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Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3

  • Nicole A. Perry-Hauser
    Affiliations
    Departments of Psychiatry and Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA

    Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA
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  • Max W. VanDyck
    Affiliations
    Department of Biochemistry, Vassar College, Poughkeepsie, New York, USA
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  • Kuo Hao Lee
    Affiliations
    Computational Chemistry and Molecular Biophysics Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, Maryland, USA
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  • Lei Shi
    Affiliations
    Computational Chemistry and Molecular Biophysics Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, Baltimore, Maryland, USA
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  • Jonathan A. Javitch
    Correspondence
    For correspondence: Jonathan A. Javitch
    Affiliations
    Departments of Psychiatry and Molecular Pharmacology and Therapeutics, Columbia University Vagelos College of Physicians and Surgeons, New York, New York, USA

    Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York, USA
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Open AccessPublished:October 13, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102594
      Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3–C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions in vivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors.

      Keywords

      Abbreviations:

      ADGRLs (adhesion G protein-coupled receptor latrophilins), aGPCR (adhesion G protein-coupled receptor), BRET (bioluminescence resonance energy transfer), CTF (C-terminal fragment), DPBS (Dulbecco’s phosphate-buffered saline), FL (full-length), GPCR (G protein-coupled receptor), GAIN (GPCR autoproteolysis-inducing domain), GPS (GPCR proteolysis site), NTF (N-terminal fragment), SRE (serum response element), TA (tethered agonist), TBS-T (tris-buffered saline with 0.1% tween-20), 7TM (seven transmembrane)
      The adhesion G protein-coupled receptor latrophilins (ADGRL1-3) are highly expressed in the central nervous system. They are best known for their role in synaptic adhesion through trans interaction with endogenous interacting partners, notably the teneurins (
      • Silva J.-P.
      • Lelianova V.G.
      • Ermolyuk Y.S.
      • Vysokov N.
      • Hitchen P.G.
      • Berninghausen O.
      • et al.
      Latrophilin 1 and its endogenous ligand Lasso/teneurin-2 form a high-affinity transsynaptic receptor pair with signaling capabilities.
      ) and fibronectin leucine-rich repeat transmembrane proteins (
      • O’Sullivan M.L.
      • de Wit J.
      • Savas J.N.
      • Comoletti D.
      • Otto-Hitt S.
      • Yates J.R.
      • et al.
      FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development.
      ), which interact with the adhesive N-terminal rhamnose-binding lectin and olfactomedin-like domains, respectively (Fig. 1A). In addition to these two adhesion modules, ADGRLs are composed of a serine-/threonine-rich region and hormone receptor motif, a conserved G protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain that encompasses the GPCR proteolysis site (GPS), and a seven transmembrane (7TM) domain (
      • Moreno-Salinas A.L.
      • Avila-Zozaya M.
      • Ugalde-Silva P.
      • Hernández-Guzmán D.A.
      • Missirlis F.
      • Boucard A.A.
      Latrophilins: a neuro-centric view of an evolutionary conserved adhesion G protein-coupled receptor subfamily.
      ,
      • Vizurraga A.
      • Adhikari R.
      • Yeung J.
      • Yu M.
      • Tall G.G.
      Mechanisms of adhesion G protein-coupled receptor activation.
      ,
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). Autoproteolytic cleavage at the GPS divides the receptor into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) that remain associated throughout receptor trafficking to the cell surface. The seven residues immediately C-terminal to the GPS (denoted P1′-P7′) (
      • Vizurraga A.
      • Adhikari R.
      • Yeung J.
      • Yu M.
      • Tall G.G.
      Mechanisms of adhesion G protein-coupled receptor activation.
      ) constitute the tethered agonist peptide (TA) (also known as the Stachel or stalk peptide), which when exposed binds to the transmembrane domain of ADGRLs and promotes the activation of heterotrimeric G proteins (
      • Liebscher I.
      • Schöneberg T.
      Tethered agonism: a common activation mechanism of adhesion gpcrs.
      ,
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ,
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ).
      Figure thumbnail gr1
      Figure 1The GPCR proteolytic site undergoes autoproteolytic cleavage to release the tethered agonist and facilitate receptor activation. A, cartoon representation of full-length (FL) ADGRL3. The N-terminal fragment (NTF) of the receptor is comprised of rhamnose-binding lectin (RBL) and olfactomedin (OLF) domains, a serine/threonine-rich region, and a hormone receptor motif (HRM). Proteolysis occurs within the GPCR autoproteolysis-inducing domain (GAIN) at the GPCR proteolytic site (GPS). Cleavage occurs between HLP1 and TP1’, resulting in exposure of the TA peptide. The C-terminal fragment (CTF) of the receptor is composed of a transmembrane GPCR fold (7 TM) that signals through heterotrimeric G proteins. B–C, construct design for the ADGRL3 mutants tested in this study. D, anticipated functional outcomes for the ADGRL3 mutants tested in this study. The upper denotation represents the receptor’s ability to undergo autoproteolytic cleavage, whereas the lower denotation represents TA-mediated receptor activation. ADGRL3, adhesion G protein-coupled receptor latrophilin 3; GPCR, G protein-coupled receptor; TA, tethered agonist; 7 TM, seven transmembrane.
      To assess how different structural elements impact the functionality of ADGRL3, we sought to design a mutation that impairs TA-mediated receptor activation but preserves normal autoproteolytic cleavage. Other groups have designed mutants with impaired autoproteolytic cleavage alone, notably with mutations of the proteolysis consensus site, HL/T, such as TP1’G in ADGRLs (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ,
      • Kordon S.P.
      • Dutka P.
      • Adamska J.M.
      • Bandekar S.J.
      • Leon K.
      • Adams B.
      • et al.
      Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder.
      ). We previously showed that mutating the conserved FP3’ and MP7’ TA residues to Ala in ADGRL3 resulted in dramatically impaired G protein activity (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ). However, here, we show that this double mutation in the full-length (FL) receptor also disrupts autoproteolysis, making it impossible to use this construct to differentiate the role of disrupted tethered agonism from that of the loss of autoproteolytic cleavage.
      Recent work revealed the high-resolution structure of TA-bound ADGRL3-CTF in complex with miniG13 heterotrimer (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ). Using the structure as a guide, the authors tested a series of critical interactions between the TA and 7TM domain. Notably, the authors observed a dramatic impairment of G protein activation when they mutated LP6’ to Ala; so we evaluated this mutant for signaling and cleavage. In addition, we also tested the double mutation of LP6’ and MP7’ to Ala. We show that ADGRL3-LP6’A/ML7’A undergoes efficient autoproteolytic cleavage but has dramatically impaired TA-mediated receptor activation, whereas the single mutant ADGRL3-LP6’A maintains substantial serum response element (SRE) activity. This double mutation therefore successfully isolates tethered agonism from receptor cleavage, providing an important molecular tool for studying latrophilins and likely other adhesion receptors.

