G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3

A Publisher Correction to this article was published on 17 August 2020

This article has been updated

Abstract

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

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Fig. 1: Exposure of the Adgrl3 TA promotes intracellular signaling.
Fig. 2: Adgrl3 CTF signals through Gq and G13.
Fig. 3: Adgrl3 couples to Gα12/13 upon acute exposure of the TA.
Fig. 4: Adgrl3 recruits β-arrestin to the plasma membrane in living cells.

Data availability

All cDNA constructs and data are available on request from the authors. Unprocessed full scans are provided for the immunoblots shown in Extended Data Figs. 5 and 8. Source data are provided with this paper.

Change history

  • 17 August 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

This work was supported by NIH grants MH112156 (J.A.J.), GM130142 (N.A.L.), GM131672 (N.O.) and T32-GM007315 (A.V.), and by the Hope for Depression Research Foundation (J.A.J.). A.I. was funded by PRIME 18gm5910013 and LEAP 18gm0010004 from the Japan Agency for Medical Research and Development (AMED) and KAKENHI 17K08264 from the Japan Society for the Promotion of Science (JSPS). T.L. was funded by the Deutsche Forschungsgemeinschaft through FOR2149 project P01 [LA2861/4-2] and CRC 1423, project number 421152132, subprojects A06, B06. We thank L. Lavis and J.B. Grimm (Janelia Research Campus) for generously providing the JF-525 and JF-646 fluorophores.

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Contributions

S.M. and J.A.J. designed the overall project strategy and experiments, which were performed by S.M., T.P. and N.A.P. GTPγS and dual luciferase SRE experiments were designed by G.G.T. and performed by H.M.S., D.P.M. and A.V. GTP-depleted BRET experiments were designed by N.A.L. and performed by N.O. A.I. provided CRISPR KO cell lines. T.L. provided Adgrl3 cDNA. S.M. and J.A.J. wrote the manuscript. T.L., A.I., N.A.L., G.G.T., S.M. and J.A.J. discussed the experimental findings, interpretation of results and edited the manuscript.

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Correspondence to Jonathan A. Javitch.

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Extended data

Extended Data Fig. 1 TA-enhanced signaling effect is also observed in SRE, NFAT, and NFκB gene expression assays.

a, SRE b, NFκB and c, NFAT. For all gene response elements (SRE, NFAT, and NFκB) signaling was increased significantly when the entire N-terminal fragment up to the GPS cleavage site (CTF) was removed; FL receptor also showed some activity in SRE (comparable to CRE in Fig. 1). Luminescence in (a-c) was measured for a range of increased receptor cDNA concentrations 24 h after transfection in HEK293T cells. All data points are normalized to an empty vector control. Data are presented as mean values ±SEM from 3 independent experimental replicates. Source data

Extended Data Fig. 2 Successive truncation of the first three ADGRL3 tethered agonist residues dramatically blunts SRE-Luciferase gene reporter activation.

SRE gene expression assay for ADGRL3 (human homolog) CTF, Δ1-CTF, Δ2-CTF and Δ3-CTF. Luminescence was measured for a range of increased receptor cDNA amounts (ng) 24 h after transfection in HEK293T cells. For the dual luciferase assay, data are presented as Firefly/Renilla luciferase units, and all data points are normalized to the corresponding ratio for the empty vector control15,52,53. Data are from one representative experiment performed 3 times. Data are presented as mean ±SD from triplicate technical replicates. Source data

Extended Data Fig. 3 Screen of Adgrl3 (FL, CTF, and Δ5-CTF constructs) signaling in the 4 major G protein signaling pathways utilizing a HEK293 CRISPR knockout cell line (HEKΔ7) and a panel of gene expression assays.

a, CRE b, NFκB c, SRE. Each Gα protein species was reintroduced one at a time (see color legend for specification) at optimized cDNA concentrations and luminescence signals were evaluated for empty vector control and receptor constructs 24 h after transfection. All data points are normalized to corresponding empty vector control. Bars indicate mean values ±SEM from 4 (a) and 5 (b-c) independent experimental replicates. Bars for Gαolf and12 are presented as mean values ±SEM from 3 independent experimental replicates. Source data

Extended Data Fig. 4 CTF Gαq signaling is detected both in CRE and NFκB.

