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W4206334976 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract G protein-coupled receptors (GPCRs) are among the most promising drug targets. They often form homo- and heterodimers with allosteric cross-talk between receptor entities, which contributes to fine-tuning of transmembrane signaling. Specifically controlling the activity of GPCR dimers with ligands is a good approach to clarify their physiological roles and validate them as drug targets. Here, we examined the mode of action of positive allosteric modulators (PAMs) that bind at the interface of the transmembrane domains of the heterodimeric GABAB receptor. Our site-directed mutagenesis results show that mutations of this interface impact the function of the three PAMs tested. The data support the inference that they act at the active interface between both transmembrane domains, the binding site involving residues of the TM6s of the GABAB1 and the GABAB2 subunit. Importantly, the agonist activity of these PAMs involves a key region in the central core of the GABAB2 transmembrane domain, which also controls the constitutive activity of the GABAB receptor. This region corresponds to the sodium ion binding site in class A GPCRs that controls the basal state of the receptors. Overall, these data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers by acting at their transmembrane interface. Editor's evaluation This manuscript builds upon recent structural insights into the GABAB receptor, an unusual and important member of the G protein-coupled receptor (GPCR) family which functions as an obligate heterodimer. The work investigates positive allosteric modulators (PAMs) of the GABAB receptor that bind to the heterodimeric interface between transmembrane helix 6 of the two protomers. Through functional characterization of a large panel of mutant receptors, a role for this binding site in conveying agonism by the PAMs is tested. The manuscript also provides evidence for a role of residues deep in the transmembrane domain in regulating both constitutive activity and allosteric agonism in GABAB receptors. These principles are likely also relevant for other family C GPCRs, suggesting a strategy for drug development targeting this important GPCR family. https://doi.org/10.7554/eLife.70188.sa0 Decision letter eLife's review process Introduction G protein-coupled receptors (GPCRs) are key players in intercellular communication. They are involved in many physiological functions (Heng et al., 2013), and many mutations (Schoneberg and Liebscher, 2021) and genetic variants (Hauser et al., 2018) of GPCR genes are associated with human diseases. Not surprisingly, GPCRs are major targets for drug development (Hauser et al., 2017). Although GPCRs are able to activate G proteins in a monomeric state, they can also form homo- and heteromers (Ferré et al., 2014), even in native tissues (Albizu et al., 2010; Rivero-Müller et al., 2010), often named homo- and heterodimer for simplicity. Such complexes allow allosteric cross-talk between receptors and contribute to a fine-tuning of transmembrane (TM) signaling. Then, modulating the activity of GPCR dimers could offer a new way of controlling physiological functions. Several approaches were developed to control GPCR dimer activity by targeting the TM domain (TMD) with ligands that could be of potential interest in vivo (Botta et al., 2020). One approach being tested was the use of bivalent ligands; that is, two ligands attached by a linker able to bind to each protomer within a dimer (Huang et al., 2021). However, each ligand still has the possibility to act on the monomers. Another possibility highly anticipated would be the development of ligands binding at the dimer interface, which would not be able to act on monomers, but instead it would specifically control the dimer activity. Hope for the possible identification of such type of ligands came from the discovery of allosteric modulators binding to sites outside the TM bundle in class A and B GPCRs (Thal et al., 2018; Wang et al., 2021), in regions possibly involved in receptor dimerization and oligomerization (Figure 1A). The first evidence for this type