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Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation

Nature volume 615, pages 945–953 (2023)Cite this article

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Abstract

Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins1, a class of surface receptors poised for mechano-proteolytic activation2,3,4, including an evolutionarily conserved mechanism of cleavage5,6,7,8. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. Here we introduce a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)9,10,11, from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)12, on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. We conclude that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases13.

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Fig. 1: Activation of the NRS for the aGPCR Cirl depends on proteolysis of the GAIN domain.
Fig. 2: Cirl-NRS is activated in a mechano-dependent manner.
Fig. 3: Cirl receptor dissociation requires interaction with its ligand Tollo.
Fig. 4: Cirl-NTF dissociates at CG cells to regulate the pool of neuroblasts.
Fig. 5: NRS for non-neural aGPCRs.

Data availability

All datasets plotted in diagrams are available in Source Data. Interactome datasets on Cirl ligands are available at ProteomeXchange with the unique identifier PXD033873. The raw western blot data are available at Figshare at https://doi.org/10.6084/m9.figshare.21930960. All other data are available upon request to the corresponding authors. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the Deutsche Forschungsgemeinschaft to N.S. and T.L. through FOR2149, project numbers 265903901 (project P01) and 265996823 (project P03) and through CRC 1423, project number 421152132 (projects A06 and B06); and by a junior research grant from the Faculty of Medicine, Leipzig University, to N.S. We thank C. Klämbt, R. Kopan, D. Montell, N. Perrimon, M. Rossner, R. Schuh, G. Struhl and W. Hütteroth for sharing materials and protocols; M. Ueffing for help with MS analyses; T. Lecuit for discussions on the Cirl–Tollo interaction; and L. Abicht, P. Beckmann, A. Böhme, H. Holzinger, K. Heise, S. Lautenschläger, M. Oppmann, S. Schmidt, U. Strobel and P. Tarlatt for technical assistance. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and Vienna Drosophila Resource Center (VDRC) were used in this study.

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  1. These authors contributed equally: Nicole Scholz, Anne-Kristin Dahse

Authors and Affiliations

  1. Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Leipzig, Germany

    Nicole Scholz, Anne-Kristin Dahse, Marguerite Kemkemer, Anne Bormann, Genevieve M. Auger, Fernando Vieira Contreras, Lucia F. Ernst, Hauke Staake, Marek B. Körner, Max Buhlan, Amelie Meyer-Mölck, Yin Kwan Chung, Beatriz Blanco-Redondo, Dmitrij Ljaschenko, Marina Bigl & Tobias Langenhan

  2. Core Facility for Medical Bioanalytics, Institute for Ophthalmic Research, Eberhard Karls University of Tübingen, Tübingen, Germany

    Franziska Klose & Mohamed Ali Jarboui

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Contributions

N.S. and T.L. conceived the study, designed, performed and analysed the experiments, prepared figures and wrote the manuscript with consent from all co-authors. N.S.: cloning, luciferase assays, transgene generation, immunohistochemistry, fly genetics, imaging, protein extraction and MS and western blot analyses. A.-K.D.: cloning, fly genetics, immunohistochemistry, imaging and t-GRASP analyses. M.K. and A.M.-M.: ELISA and luciferase experiments. A.B.: protein extraction, MS and western blot analyses and luciferase experiments. G.M.A.: cell marker expression analyses. M.B.K.: immunohistochemical stainings. F.V.C.: Mayo-NRS and Ketchup-NRS expression profiles. L.F.E.: fly genetics and imaging. H.S.: fly genetics. M. Buhlan and D.L.: joint bending and TransTimer analyses. Y.K.C.: cloning and ELISA experiments. B.B.-R.: Mayo-NRS and Ketchup-NRS protein analyses. F.K. and M.A.J.: MS analyses. M. Bigl: protein extraction, MS and western blot analyses and ELISA experiments. T.L.: cloning, luciferase assays, transgene generation, fly genetics, imaging and quantification of cell marker expression.

Corresponding authors

Correspondence to Nicole Scholz or Tobias Langenhan.

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Competing interests

N.S. and T.L. are co-inventors of a pending patent covering NTF release sensors for aGPCRs (WO/2022/063915; priority application: EP 3974535; applicant: Leipzig University) covered in this manuscript. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Structure–function relationships of aGPCRs and functionality of the NRS technique.

a, aGPCRs are composed of extra- (ECR) and intracellular regions (ICR) as well as a heptahelical transmembrane-spanning domain (7TM). Owing to autocatalytic cleavage by the GPCR autoproteolysis-inducing (GAIN) domain, most aGPCRs exist as non-covalently stabilized heterodimers composed of an N- (NTF) and C-terminal fragment (CTF), which are affixed to each other by the GAIN domain that contains the tethered agonist (TA)/Stachel. The latrophilin-like Cirl receptor contains rhamnose-binding lectin (RBL) and hormone-receptor motif (HRM) domains in its ECR. b, Two principle aGPCR activation modes have received evidence and either do (Dissociation model) or do not (Non-Dissociation model) rely on aGPCR heterodimer separation. c, The NRS consists of the ECR of a given adhesion GPCR including the autoproteolytically active GAIN domain with its GPCR proteolysis site (GPS) fused to the juxta- and transmembrane segment (JTS) of the Drosophila Notch receptor and an intracellular heterologous transcription-factor (TF) unit. The JTS contains the recognition sites for cleavage by metallo- and intramembrane proteases (S2–S4). The protein sequence at the GPS used in the Cirl-NRS is shown.