CTF signaling in CRE, NFκB, and SRE was evaluated after 18 h of treatment with either vehicle or a potent Gαq inhibitor (YM-254890, 1 μM). Data was collected in regular HEK293T cells. Data points are normalized to empty vector control and displayed as the fold decrease with YM-254890. Bars show mean ±SEM from 4 independent experimental replicates. Source data

Extended Data Fig. 5 Urea-mediated ADGRL3 N-terminal Fragment dissociation.

For the membrane urea treatment experiments presented in this figure, a FLAG- (N-terminal) and His8- (C-terminal) tagged ADGRL3 construct that was truncated N-terminally to the HormR domain was used21 (See Fig. 1 for Adgrl3 architecture). Insect cell membranes (High-Five) with expressed ADGRL3 were mock treated or extracted with urea. The presence of the ADGRL3 NTF and CTF in the membrane (Mem) and extract (Soluble, Sol) fractions was determined by immunoblotting with an anti-FLAG antibody to detect the NTF and an anti-penta-His antibody to detect the CTF. The NTF (apparent MW ~50 kDa) was partially solubilized with the urea, whereas the CTF (apparent MW ~27 kDa) was not. The penta-His blot panels are from one contiguous blot, but broken to avoid oversaturation of the ~70 kDa band (unprocessed receptor) and to show a higher exposure of low MW panel (~27 kDa CTF). Data from one representative experiment that was repeated three times. Source data

Extended Data Fig. 6 CRE, NFκB, and SRE gene expression assays for PAR1-CTF and corresponding T923S/ΔN924-CTF control construct.

CRE a, NFκB b, and SRE c, signaling was increased significantly for T923S/ΔN924-CTF to levels comparable with CTF, whereas PAR1-CTF signals were comparable to FL levels. CTF and FL are replotted from Fig. 1c and Extended Data Fig. 1 for direct comparison. Luminescence was measured for a range of receptor cDNA concentrations 24 h after transfection in HEK293T cells. All data points are normalized to an empty vector control. Data are shown as mean ±SEM from 3 independent experimental replicates. Source data

Extended Data Fig. 7 TA-exposed Adgrl3 does not activate the Gαi/o family.

a, Gβγ release assay testing D2R activation of Gαi1, Gαi2, Gαi3, GαoA and GαoB in HEK full G protein KO cells. In comparison to the HEKΔ7 CRISPR knockout, this cell line also lacks the full Gαi/o family. Luminescence was read 10 min after stimulation with 10 μM quinpirole. b, Gβγ release assay testing the T923S/ΔN924-CTF, PAR1-CTF, and PAR1 activation of Gαi1, Gαi2, Gαi3, GαoA and GαoB in HEK full G protein KO cells. Luminescence was read 10 min after stimulation with 1 μM thrombin. All data are normalized to buffer controls and show the BRET effect induced by ligands. Bars show mean ±SEM from 3 independent experimental replicates. One-way ANOVA with Dunnett’s multiple-comparison post-hoc test was performed for each cDNA construct individually, (no receptor (empty vector), T923S/ΔN924-CTF, PAR1-CTF, and PAR1) to determine statistical significance between the No Gα control and each Gα subtype (For Gαi3 **p = 0.0064, for GαoB **p = 0.0032). See Supplementary Data for the full set of p-values. Source data

Extended Data Fig. 8 β-arrestin-2 decreases G protein-dependent ERK1/2 phosphorylation.

HEK Δβarr1/2 cells were transfected with PAR1-CTF or PAR1-CTF with β-arrestin-2. After 48 hr, the cells were acutely activated with 1 μM thrombin over a time course of 45 min. a, Representative immunoblotting analysis with antibodies against phosphoERK1/2 (#9101 S), total ERK1/2 (#9102 S), and HA (#2367 S). Each sample was derived from the same experiment and the blots were processed in parallel. The HA blot was used as a sample processing control to ensure uniform β-arrestin-2 expression. b, The level of phosphoERK1/2 was normalized to total ERK and the baseline at 0 min was subtracted to produce the time-dependent change in pERK1/2. Data are presented as mean ± SEM from 3 (PAR1-CTF) 4 (PAR1-CTF, β-Arrestin-2) independent experimental replicates. Source data

Supplementary information

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Supplementary Figs. 1–8 and Table 1.

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Supplementary Data

Source Data for Supplementary Figs. 1–8 and P values for relevant figures.

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Mathiasen, S., Palmisano, T., Perry, N.A. et al. G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3. Nat Chem Biol 16, 1343–1350 (2020). https://doi.org/10.1038/s41589-020-0617-7

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