      Results

      The ADGRL3-LP6’A/MP7’A mutations in the TA impair activity

      To find an ADGRL3 construct with impaired TA-dependent activation but preserved autoproteolytic cleavage, we designed three constructs (Fig. 1, B and C): 1) ADGRL3-FP3’A/MP7’A (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ), 2) ADGRL3-LP6’A (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ), and 3) ADGRL3-LP6’A/MP7’A (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ). We chose ADGRL3-FP3’A/MP7’A as a positive control for impaired TA activity (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ) and ADGRL3-TP1’G as a positive control for impaired autoproteolytic cleavage (Fig. 1D) (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ,
      • Kordon S.P.
      • Dutka P.
      • Adamska J.M.
      • Bandekar S.J.
      • Leon K.
      • Adams B.
      • et al.
      Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder.
      ). We first tested our receptor constructs for impaired receptor activation using a dual-glo SRE luciferase reporter (Fig. 2A). We validated the assay by cotransfecting cells with the SRE-luciferase plasmid and increasing concentrations of either FL ADGRL3 or the constitutively active CTF construct (ADGRL3-CTF) (Fig. 2, B and C). FL ADGRL3 showed increased SRE activity with increasing concentrations of transfected DNA (ranging from ∼5–10 fold). While it is conceivable that this increase in signal may be due to a small fraction of receptors from which the NTF has dissociated, it is also possible that NTF dissociation is not absolutely required for receptor activation. The signal was also greatly enhanced by ADGRL3-CTF (∼25–30 fold), largely independent of DNA concentration in the range tested, consistent with our published work (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ).
      Figure thumbnail gr2
      Figure 2SRE-luciferase activity is disrupted in ADGRL3-LP6’A/MP7’A. A, schematic of the dual-glo serum response element (SRE)-luciferase reporter assay. To activate ADGRL3, the tethered agonist (TA) binds to the orthosteric pocket of the receptor, resulting in the release of Gα and Gβᵧ subunits from the heterotrimer. This release initiates a cascade of downstream second messenger pathways and eventual gene transcription of firefly luciferase by SRE. As an internal control, Renilla luciferase is expressed downstream of the constitutive promoter CMV. B, SRE-luciferase assay for full-length ADGRL3 and mutant constructs ADGRL3-FP3’A/MP7’A, ADGRL3-TP1’G, ADGRL3-LP6’A, and ADGRL3-LP6’A/MP7’A. C, SRE-luciferase assay for ADGRL3-CTF and mutant constructs ADGRL3-FP3’A/MP7’A-CTF, ADGRL3-TP1’G-CTF, ADGRL3-LP6’A-CTF, and ADGRL3-LP6’A/MP7’A-CTF. Data in (B) and (C) are expressed relative to the absence of receptor (control). Statistics were calculated using the two-way ANOVA with Tukey’s multiple comparison test. The mean of the WT receptor control at 200 ng or 600 ng was compared to the mean of the corresponding mutant receptors (∗p < 0.1; ∗∗∗∗p < 0.0001). ADGRL3, adhesion G protein-coupled receptor latrophilin 3; CTF, C-terminal fragment.
      Mutation of TP1’G in the proteolysis consensus site of the GAIN domain of ADGRL1 was shown to abrogate cleavage (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). The same mutation in human ADGRL3 also abolished autoproteolysis but preserved activity in an SRE-luciferase assay (
      • Kordon S.P.
      • Dutka P.
      • Adamska J.M.
      • Bandekar S.J.
      • Leon K.
      • Adams B.
      • et al.
      Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder.
      ). We found that FL ADGRL3-TP1’G showed measurable but diminished receptor activation, whereas ADGRL3-TP1’G-CTF demonstrated robust SRE activity, on par or even greater than WT ADGRL3-CTF (Fig. 2, B and C). Thus, ADGRL3-TP1’G retains a functional TA.
      Using the SRE-luciferase assay, we showed that ADGRL3-FP3’A/MP7’A and ADGRL3-FP3’A/MP7’A-CTF had disrupted SRE activity compared to WT receptor, suggesting that this receptor construct cannot activate Gα12/13 (Fig. 2, B and C). In contrast, while a single mutation of LP6’ to Ala led to somewhat diminished signaling in the FL construct, it led to robust SRE activation in the CTF (Fig. 2, B and C). This is unlike the result previously published for LP6’A using GTPγS turnover with reconstituted Gα13 (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ). However, unlike the turnover assay, which is an acute readout of G protein coupling, the SRE activity assay is downstream of G protein activation and accumulates luciferase over a period of 24 h. Thus, our findings highlight the importance of using multiple assays to assess different aspects of signaling (i.e., acute versus extended response). As hypothesized, double mutation of LP6’A and MP7’A fully disrupted SRE activity in both the FL and CTF formats (Fig. 2, B and C), suggesting the receptors are unable to activate Gα12/13.