of ligands came from the structure of the class C GABAB receptor, where two different positive allosteric modulators (PAMs) were reported to bind at the TM interface of this heterodimer made of the two homologous subunits GABAB1 (GB1) and GABAB2 (GB2) (Kim et al., 2020; Mao et al., 2020; Shaye et al., 2020; Figure 1B). No other example has been described yet, including in the other class C GPCRs, such as the metabotropic glutamate (mGlu) receptors where the allosteric modulators that target the TMD bind in the ancestral GPCR cavities within the TMD core (Doré et al., 2014; Wu et al., 2014; Figure 1B). Figure 1 Download asset Open asset Allosteric binding sites in the different classes of G protein-coupled receptors (GPCRs). (A, B) Scheme and structure of the transmembrane domain (TMD) representative of the diversity of the binding sites for allosteric modulators (filled blue circles or blue spheres) in selected human class A and B GPCRs (muscarinic M2 receptor PDB 4MQT [1], purinergic P2Y1 receptor PDB 4XNV [2], corticotropin-releasing factor receptor 1 PDB 4K5Y [3], and β2 adrenergic receptor PDB 5X7D [4]) (A), as well as in the class C homodimer mGluR5 (PDB 6N51) bound to a NAM (PDB 4OO9) and the heterodimer GABABR bound to GS39783 (PDB 6UO8) (B). In classes A and B, the allosteric modulators were shown to bind to different sites within and outside of the TM bundle, in contrast to the orthosteric ligand (black triangle). The GABAB is activated by γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central system linked to various neurological diseases. This receptor is an attractive drug target for brain diseases (Bowery, 2006; Gassmann and Bettler, 2012) with therapeutic drugs such as baclofen (Lioresal) and β-phenyl-γ-aminobutyric acid (phenibut) used to treat spasticity (Chang et al., 2013), alcohol addiction (Agabio et al., 2018), anxiety, and insomnia (Lapin, 2001). Auto-antibodies that target GABAB have previously been identified at the origin of epilepsies and encephalopathies (Dalmau and Graus, 2018), and genetic mutations have previously been associated with Rett syndrome and epileptic encephalopathies (Hamdan et al., 2017; Vuillaume et al., 2018; Yoo et al., 2017). GABAB has a unique allosteric mechanism for signal transduction, in which the binding of an agonist in the extracellular domain of GB1 leads to G protein activation through a rearrangement of the intracellular interface of the TMD of GB2 (Monnier et al., 2011; Shaye et al., 2020; Xue et al., 2019). PAMs offer a number of advantages over the agonists since they temporally and spatially control enhanced signaling only when the natural ligand is present (Conn et al., 2014; Pin and Prézeau, 2007; Schwartz and Holst, 2007). However, many of these PAMs have an intrinsic agonist activity as they can increase receptor activity in the absence of an orthosteric agonist. These allosteric ligands are called ago-PAMs (Conn et al., 2014), in contrast to pure PAMs that have no intrinsic agonist effect. The ago-PAMs could be of tremendous interest for the treatment of patients with genetic variants of GPCRs as more and more are discovered in the exome sequence studies (Hauser et al., 2018). Finally, the allosteric agonist activity of these molecules is not predictable yet, and the molecular basis of such activity remains unknown. In this study, we examined if the commonly available GABAB PAMs all bind in the GB1-GB2 TM interface and how these PAMs can control the GABAB heterodimer activity. We show that, indeed, all PAMs bind at the same site, at the active interface of these TMDs, despite their different structures. We also reveal that the agonist activity of these PAMs involves a key region in the central core of the GB2 TMD that also controls the constitutive activity of the receptor. This region is functionally conserved in most GPCRs as it corresponds to the Na+ site found in class A receptors (Zarzycka et al., 2019). These data reveal the possibility of developing allosteric compounds able to specifically modulate the activity of GPCR homo- and heterodimers. Results Different functional properties of the PAMs for GABAB receptor We have evaluated the agonist activity and the allosteric