-Lumi4-Tb (blue star). Then, 100 nM DN45 containing a c-Myc epitope was labeled with 200 nM anti-c-Myc-antibody (gray) coupled to d2 (red star). (B) Specific binding of DN45 to human mGlu4 receptor without (black) and with (pink) a saturating concentration of agonist (mGlu group I: 1 µM quisqualic acid; group II: 100 nM LY379268; group III: 10 µM L-AP4), represented by an increase of signal compared to an irrelevant nanobody containing the c-Myc sequence. (C) No specific binding was observed between the nanobody and any rat mGlu receptors in the absence (black) or presence (pink) of agonist. (D) Binding of an increasing concentration of DN45 on hmGlu4 under basal condition (black) in the presence of the agonist L-AP4 (10 µM) or in the presence of the antagonist LY341495 (100 µM). Data in B–D are mean ± SEM of three individual experiments (SI Appendix, Table S1).

Fig. 2.

DN45 is a full agonist of the hmGlu4 receptor. (A) Measurement of the change in TR-FRET induced upon stimulation with increasing concentrations of L-AP4 (blue) or DN45 (red). (B) Measurement of the fraction of activated receptor by single-molecule FRET in basal/apo state (black) in the presence of LY341495 (green), DN45 (red), or L-AP4 (blue). (C) Measurement of the rearrangement of the Gi protein in a BRET experiment upon stimulation with increasing concentrations of L-AP4 (blue) and DN45 (red). (D) Measurements of the activation of the Gq pathway via transient overexpression of Gqi9 upon stimulation with increasing concentrations of L-AP4 (blue), DN45 (red), or glutamate (black). (E) Measurements of the activation of the Gq pathway via transient overexpression of Gqi9 upon stimulation with increasing concentrations of DN45 alone (red) or in the presence of LY341495 (i.e., 100 or 400 µM). (F) Measurement of the activation of the Gq pathway upon stimulation with increasing concentrations of L-AP4 alone (blue) or L-AP4 with nonsaturating concentrations (i.e., 5, 10, or 100 nM) of DN45. Data in A–F are mean ± SEM of three or more individual experiments. Statistical analysis for A–D was performed using unpaired two-tailed t tests, and the statistical analysis of F was an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (SI Appendix, Tables S2 and S3).

Fig. 3.

Residues in lobe 2 of the human mGlu4 VFT confer subtype selectivity to DN45. (A) Front view of the closed conformation of the VFT of the human mGlu4 receptor, stabilized by DN45 (red) in the best-scored nonconstrained docking followed by the 10-ns molecular dynamics simulation. Residues on the surface of the mGlu4 VFT that are specific for the human ortholog are highlighted in purple (side chains). (B) Binding of DN45 was determined in the TR-FRET binding assay as illustrated in Fig. 1A with human wild-type (hmG4) and mutated mGlu4 receptors (i.e., hmG4 I318S, hmG4 H323R, or hmG4 D485G) in the presence of 1 µM L-AP4. (C) Activation of the Gq pathway by hmG4, hmG4 I318S, hmG4 H323R, and hmG4 D485G with increasing concentrations of DN45. (D) Binding of DN45 to mutated rat mGlu4 receptors rmG4-5M (K_D = 22.8 nM), rmG4-4MA (K_D = 10.4 nM), and rmG4-4MB (K_D = 13.6 nM) is not significantly different from binding to hmG4 (K_D = 5.4 nM), and no binding is observed for rmG4-3M, rmG4-2M, rmG4-M1, or rmG4. (E) DN45 activation of the Gq pathway by rmG4-5M, rmG4-4MA, and rmG4-4MB. Data in B–E are mean ± SEM of three or more individual experiments. The K_D values and statistical analysis for B–E was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test and are presented in SI Appendix, Tables S1 and S2.

Fig. 4.

DN45 agonist activity needs interactions with lobe 1 of the VFT. (A) A front view of the interactions between DN45 and lobe 1 and lobe 2 of the human mGlu4 receptor. Residues on lobe 1 in the loop spanning from H371 to E401 and residues I318, H323, and D485 are highlighted in purple (side chains). (B) A zoomed-in view of the loop comprising residues H371 to E401. (C) IP-1 production by L-AP4 (blue) and DN45 (red) of a human mGlu4 receptor bearing an N-glycosylation site at N399. (D) Effect of L-AP4 (blue) and DN45 (red) on IP-1 production by a human mGlu4 receptor bearing three mutations (i.e., R391M, R393W, and A399K). (E) Effect of L-AP4 (blue) and DN45 (red) on a human mGlu4 receptor bearing four mutations (i.e., H371E, R391M, R393W, and A399K). (F) Effect of L-AP4 alone (filled circles) and in the presence of 100 nM DN45 (open squares) on the activity of the quadruple hmGlu4 receptor mutant. Data of C–F are mean ± SEM of three individual experiments. Statistical analysis of C and F was performed using an unpaired one-tailed t test (SI Appendix, Table S2).

Fig. 5.

DN45 acts as a positive allosteric modulator on heterodimeric mGlu2-4. (A) A cartoon representing a TR-FRET–based assay to measure binding of DN45 to the mGlu2-4 heterodimer using CLIP-mGlu2-C1KKXX and SNAP-mGlu4-C2KKXX. (B) Binding of DN45 to mGlu2-4 alone (red), in the presence of 100 µM mGlu4 antagonist LY341495 (light blue), of 1 mM glutamate (green), of 10 µM LY379268 (orange), or of 10 µM L-AP4 (blue). (C) Activation of the mGlu2-4 heterodimer by DN45 alone (red) or in the presence of the mGlu2 agonist LY379268 (50 nM) measured by a TR-FRET biosensor using CLIP-mGlu2-C1KKXX and SNAP-mGlu4-C2KKXX, labeled with 1 µM BC-Green and 100 nM BG-Lumi4Tb. Values are normalized to the maximal response to LY379268. (D) The response of LY379268 (filled circles) on the TR-FRET biosensor is potentiated by 10 µM DN45 (open squares). Values are normalized to the maximal response to LY379268. Data of B–D are mean ± SEM of three or more individual experiments. Statistical analysis of B was performed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test, and analysis of D was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test (SI Appendix, Tables S1 and S2).