      ADGRL3-LP6’A/MP7’A does not couple to Gɑ13 in an acute activation assay

      To confirm these results with the double mutant at the level of G protein activation, we used a Gβγ release bioluminescence resonance energy transfer (BRET) assay (
      • Hollins B.
      • Kuravi S.
      • Digby G.J.
      • Lambert N.A.
      The c-terminus of GRK3 indicates rapid dissociation of G protein heterotrimers.
      ) with acute TA exposure using a protease-activatable ADGRL3 construct (Fig. 3A). In contrast to our previous work using thrombin for cleavage (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ), which leaves a single residue “scar” at the start of the TA, we adapted a recently published method that uses enterokinase and leaves a native TA (
      • Lizano E.
      • Hayes J.L.
      • Willard F.S.
      A synthetic method to assay adhesion-family G-protein coupled receptors. Determination of the G-protein coupling profile of ADGRG6(GPR126).
      ). Enterokinase recognizes the trypsinogen substrate sequence DDDDK and cleaves after the lysine residue. Thus, we cloned an ADGRL3 construct with the endogenous ADGRL3 signal peptide, a self-labeling protein (SNAP-tag), a flexible linker (GGSGGSGGS), the enterokinase recognition site (DDDDK), and the truncated ADGRL3-CTF sequence. We expressed this receptor construct in a HEK293 cell line with targeted deletion of all G proteins (
      • Alvarez-Curto E.
      • Inoue A.
      • Jenkins L.
      • Raihan S.Z.
      • Prihandoko R.
      • Tobin A.B.
      • et al.
      Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signaling.
      ) and monitored energy transfer after the addition of enterokinase in the presence and absence of Gα13. As with our thrombin-activatable construct (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ), the enterokinase-cleavable WT ADGRL3 construct gave a robust BRET response after the addition of enterokinase in the presence of Gα13 compared to its absence (Fig. 3B). However, for the cleavable ADGRL3-LP6’A and ADGRL3-LP6’A/MP7’A constructs, enterokinase treatment failed to produce a BRET response even in the presence of Gα13 (Fig. 3B). This suggests that ADGRL3-LP6’A and ADGRL3-LP6’A/MP7’A have greatly impaired acute TA-mediated activation of Gα13.
      Figure thumbnail gr3
      Figure 3ADGRL3-LP6’A/MP7’A cannot activate Gα13. A, schematic of the Gβγ release bioluminescence energy resonance transfer (BRET) assay. HEK293 cells with targeted deletion of all G proteins (
      • Alvarez-Curto E.
      • Inoue A.
      • Jenkins L.
      • Raihan S.Z.
      • Prihandoko R.
      • Tobin A.B.
      • et al.
      Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signaling.
      ) were transfected with ADGRL3 cDNA, Gα13, Gβ1, Gγ2-Venus, membrane-anchored GRK3ct-Rluc8, and empty vector pCDNA5/FRT to balance. The protease activatable ADGRL3 constructs contain an ADGRL3 signal peptide, followed by a SNAP-tag, flexible linker, Flag tag, and the ADGRL3-CTF. Upon addition of 5.5 units of enterokinase, the construct is cleaved to acutely expose the tethered agonist and activate G protein. B, Gβγ release testing ADGRL3-CTF, ADGRL3-LP6’A-CTF, and ADGRL3-LP6’A/MP7’A-CTF activation of Gα13. Statistics were calculated using ordinary one-way ANOVA with Sidak’s multiple comparison test to compare each cell mean with the other cell mean in that column. This analysis was then followed by multiple unpaired t tests to compare the WT receptor to the two mutant constructs (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). C, cartoon representation of the full-length (FL) ADGRL3 receptor labeled with impermeant Janelia Fluor 646. D, fluorescent counts for FL ADGRL3, SNAP-ADGRL3, and SNAP-ADGRL3-LP6’A/MP7’A. E, fluorescent counts for FL ADGRL3, SNAP-ADGRL3-CTF, and SNAP- ADGRL3-LP6’A/MP7’A-CTF. The same negative control values were used for FL and CTF expression comparisons. Statistics for SNAP-tag labeling were calculated using ordinary one-way ANOVA with Tukey’s multiple comparison test (ns, not significant; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). ADGRL3, adhesion G protein-coupled receptor latrophilin 3; CTF, C-terminal fragment.