modulation of the most studied PAMs (Urwyler, 2011) on the recombinant wild-type GABAB receptors, rac-BHFF (Malherbe et al., 2008), CGP7930 (Urwyler et al., 2001), and GS39783 (Urwyler et al., 2003; Figure 2A). These compounds had showed pure PAM or ago-PAM effect on GABAB activity in different studies both in vitro and in vivo (Urwyler, 2011). The binding site of both rac-BHFF (Kim et al., 2020; Mao et al., 2020) and GS39783 (Shaye et al., 2020) in the purified full-length GABAB receptor was recently reported from structural analyses, including when the receptor is in complex with the G protein (Shen et al., 2021). They bind into a pocket formed in the active heterodimer by its TM6s at the interface of the TMDs. Figure 2 with 1 supplement see all Download asset Open asset Different functional properties of the positive allosteric modulators (PAMs) for GABAB receptor. (A) Chemical structures of the three PAMs of GABAB receptor used in the study and commercially available. (B, C) Intracellular Ca2+ responses (B) and inositol-phosphate-1 (IP1) accumulation (C) mediated by the indicated compounds. (D) Schematic representation of the Go protein BRET sensor. (E, F) Kinetics of BRET ratio changes of this sensor (E) upon addition (arrow) of buffer (control condition), 100 μM GABA or 100 μM of the indicated PAMs (blue). Data are from a typical experiment performed three times independently. Changes in BRET ratio (F) were measured 150 s after drug application. Data are shown as means ± SEM of three biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the buffer condition) with ***p<0.0005, ****p<0.0001. (G–I) Intracellular Ca2+ responses mediated by GABA in the absence or presence of the indicated concentrations of rac-BHFF (G), CGP7930 (H), or GS39783 (I). Data are normalized by the response of 1 mM GABA and shown as means ± SEM of 4–15 biologically independent experiments. Figure 2—source data 1 Source data for Figure 2B–C and F–I, Figure 2—figure supplement 1, and Supplementary files 1 and 2. https://cdn.elifesciences.org/articles/70188/elife-70188-fig2-data1-v3.xlsx Download elife-70188-fig2-data1-v3.xlsx In the present study, we show that rac-BHFF and CGP7930 have intrinsic agonist and PAM activity in different functional assays (Figure 2B–F, Figure 2—figure supplement 1). In contrast, the agonist activity of GS39783 is weaker, acting more like a pure PAM. These results are in line with our previously reported data (Lecat-Guillet et al., 2017). The strong agonist effect of rac-BHFF was revealed by its capacity to activate the GABAB receptor even in the absence of GABA. It was measured by intracellular calcium release and inositol-phosphate-1 (IP1) accumulation assays in cells coexpressing the chimeric G protein Gαqi9 (a Gαq protein in which the last nine C-terminal residues have been replaced by those of Gαi2), which allows the coupling of Gi/o-coupled receptors to phospholipase C (Figure 2B–F and Supplementary files 1 and 2; Supplementary file 3). rac-BHFF alone reached more than 60% of the maximal effect of the full agonist GABA (Figure 2B and C, Figure 2—figure supplement 1A, and Supplementary file 1). CGP7930 also has agonist activity in absence of GABA (Figure 2B and C and Supplementary file 1). In contrast to the two other PAMs, the intrinsic agonist activity of GS39783 was only observed in the IP1 accumulation assays (Figure 2C), and not in the calcium release assay (Figure 2B and Supplementary file 1), and using a BRET sensor for the activation of GαoA protein (Figure 2F). The observed discrepancy is most likely related to the nature of these assays, the IP1 accumulation assay being an equilibrium assay while the two other are highly dependent on the kinetics of ligand binding to the receptor (Bdioui et al., 2018). The IP1 assay is thus proposed to be the most sensitive assay for evaluating slow binders and low-efficacy compounds such as the GS39783. GB1 and GB2 TMDs are sufficient for the agonist activity of the PAMs Our recent studies have shown that there is strong positive cooperativity between GB1 and GB2 TMDs for receptor activation (Monnier et al., 2011; Xue et al., 2019). Here, we demonstrate that both GB1 and GB2 TMDs are required for the