      ADGRL3-LP6’A/MP7’A is expressed at the cell surface

      To ensure that the ADGRL3-LP6’A/MP7’A construct was normally expressed, we performed a cell surface–labeling experiment with an impermeant dye targeted to the extracellular SNAP-tag (
      • Keppler A.
      • Kindermann M.
      • Gendreizig S.
      • Pick H.
      • Vogel H.
      • Johnsson K.
      Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro.
      ,
      • Keppler A.
      • Pick H.
      • Arrivoli C.
      • Vogel H.
      • Johnsson K.
      Labeling of fusion proteins with synthetic fluorophores in live cells.
      ) (Fig. 3C). We did not detect a significant difference in expression between FL WT ADGRL3 and FL ADGRL3-LP6’A/MP7’A (Fig. 3D). ADGRL3-LP6’A/MP7’A-CTF was expressed at a somewhat greater level than ADGRL3-CTF (Fig. 3E), but even with this higher expression, it was unable to activate Gα13 (Fig. 3B).3

      ADGRL3-LP6’A/MP7’A retains normal autoproteolytic cleavage

      We next used an immunoblot assay to test whether the FL versions of our ADGRL3 mutants undergo autoproteolytic cleavage (Fig. 4). We used a primary antibody against the FLAG tag positioned on the C-terminus of the receptor. We expected our FL ADGRL3 constructs to run at ∼173 kDa and the cleaved receptor at ∼71 kDa (Fig. 4A). Both our FL ADGRL3-Flag and truncated ADGRL3-CTF-FLAG ran as expected (Fig. 4B). The ADGRL3-TP1’G-Flag showed banding at only the uncleaved molecular weight, confirming that it does not undergo autoproteolytic cleavage. The ADGRL3-FP3’A/MP7’A-FLAG construct also showed banding only at the uncleaved position. This was not completely unexpected, as previous work in ADGRL1 showed that a single mutation of FP3’ impairs autoproteolytic cleavage (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ). Finally, the ADGRL3-LP6’A/MP7’A-Flag construct showed banding only at the position of cleaved receptors, consistent with robust autoproteolytic cleavage.
      Figure thumbnail gr4
      Figure 4ADGRL3-LP6’A/MP7’A retains normal autoproteolytic cleavage. A, expected molecular weights for the uncleaved and cleaved receptors are 172.8 kDa and 70.5 kDa, respectively. B, representative immunoblot against primary Flag (1:500, Thermo Fisher Scientific, PA1-984B) and secondary anti-rabbit IgG-HRP (1:10,000, Thermo Fisher Scientific, Cat #31458). ADGRL3, adhesion G protein-coupled receptor latrophilin 3.

      Structural basis for disrupted TA-mediated activation and cleavage

      To build a structural context for understanding the impaired TA activation of ADGRL3-LP6’A/MP7’A, we carried out molecular dynamic (MD) simulations based on the cryo-EM structure of the ADGRL3/Gα13 complex (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ), both for the WT and the double mutant receptor. In comparison to WT, the LP6’A/MP7’A mutation altered the orientation of W1158 at the bottom of the binding pocket (Fig. 5, A–C). This conformational rearrangement is likely due to the space created by the mutations, which allows the χ1 rotamer of W1158 to rotate from gauche+ to trans, whereas the interaction with MP7’ retains the gauche+ rotamer of W1158 in WT (Fig. 5E). Consequently, and in combination with other disruptive effects of the mutations, the TA was shifted upwards in the binding pocket, as demonstrated by the increased distance between residues LP6′ and F1092 and the decreased distance between residues MP7′ and F995 (Fig. 5D). To validate the importance of the interaction of the TA with W1158, we generated a W1158A mutant and assessed signaling in the SRE activity and BRET assays (Fig. 5, F and G). Both assays showed greatly impaired signaling, suggesting that W1158 plays a role as a toggle switch, similar to the role of Trp6.48 in class A GPCRs (
      • Shi L.
      • Liapakis G.
      • Xu R.
      • Guarnieri F.
      • Ballesteros J.A.
      • Javitch J.A.
      Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch.
      ).
      Figure thumbnail gr5
      Figure 5The LP6’A/MP7’A mutation weakens the interactions of the TA in the binding pocket. A, the molecular dynamics (MD) simulation system of the human ADGRL3/G protein complex embedded in a lipid bilayer. The receptor, G protein, and lipid bilayer are colored in gray, gold, and cyan, respectively. The tethered agonist (TA) is highlighted with dark gray. B–C, the resulting conformations of the TA and several key residues in the binding pocket of ADGRL3 (cyan) and ADGRL3-LP6’A/MP7’A (magenta), respectively. The arrows in (C) indicate the rearrangements of these residues. D–E, scatter plots of the distances between residues P7′-F995 and P6′-F1092, and the χ1 and χ2 dihedral angles of W1158, respectively, showing the upward movement of the TA in the binding pocket. F, SRE-luciferase assay for ADGRL3-CTF-nluc and mutant construct ADGRL3-W1158A-CTF-nluc. Statistics were calculated using an unpaired t test (∗∗∗p < 0.001). G, Gβγ release testing ADGRL3-CTF and ADGRL3-W1158A-CTF activation of Gα13. Statistics were calculated using an unpaired t test (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). H, the structure of the ADGRL1 GAIN domain (PDB 4DLQ) (
      • Araç D.
      • Boucard A.A.
      • Bolliger M.F.
      • Nguyen J.
      • Soltis S.M.
      • Südhof T.C.
      • et al.
      A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.
      ), with the TA highlighted with dark gray. I, a zoom-in view of the autoproteolysis site indicated by the dotted box in panel H. FP3’ located at the turn between the last β-strand of the GPS and the TA is expected to stabilize the conformation necessary for the autoproteolysis occurring between LP1 and TP1’; TP1’ also forms a hydrophobic-aromatic interaction with the FP3’, indicated by the cyan space-filling representation. The “∗” indicates the location of autoproteolysis. Orange and green residues are those within 5 Å of FP3’ and LP6’, respectively. ADGRL3, adhesion G protein-coupled receptor latrophilin 3; CTF, C-terminal fragment; GAIN, GPCR autoproteolysis-inducing domain; GPS, GPCR proteolysis site; SRE, serum response element.
      Structural analysis of the ADGRL1 GAIN domain (Fig. 5H) suggests that FP3’ likely stabilizes the turn between the two β-strands in the GAIN domain where autoproteolysis occurs between LP1 and TP1’ and forms an aromatic-hydrophobic interaction with TP1’ (Fig. 5I). Disruption of these interactions by mutation of FP3’, therefore, disrupts cleavage, whereas mutation of the LP6’ likely preserves essential hydrophobic interactions and allows cleavage.