efficient agonist activity of the PAMs. This is consistent with the binding site of rac-BHFF and GS39783 involving both GB1 and GB2 TMD in the active conformation of the receptor (Kim et al., 2020; Mao et al., 2020; Shaye et al., 2020; Shen et al., 2021). We measured the rac-BHFF-induced intracellular calcium signaling of different GABAB constructs expressed at the cell surface (Figure 3, Figure 3—figure supplement 1A, and Supplementary file 3). The presence of both GB1 and GB2 TMDs was sufficient for its activity (Figure 3A–D). Accordingly, the presence of GB1 or GB2 ECD or both is dispensable for the ago-PAM activity since the relevant constructs in which one or both ECDs were deleted could still be activated by rac-BHFF alone efficiently (Figure 3B–D). However, no agonist activity of rac-BHFF could be measured in GABAB receptors in which the GB1 TMD was replaced by a GB2 TMD or only the seventh helix of the GB1 (GB1-TM7) (Monnier et al., 2011; Figure 3E–F, Figure 3—figure supplement 1B and C), even though these constructs could still be activated by GABA efficiently (Figure 3E–F, Figure 3—figure supplement 1D). Of note, the conformational state of the GB1 TMD is not critical for both orthosteric and allosteric activation since similar results were obtained with the mutant GB1DCRC (Figure 3—figure supplement 1E and F). This mutant is activated by GABA similarly to the wild-type receptor (Figure 3—figure supplement 1E) as previously reported (Monnier et al., 2011). But it was engineered to create a disulfide bond in GB1 TMD, thus expecting to limit the conformational change of this domain upon ligand stimulation of the GABAB receptor. The importance of the intact GB1 TMD in the agonist activity of rac-BHFF was also confirmed by measuring the Go protein activation by BRET (Figure 3—figure supplement 2A). Finally, we obtained a similar conclusion regarding the requirement of both GB1 and GB2 TMDs for the ago-PAM activity of the two other PAMs by comparing all of them in the most sensitive IP1 assays (Figure 3—figure supplement 2B). Of note, for CGP7930 there is an apparent controversy between our data where both GB1 and GB2 TMD are required to observe an agonist effect, while Binet et al., 2004 found GB2 TMD alone was sufficient. It might be due to the endogenous expression of the GB1 subunit that may exist in some cell lines including the HEK293 cells used in both studies (Xu et al., 2014). Figure 3 with 2 supplements see all Download asset Open asset Both GB1 and GB2 transmembrane domains (TMDs) are sufficient for the agonist activity of the positive allosteric modulators (PAMs). (A–F) Intracellular Ca2+ responses mediated by the indicated subunit compositions (pictograms) upon stimulation with rac-BHFF. The inserted graphs correspond to the responses upon stimulation with GABA. Data are normalized by using the response of 100 μM rac-BHFF or 1 mM GABA, for rac-BHFF and GABA treatment, respectively, on wild-type GABAB receptor and shown as means ± SEM of 3–8 biologically independent experiments. The dotted lines in the main and inserted graphs indicate the dose–responses of the wild-type receptor determined in panel (A). Figure 3—source data 1 Source data for Figure 3, Figure 3—figure supplement 1, Figure 3—figure supplement 2, and Supplementary file 3. https://cdn.elifesciences.org/articles/70188/elife-70188-fig3-data1-v3.xlsx Download elife-70188-fig3-data1-v3.xlsx GB1 and GB2 TM6s interface is the binding site for the different PAMs A similar binding site in the structure of the purified GABAB was reported for rac-BHFF and GS39783 (Figure 4A; Kim et al., 2020; Mao et al., 2020; Shaye et al., 2020; Shen et al., 2021). This binding site of GS39783 was investigated using receptor mutants expressed at the surface of live cells (Shaye et al., 2020), and the mutagenesis data were consistent with the binding site observed in the structure. In the present study, we have analyzed both the potency and agonist efficacy of the three PAMs on a series of both GB1 and GB2 bearing single mutations in their TM6s. First, the potency of the rac-BHFF for each mutant in the intracellular calcium assay was measured in the presence of GABA at a concentration equivalent to