      Discussion

      Adhesion GPCRs are challenging to study, largely due to their structural complexity and the lack of robust pharmacological tools to activate or inhibit their actions. Specific to ADGRL3, gene disruption across animal species causes hyperactivity and alters dopaminergic neurotransmission (
      • Regan S.L.
      • Hufgard J.R.
      • Pitzer E.M.
      • Sugimoto C.
      • Hu Y.-C.
      • Williams M.T.
      • et al.
      Knockout of latrophilin-3 in Sprague-Dawley rats causes hyperactivity, hyper-reactivity, under-response to amphetamine, and disrupted dopamine markers.
      ,
      • Mortimer N.
      • Ganster T.
      • O’Leary A.
      • Popp S.
      • Freudenberg F.
      • Reif A.
      • et al.
      Dissociation of impulsivity and aggression in mice deficient for the ADHD risk gene Adgrl3: evidence for dopamine transporter dysregulation.
      ,
      • Wallis D.
      • Hill D.S.
      • Mendez I.A.
      • Abbott L.C.
      • Finnell R.H.
      • Wellman P.J.
      • et al.
      Initial characterization of mice null for Lphn3, a gene implicated in ADHD and addiction.
      ,
      • van der Voet M.
      • Harich B.
      • Franke B.
      • Schenck A.
      ADHD-associated dopamine transporter, latrophilin and neurofibromin share a dopamine-related locomotor signature in Drosophila.
      ,
      • Orsini C.A.
      • Setlow B.
      • DeJesus M.
      • Galaviz S.
      • Loesch K.
      • Ioerger T.
      • et al.
      Behavioral and transcriptomic profiling of mice null for Lphn3, a gene implicated in ADHD and addiction.
      ,
      • Regan S.L.
      • Cryan M.T.
      • Williams M.T.
      • Vorhees C.V.
      • Ross A.E.
      Enhanced transient striatal dopamine release and reuptake in lphn3 knockout rats.
      ,
      • Lange M.
      • Norton W.
      • Coolen M.
      • Chaminade M.
      • Merker S.
      • Proft F.
      • et al.
      The ADHD-linked gene Lphn3.1 controls locomotor activity and impulsivity in zebrafish.
      ). Thus, ADGRL3 may offer a novel target for modulating dopaminergic neurotransmission, but the molecular mechanisms underlying this regulation remain unknown. To elucidate these mechanisms in vivo requires receptor constructs that selectively disrupt the various structural and functional elements of the receptor. While several studies have reported ADGRL3 constructs that disrupt either cell–cell adhesion (
      • Jackson V.A.
      • del Toro D.
      • Carrasquero M.
      • Roversi P.
      • Harlos K.
      • Klein R.
      • et al.
      Structural basis of latrophilin-FLRT interaction.
      ,
      • Del Toro D.
      • Carrasquero-Ordaz M.A.
      • Chu A.
      • Ruff T.
      • Shahin M.
      • Jackson V.A.
      • et al.
      Structural basis of teneurin-latrophilin interaction in repulsive guidance of migrating neurons.
      ,
      • Lu Y.C.
      • Nazarko O.V.
      • Sando R.
      • Salzman G.S.
      • Li N.-S.
      • Südhof T.C.
      • et al.
      Structural basis of latrophilin-FLRT-UNC5 interaction in cell adhesion.
      ) or autoproteolytic cleavage (
      • Kordon S.P.
      • Dutka P.
      • Adamska J.M.
      • Bandekar S.J.
      • Leon K.
      • Adams B.
      • et al.
      Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder.
      ), we were unable to find a published construct that impaired TA-mediated receptor activation without impacting autoproteolysis. Here, we describe a double mutation in ADGRL3, ADGRL3-LP6’A/MP7’A, that retains normal cleavage but has impaired TA activity and G protein coupling and provides a structural context for our findings. This engineered receptor will be useful in determining how autoproteolytic cleavage and TA activity individually impact ADGRL3 function in vivo in the context of the FL receptor, and its design can likely be applied to other adhesion G protein-coupled receptors (aGPCRs).
      The relative contributions and/or necessity of autoproteolytic cleavage and the tethered agonist to aGPCR activity remain an area of active study and substantial contention. Several published studies have attempted to unravel the mechanistic details of aGPCR action in vivo (
      • Pederick D.T.
      • Lui J.H.
      • Gingrich E.C.
      • Xu C.
      • Wagner M.J.
      • Liu Y.
      • et al.
      Reciprocal repulsions instruct the precise assembly of parallel hippocampal networks.
      ,
      • Bridges J.P.
      • Safina C.
      • Pirard B.
      • Brown K.
      • Filuta A.
      • Panchanathan R.
      • et al.
      Regulation of pulmonary surfactant by the adhesion GPCR GPR116/ADGRF5 requires a tethered agonist-mediated activation mechanism.
      ,
      • Sakurai T.
      • Kamakura S.
      • Hayase J.
      • Kohda A.
      • Nakamura M.
      • Sumimoto H.
      GPR125 (ADGRA3) is an autocleavable adhesion GPCR that traffics with Dlg1 to the basolateral membrane and regulates epithelial apico-basal polarity.
      ,
      • Pederick D.T.
      • Perry-Hauser N.A.
      • Meng H.
      • He Z.
      • Javitch J.A.
      • Luo L.
      Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons.
      ). However, current reports that use mutagenesis to impair tethered agonist activity have largely ignored the effect of mutations on autoproteolytic cleavage. Essentially, all published mutations used to disrupt TA activity involve the P3′ position and thus are likely to prevent cleavage in the FL constructs. While these mutations are useful in the context of studying the function of the isolated CTF, they create an unappreciated confound in the context of the FL receptor. Isolating tethered agonism from autoproteolytic cleavage, which we have accomplished here for the first time, will simplify analysis of in vivo results and provide powerful support for how these receptor features impact biological systems.
      There are several adhesion GPCRs that cannot undergo autoproteolysis: ADGRE1, ADGRA2/A3, ADGRC1/C3, ADGRD2, ADGRF2, ADGRF4, and ADGRG7 (
      • Vizurraga A.
      • Adhikari R.
      • Yeung J.
      • Yu M.
      • Tall G.G.
      Mechanisms of adhesion G protein-coupled receptor activation.
      ). Impaired autoproteolysis is typically the result of an altered GPS. For example, minimal to no autoproteolysis occurs for receptors that lack a basic residue at P2 (e.g., Arg/Lys/His) or a polar residue at P1’ (e.g., Ser/Tyr/Asn/Gln/Cys/Thr). Some of these noncleaved aGPCRs are still capable of signaling; therefore, activation does not seem to be completely dependent on tethered agonist exposure through removal of the NTF (
      • Liebscher I.
      • Schöneberg T.
      Tethered agonism: a common activation mechanism of adhesion gpcrs.
      ,
      • Kishore A.
      • Purcell R.H.
      • Nassiri-Toosi Z.
      • Hall R.A.
      Stalk-dependent and stalk-independent signaling by the adhesion G protein-coupled receptors GPR56 (ADGRG1) and Bai1 (ADGRB1).
      ). MD simulations of spontaneous TA exposure have recently been reported for five intact aGPCR homologs (ADGRB3, ADGRE2, ADGRE5, ADGRG1, and Lphn1) (
      • Beliu G.
      • Altrichter S.
      • Guixà-González R.
      • Hemberger M.
      • Brauer I.
      • Dahse A.-K.
      • et al.
      Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.
      ). This study used biorthogonal labeling of conserved positions within the TA to show that large portions (+6 residues) of the TA can become solvent accessible in the context of the GAIN domain. Thus, it is possible that an intact aGPCR heterodimer could unmask the TA sufficiently for interaction with the 7 TM, resulting in receptor activation, and this might also occur for an uncleaved construct to an extent sufficient for signaling. A recent report indicates that an uncleaved knock-in construct of ADGRF5 fails to rescue function in vivo (
      • Bridges J.P.
      • Safina C.
      • Pirard B.
      • Brown K.
      • Filuta A.
      • Panchanathan R.
      • et al.
      Regulation of pulmonary surfactant by the adhesion GPCR GPR116/ADGRF5 requires a tethered agonist-mediated activation mechanism.
      ). This contrasts with our work using ADGRL2, in which we show that an uncleaved receptor with an intact TA retains intermediate function relative to WT, whereas an uncleaved TA with a dead TA is without function (
      • Pederick D.T.
      • Perry-Hauser N.A.
      • Meng H.
      • He Z.
      • Javitch J.A.
      • Luo L.
      Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons.
      ). Thus, the field is complex and requires constructs like those we have developed here to evaluate these questions systematically in vitro and in vivo.