its 20% maximal efficacy concentration (EC20). Two single mutants of GB1 (K792 ICL3A and Y8106.44A) and three single mutants of GB2 (M6946.41A, Y6976.44A, and N6986.45A) strongly impaired the potency of rac-BHFF for the GABAB in the calcium assay (Figure 4B and C). Residues were named according to the class C GPCR TMD nomenclature (Isberg et al., 2015). This loss of activity was not due to a loss of expression of the GABAB mutants at the cell surface as shown by the cell surface quantification of HALO-tagged GB1 labeled with the fluorophore Lumi4-Tb when coexpressed with GB2 (Figure 4D). In addition, the receptor remained functional for all constructs (Figure 4B and C, Figure 4—figure supplement 1A), although GABA had an impaired activity for the GB1 mutant K792 ICL3A (Figure 4B) and the GB2 mutants M6946.41A and Y6976.44A (Figure 4C). Finally, the critical importance of the two TM6s for the agonist activity of rac-BHFF was confirmed by IP1 measurements. rac-BHFF had a strong impaired efficacy on two GB1 mutants (Y8106.44A and 6.43MYN6.45-AAA) and one GB2 mutant (6.43MYN6.45-AAA) (Figure 4E). The data obtained in the intracellular calcium release assay were also consistent (Figure 4—figure supplement 2). This demonstrated that the interface of TM6s is crucial for the agonist activity of rac-BHFF effect. These mutagenesis data are consistent with the binding site of rac-BHFF at the interface between the two GABAB subunits, similar to GS39783 (Shaye et al., 2020), as reported in the GABAB structures (Kim et al., 2020; Mao et al., 2020; Shen et al., 2021). Figure 4 with 2 supplements see all Download asset Open asset GB1 and GB2 TM6s interface is the binding site for the different positive allosteric modulators (PAMs). (A) Structure of the GABAB receptor (PDB 6UO8) where the binding site of GS39783 (PDB 6UO8) and rac-BHFF (PDB 7C7Q) in the receptor is highlighted, and close-up view of the molecules bound (PAMs shown as sticks, and hydrogen bonds between PAMs and receptor are depicted as dashed yellow lines). The α-carbon of the main residues involving in the binding site for these PAMs is highlighted as a sphere in GB1 (blue) and GB2 (light blue). (B, C) Intracellular Ca2+ responses mediated by the indicated constructs upon stimulation with rac-BHFF in the presence of EC20 GABA, or GABA alone. Data are normalized by wild-type response of 100 μM rac-BHFF + EC20 GABA or 100 μM GABA, for rac-BHFF and GABA treatment, respectively, and shown as means ± SEM of 4–5 biologically independent experiments. (D) Quantification of cell surface-expressed GB1 in HEK293 cells transfected with the indicated HALO-tagged GB1 and SNAP-tagged GB2 constructs after labeling with HALO-Lumi4-Tb. Data are normalized by wild-type receptor and expressed as means ± SEM. (E, F) Inositol-phosphate-1 (IP1) production induced by the indicated PAMs (E) or basal IP1 accumulation (F) in intact HEK293 cells expressing the indicated subunit combinations. Data are normalized by wild-type response and shown as means ± SEM of 4–5 biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) with *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001. Figure 4—source data 1 Source data for Figure 4B–F, Figure 4—figure supplement 1, and Figure 4—figure supplement 2. https://cdn.elifesciences.org/articles/70188/elife-70188-fig4-data1-v3.xlsx Download elife-70188-fig4-data1-v3.xlsx By using these GB1 and GB2 mutants bearing single mutations in their TM6s, we investigated the previously unknown mode of action of CGP7930. Interestingly, most of the mutations in GB2 strongly impaired the efficacy of CGP7930, whereas such effect was not observed for the mutations in GB1 (Figure 4E). Thus, the GB2 TM6 seems more important than the GB1 TM6 for CGP7930 compared to rac-BHFF. Of note, GS39783 is more sensitive to mutations than the two other PAMs. It might be because the mutated residues are highly important for the binding of GS39783 or alternatively to its weakest agonist activity compared to Rac-BHFF and CGP7930, then resulting in a stronger loss of agonist activity of GS39783 on these mutants. Altogether, the three PAMs have a different agonist activity on the GABAB mutants. Moreover, our mutagenesis data are