      Experimental procedures

      Plasmid DNA constructs

      FL mouse ADGRL3 cDNA was used as a template in PCR to make the described ADGRL3 constructs on a pcDNA3.1+ backbone. Plasmids were assembled by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). All engineered cDNAs were sequenced by Genewiz from Azenta Life Sciences.

      Cell culture

      HEK293T cells (American Type Culture Collection) and HEK293 cells with targeted deletion via CRISPR-Cas9 of GNAS, GNAL, GNAQ, GNA11, GNA12, GNA13, and GNAZ (HEKΔ7) (
      • Alvarez-Curto E.
      • Inoue A.
      • Jenkins L.
      • Raihan S.Z.
      • Prihandoko R.
      • Tobin A.B.
      • et al.
      Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signaling.
      ) were maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (10,000 U/ml) at 37 °C in a 5% CO2 humidified incubator.

      Gene expression assay

      The experimental setup for this assay was described previously (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ). Briefly, HEK293T cells were transfected with Lipofectamine 2000 (2.5 μl/1 μg cDNA) and Opti-MEM using two concentrations of ADGRL3 constructs (200 or 600 ng), 600 ng of reporter dual-glo SRE-luciferase/renilla plasmid (
      • Nazarko O.
      • Kibrom A.
      • Winkler J.
      • Leon K.
      • Stoveken H.
      • Salzman G.
      • et al.
      A comprehensive mutagenesis screen of the adhesion GPCR latrophilin-1/ADGRL1.
      ), and balancer pcDNA5/FRT to 1200 ng. After 24 h, cells were aliquoted into a 96-well black/white isoplate (PerkinElmer Life Sciences) in technical replicates at 80 μl/well. Lysis buffer (40 μl/well) containing D-luciferin (NanoLight Technologies) was prepared as previously described (
      • Baker J.M.
      • Boyce F.M.
      High-throughput functional screening using a homemade dual-glow luciferase assay.
      ). After 10 min, firefly luciferase emission was read at 535 nm on a PHERAstar FS microplate reader (BMG LABTECH). Renilla salts buffer (60 μl/well) containing coelenterazine-h (NanoLight Technologies) was prepared as previously described (
      • Baker J.M.
      • Boyce F.M.
      High-throughput functional screening using a homemade dual-glow luciferase assay.
      ). Renilla luciferase emission was read at 475 nm after 10 min. Data were normalized by dividing the 525 nm firefly emission by the 475 nm Renilla emission. Fold change was calculated by dividing these normalized values by the empty vector control.
      For assays using the ADGRL3-CTF-nluc constructs, the cell media was exchanged to DMEM approximately 6 h after transfection. After 24 h, the media was aspirated from the cells, and each well was gently rinsed with Dulbecco’s phosphate-buffered saline (DPBS). Cells were then mechanically detached using 200 μl DPBS, and 60 μl of the resuspension was distributed in triplicate to a 96-well black/white isoplate. Next, 30 μl of D-luciferin dissolved in the assay buffer was added to each well for a final concentration of 2 mM. Emission was read at 525 nm after 30 min incubation using a PHERAstar FS microplate reader.

      BRET assay

      The experimental setup for this assay was described previously (
      • Mathiasen S.
      • Palmisano T.
      • Perry N.A.
      • Stoveken H.M.
      • Vizurraga A.
      • McEwen D.P.
      • et al.
      G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.
      ). Briefly, HEKΔ7 cells were cotransfected with receptor cDNA (200 ng), Gα (0 or 720 ng), Gβ1 (250 ng), Gγ2-Venus (250 ng), membrane-anchored GRK3ct-Rluc8 (50 ng), and empty vector pCDNA5/FRT to 1470 ng. The transfected cells were resuspended after 24 h, and 45 μl/well was distributed into a 96-well OptiPlate black-white plate. Cells were incubated for 10 min with 10 μl coelenterazine-h (final 5 μM). Then, 45 μl/well of enterokinase (5.5 units) was added to initiate receptor cleavage. Donor (Rluc8) and acceptor (mVenus) emission were read using a PHERAstar FS microplate reader at 485 nm and 525 nm, respectively. The BRET signal was calculated as the ratio of light emitted at 525 nm over that emitted at 485 nm. Enterokinase-induced BRET was obtained by subtracting baseline BRET (DPBS) for each condition.

      Surface expression measurements using SNAPfast-tag

      HEK293T cells were seeded at a density of 900,000 cells/well in a 6-well plate. After 24 h, the cells were transfected using FuGENE transfection reagent (8 μl/2 μg cDNA) and Opti-MEM with SNAPfast-tagged receptor cDNA (2 μg). At 24 h posttransfection, cells were incubated with 500 μl 1 μM impermeant Janelia Fluor 646 dissolved in complete DMEM for 30 min. The cells were washed 3 times with complete DMEM and once with DPBS. Cells were then resuspended in 500 μl DPBS. The resuspension was added in technical replicates to a 96-well OptiPlate black plate (PerkinElmer Life Sciences) at a volume of 100 μl/well. Emission was read using a PHERAstar FS microplate reader with the filter 640/680 at a gain of 2000.

      Immunoblot analysis

      HEK293T cells were seeded in a 6-well plate at a density of 400,000 cells/ml. After 24 h, cells were transfected with receptor cDNA (2 μg) using FuGENE transfection reagent (8 μl/2 μg cDNA) and Opti-MEM. After 24 h, cells were placed on ice and lysed with 500 μl RIPA buffer for 30 min. After lysis, cells were detached and spun at 15,000g for 30 min at 4 °C to pellet debris. Cells were then treated at 37 °C for 1 h with PNGase F (New England Biolabs). Then, 60 μl of PNGase-treated lysate was transferred to a 1.5 ml microcentrifuge tube containing 60 μl 2X SDS Laemmli sample buffer (Sigma-Aldrich). Proteins were then separated via SDS-PAGE (Mini-PROTEAN TGX, 4–15%, Bio-Rad Laboratories, Inc). The gel was then transferred to a PVDF membrane (Immobilon-P Membrane, Merck Millipore Ltd) and placed in a 5% milk tris-buffered saline with 0.1% tween-20 (TBS-T) solution for 1 h at RT. The membrane was washed 5 × 5 min in TBS-T and incubated at 4 °C overnight with 1° rabbit anti-FLAG antibody (1:500, Thermo Fisher Scientific, PA1-984B). Following this incubation, the membrane was washed 5 × 5 min in TBS-T and then incubated for 1 h with 2° anti-rabbit HP antibody (1:10,000, Thermo Fisher Scientific, Cat #31458). The membrane was then washed 5 × 5 min with TBS-T and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Bands were visualized using an Azure Biosystems c600 Imaging System (Azure Biosystems Inc).

      Molecular modeling and MD simulations

      The cryo-EM structure of human ADGRL3 in complex with Gα13 protein (PDB 7SF7) (
      • Barros-Álvarez X.
      • Nwokonko R.M.
      • Vizurraga A.
      • Matzov D.
      • He F.
      • Papasergi-Scott M.M.
      • et al.
      The tethered peptide activation mechanism of adhesion GPCRs.
      ) was used as the structural template to build the homology model of the mouse ADGRL3/Gα13 complex with MODELLER (version 10.0) (
      • Webb B.
      • Sali A.
      Protein structure modeling with MODELLER.
      ). The resulting model with the lowest DOPE score was selected. Based on the pKa prediction with PROPKA for the titratable residues, which found the side chain (
      • Olsson M.H.M.
      • Søndergaard C.R.
      • Rostkowski M.
      • Jensen J.H.
      PROPKA3: consistent treatment of internal and surface residues in empirical pK predictions.
      ) carboxyl group of Glu992 of ADGRL3 to have a pKa of 8.02, we protonated Glu992 to its neutral form. The N-terminus of the tethered peptide of ADGRL3 was neutrally capped with NH2-. To assemble the MD simulation systems for both of the ADGRL3/Gα13 complexes (WT and LP6’A/MP7’A mutant), the CHARMM-GUI server (
      • Wu E.L.
      • Cheng X.
      • Jo S.
      • Rui H.
      • Song K.C.
      • Dávila-Contreras E.M.
      • et al.
      CHARMM-GUI Membrane Builder toward realistic biological membrane simulations.
      ) was used to embed each complex in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer with a water phase on both sides. Na+ and Cl were added to neutralize the system and to reach a final concentration of 0.15 M. Each simulation system includes ∼137,000 atoms and has equilibrated dimensions of ∼101 × 101 × 131 Å3.
      The MD simulations were carried out using NAMD 2.14 (
      • Phillips W.J.
      • Wong S.C.
      • Cerione R.A.
      Rhodopsin/transducin interactions. II. Influence of the transducin-beta gamma subunit complex on the coupling of the transducin-alpha subunit to rhodopsin.
      ) with the CHARMM36 m force field for both protein and POPC (
      • Beliu G.
      • Altrichter S.
      • Guixà-González R.
      • Hemberger M.
      • Brauer I.
      • Dahse A.-K.
      • et al.
      Tethered agonist exposure in intact adhesion/class B2 GPCRs through intrinsic structural flexibility of the GAIN domain.
      ,
      • Nazarko O.
      • Kibrom A.
      • Winkler J.
      • Leon K.
      • Stoveken H.
      • Salzman G.
      • et al.
      A comprehensive mutagenesis screen of the adhesion GPCR latrophilin-1/ADGRL1.
      ), and the TIP3 model (
      • Jorgensen W.L.
      • Chandrasekhar J.
      • Madura J.D.
      • Impey R.W.
      • Klein M.L.
      Comparison of simple potential functions for simulating liquid water.
      ) for water. The NPγT ensemble was used at constant temperature (310 K) maintained with Langevin dynamics and 1 atm constant pressure achieved with the hybrid Nose–Hoover Langevin piston method (
      • Grønbech-Jensen N.
      • Farago O.
      Constant pressure and temperature discrete-time Langevin molecular dynamics.
      ) on an anisotropic flexible periodic cell with a constant-ratio constraint applied in the X − Y plane. Simulations were performed with a cutoff of 12 Å for the nonbonded interactions. The particle mesh Ewald method (
      • Darden T.
      • York D.
      • Pedersen L.
      Particle mesh Ewald: an N⋅log(N) method for Ewald sums in large systems.
      ) was used to evaluate long-range electrostatic effects. The systems were initially minimized for 10,000 steps and then equilibrated with restraints on the protein heavy atoms for 1875 ps. The time step of 1 fs was used for the first 375 ps, which was then increased to 2 fs for the rest of simulations. Another 30 ns equilibrating simulation with restraints only on the protein backbone atoms was performed afterward. All restraints on the receptor were released in production runs. We collected six WT and five mutant MD simulation trajectories starting from different random number seeds, resulting in total simulation lengths of 1893 and 1998 ns, respectively.
      In-house python scripts and MD analysis (
      • Michaud-Agrawal N.
      • Denning E.J.
      • Woolf T.B.
      • Beckstein O.
      MDAnalysis: a toolkit for the analysis of molecular dynamics simulations.
      ) were used to process the trajectories and calculate the geometric measures shown in Figure 5. The distances of L/AP6’-F1092 and M/AP7’-F995 are the minimum distances between the backbone heavy atoms of residues P6′ or P7′ and the sidechain heavy atoms of F995 or F1092.

      Data availability

      Data will be shared upon request. Contact the corresponding author here: [email protected] .

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Dr Demet Araç-Ozkan (University of Chicago, IL) for the generous gift of the Dual-Glo SRE-luciferase reporter plasmid, Dr Luke Lavis (Janelia Research Campus) for supplying the Fluor 646 dye, and Dr Asuka Inoue (Tohoku University, Japan) for the HEKΔ7 cell line. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov).

      Author contributions

      N. A. P.-H. and J. A. J. conceptualization; N. A. P.-H. formal analysis; L. S. software; L. S. and J. A. J. supervision; L. S. and J. A. J. funding acquisition; N. A. P.-H. validation; N. A. P.-H. visualization; N. A. P.-H., M. W. V. D., and K. H. L. investigation; N. A. P.-H. and L. S. methodology; N. A. P.-H., M. W. V. D., K. H. L., L. S., and J. A. J. writing–original draft; N. A. P.-H., M. W. V. D., K. H. L., and J. S. and J. A. J. writing–review & editing.

      Funding and additional information

      This work was supported by T32 MH015144 (N. A. P.-H.) and MH054137 , the Hope for Depression Research Foundation and Miriam’s Magical Memorial Mission (J. A. J.). This work was partially supported by the National Institute on Drug Abuse —Intramural Research Program ( Z1A DA000606 , L. S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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