consistent with the PAMs sharing the same binding pocket at the TM6 TM interface, even though their mode of binding does not seem to involve the same residues in GB1 and GB2. Finally, we have analyzed the effect of the mutations on the constitutive activity of the GABAB receptor that was reported in transfected cell lines (Grünewald et al., 2002; Lecat-Guillet et al., 2017). Indeed, TM6 is expected to control the conformational landscape of the receptor and to play a key role for G protein activation in class A and B GPCRs (Weis and Kobilka, 2018). In GABAB receptor, contacts between the two TM6s were shown to stabilize the active state of the heterodimer (Kim et al., 2020; Mao et al., 2020; Shaye et al., 2020; Shen et al., 2021; Xue et al., 2019). These contacts are consistent with the allosteric interactions between the GB1 and GB2 TMDs during receptor activation (Monnier et al., 2011). In addition, genetic mutations in the GB2 TM6 (S6956.42I, I7056.52N, and A7076.54T) were reported to induce a high constitutive activity of the receptor (Vuillaume et al., 2018). Interestingly, in the present study most of the GB1 mutants produced a lower constitutive activity of the receptor compared to the wild-type (Figure 4F), for a similar cell surface expression (Figure 4D). In contrast, GB2 mutants had a similar constitutive activity as the wild-type receptor, even though the cell surface expression was lower. Of interest, this constitutive activity was blocked by the competitive antagonist CGP54626, on wild-type and most mutated receptors, but it remained unchanged for the triple mutant (6.43MYN6.45-AAA) in GB2 (Figure 4—figure supplement 1B). This suggested that these three mutations in GB2 have impaired the allosteric coupling between the two TMDs in the receptor. Altogether, these results confirmed the important role of TM6s in controlling the landscape of conformations of the receptor and its basal state. Moreover, it suggests a key role for TM6 allosteric interactions between the GB1 and GB2 TMDs. Exploring the GB2 TMD core to clarify the mechanism for agonist activity of the PAMs How could ago-PAM binding at the active heterodimer interface induce G protein activation by GB2? Binding in the TM6s interface could directly stabilize the active state of the GB2 TMD. Alternatively, and not incompatible with this first mechanism, a second binding site in the GB2 TMD could exist. A second binding site for the PAM, not yet discovered, could exist in the central core of GB2 TMD as previously proposed (Dupuis et al., 2006; Evenseth et al., 2020). Binding at this second site per se could be responsible for its agonist activity or alternatively could favor agonist activity of the compound bound in TM6s interface. Possible cavities in GB2 TMD have been revealed by the structures of the receptor, such as those occupied by one phospholipid molecule that covers nearly the entire range of ligand binding positions previously reported in class A, B, C, and F GPCRs (Kim et al., 2020; Papasergi-Scott et al., 2020; Park et al., 2020). This lipid is present only in the inactive conformation, and it was proposed to be important for intramolecular signal transduction in GABAB (Papasergi-Scott et al., 2020; Figure 5A). Mutations designed to destabilize phospholipid binding in GB2 TMD resulted in increases in both GABAB basal activity and receptor response to GABA. Therefore, to identify a potential second binding site for the PAM, we have introduced mutations in the upper part of the GB2 TMD, the region involved in this phospholipid binding (Figure 5—figure supplement 1A). Mutations in this region were shown to confer agonistic activity to GS39783 (Dupuis et al., 2006). Single and multiple amino acid substitutions were performed at seven positions in the GB2 (see details in Figure 5A, Figure 5—figure supplement 1A). We have also changed GB2 L5593.36, a residue critical in the ancestral binding pocket of GPCRs, into alanine. It is equivalent to the residue 3.32 in class A GPCRs (Ballesteros–Weinstein numbering scheme [Ballesteros and Weinstein, 1995]) that was shown to be conserved for direct interactions with agonists and antagonists (Venkatakrishnan et al., 2013). It is also well conserved in the class C GPCRs, including GABAB, where it plays an important role for the NAM and PAM binding and function (Doré et al., 2014; Farinha et al., 2015; Feng et al., 2015; Leach et al., 2016; Wu et al., 2014). Figure 5 with 6 supplements see all Download asset Open asset A deep region in GB2 transmembrane domain (TMD) is responsible for agonist activity of the positive allosteric modulator (PAM). (A) Cartoon highlighting a possible second binding site (dotted oval) for the PAMs in the ancestral ligand binding pocket of the GB2 TMD. In the structure of the inactive state, this pocket is occupied by one molecule of phospholipid (shown as yellow sticks). The residues (α-carbon) of this phospholipid binding pocket (green) and the residues underneath (red) were changed to evaluate their importance in the agonist activity of rac-BHFF. The highly conserved residues L3.36 and Y5.44 were mutated into Ala; G6.53 conserved in GB2 were changed to Thr conserved at this position in GB1, or Phe conserved at this position for other class C GPCRs such as mGlu and CaSR; in Mut 8, the non-conserved residues 7.26NVQ7.28 were mutated in their equivalent in GB2 Drosophila; V7.35 conserved in GB2 (Val or Phe) was changed to Phe. (B) Intracellular Ca2+ responses mediated by the indicated GB2 mutants (M1 to M16) coexpressed with the wild-type GB1 subunit upon stimulation with 30 μM rac-BHFF or 1 mM GABA. Data are normalized by wild-type response and expressed as means ± SEM of three biologically independent experiments. (C) Intracellular Ca2+ responses mediated by the indicated constructs upon stimulation with rac-BHFF in the presence of EC20 GABA of each combination. Data are normalized by wild-type response of 100 μM rac-BHFF + EC20 GABA and shown as means ± SEM. (D) Basal inositol-phosphate-1 (IP1) accumulation mediated by the indicated constructs. Data are normalized by the response of the wild-type and shown as means ± SEM of five biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) with ***p<0.0005, ****p<0.0001. (E) Correlation between the GABAB constitutive activity measured using the Go protein BRET sensor and the rac-BHFF agonist effect for the WT GB1 subunit coexpressed with the indicated GB2 mutants. Data are normalized by the response of the wild-type and shown as means ± SEM. (F) Basal IP1 accumulation mediated by the indicated constructs, including the genetic mutation A567T identified in human GB2 that is equivalent to the mutation A566T in rat. Data are normalized by the response of the WT and shown as means ± SEM of 3–4 biologically independent experiments. Data are analyzed using an unpaired t-test for human, and one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT) for the rat, with *p<0.05, ***p<0.0005, ****p<0.0001. (G) Top view of the sodium binding pocket within the structure of human A2A adenosine receptor (PDB 4EIY) where the three residues important for Na+ interactions are highlighted (Cα in red), the equivalent residues identified in human GB2 TMD (PDB 6UO8), and human mGluR5 TMD (PDB 4OO9); the X.50 numbers shown for A2AAR are equivalent to the numbers in mGluR5 on the basis of X.50 residues defined in Doré et al., 2014; TM2, TM3, and TM7 that contain the residues involving the identified region are highlighted in cyan. (H) Basal IP1 accumulation mediated by the indicated WT and mutated mGlu5 receptors in the presence of the co-transfected glutamate transporter EAAT3. Data are normalized by the response of the WT and shown as means ± SEM of four biologically independent experiments. Data are analyzed using one-way ANOVA test followed by a Dunnett’s multiple comparison test to determine significance (compared with the WT), with **p<0.005. For clarity, the residue numbers for GB2 subunit are base" @default.
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W4206334976 date "2021-07-21" @default.
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W4206334976 title "Decision letter: Allosteric ligands control the activation of a class C GPCR heterodimer by acting at the transmembrane interface" @default.
W4206334976 doi "https://doi.org/10.7554/elife.70188.sa1" @default.
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