Extended Data Fig. 2 Activation of the Notch receptor pathway.

a, Structural layout of the Drosophila Notch receptor protein with its ECR containing numerous epidermal growth factor (EGF) domains, the negative regulatory region (NRR), which physiologically suppresses the staggered proteolytic processing at various cleavage sites (S2–S4) until receptor stimulation, and the ICR with sequences involved in nuclear import of the Notch intracellular domain (NICD) and co-activation of gene transcription. b, Sequence of events that correspond to Notch processing and transmembrane signal transduction and involved proteases31. c, Amino acid sequence and domain and motif annotation of the Cirl-NRS-LexA protein. ADAM, A disintegrin and metalloproteinase; ECR, extracellular region; ICR, intracellular region; TM, transmembrane domain.

Extended Data Fig. 3 In vitro characterization of NRS activation.

a, Characterization of hybrid transmembrane sensors containing the ECR of the human CD4 receptor fused to the NotchJTS-LexA module (CD4-NRS-LexA) in Drosophila Schneider-2 cells using a luciferase-based assay. Addition of the CD4-ECR to the NRS basis (CD4-NRS-LexA) suppresses NRS activity. When the CD4-ECR is severed by secTEVp at cognate TEVp at TEVs interposed between CD4 and NRS-LexA components of the sensor (CD4-3TEVs-NRS-LexA, CD4-6TEVs-NRS-LexA), it becomes activated (magenta). Co-expression of cleavable sensors and intraTEVp does not result in sensor activation (grey). NΔEGF-LexA/NΔECN-LexA set, CD4-3TEVs-NRS-LexA set and CD4-6TEVs-NRS-LexA set were tested in separate assays but are displayed in the same graph. Data (n = 10 biological replicates from three independent experiments for all groups, except CD4-3TEVs-NRS-LexA group n = 3 from one experiment) were normalized and presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). NΔEGF-LexA/NΔECN-LexA groups were compared with two-tailed Mann–Whitney U test, CD4-3TEVs-NRS-LexA dataset by ordinary one-way ANOVA with Tukey’s test, CD4-6TEVs-NRS-LexA dataset with Kruskal–Wallis one-way ANOVA with Dunn’s test (confidence interval = 95 % for all comparisons). P values are displayed above data. See also Source Data. b, NRS-LexA activity of the same sensor set as in a, visualized through expression of a lexAop-DsRed reporter (CD4-3TEVs-NRS-LexA not shown). Representative confocal images of Schneider-2 cell cultures with NRS-LexA signals (magenta, arrows) counterstained with Hoechst (blue). Scale bar = 100 µm. Experiment was independently repeated 3x with similar results. c, Protein sequence alignment of the JTS of Drosophila (Uniprot: P07207) and human Notch1 receptors (Uniprot: P46531). Positions of the TM helix (grey box) and S2, S3 and S4 protease cleavage sites are indicated. For control sensors in this study the critical valine residue at the S3 cleavage site (light brown box) was point mutated (V1763K). Black boxes delineate highly conserved residues. d, Function of NΔECN-LexA and CD4-6TEVs-NRS-LexA variants (grey circles) requires γ-secretase activity as application of 10 µM DAPT suppresses their activation (white circles). Data (n = 3 biological replicates from one experiment for all groups) are presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, whiskers minimum and maximum values). Data groups (-DAPT/+DAPT for each sensor) were compared with two-tailed unpaired t-test (confidence interval = 95 %). P values are displayed above data. See also Source Data. e, Surface and total expression quantified by ELISA shows that Cirl-NRS-LexA variants as shown in b are delivered to the cell surface. Surface (n = 24 biological replicates from six independent experiments for all groups, except Cirl-NRSΔS3-LexA group n = 12 biological replicates from three independent experiments) and total ELISA data (n = 28 biological replicates from seven independent experiments for all groups, except Cirl-NRSΔS3-LexA group n = 12 biological replicates from three independent experiments) were normalized and presented as multiples of control dataset in box-whisker plots (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Data were analysed with Kruskal–Wallis one-way ANOVA with Dunn’s test (confidence interval = 95 % for all comparisons). P values are displayed above and below data. Surface/total expression ratio (right panel) normalized to Cirl-NRS-LexA ratio indicates degree of surface trafficking of each Cirl-NRS-LexA variant and Cirl. See also Source Data.

Source data

Extended Data Fig. 4 Comparison of Cirl-NRS activity with different binary expression system readouts.

a, Organization of the Cirl locus, Cirl-NRS alleles and their gene products. b–d, Cirl-NRS-LexA (b), Cirl-NRS-GAL4 (c) and Cirl-NRS-QF2 (d) sensors display comparable activity in adults in neurons of the proboscis (chevron), eyes (double chevron) and leg joints (arrowheads). Reporter transgene are: 13xLexAop2-6xmCherry-HA (b), 20xUAS-6xmCherry-3xHA (c) and QUAS-mtdTomato-3xHA (d). Scale bars = 0.5 mm. e–g, Cirl-NRS dissociation signals reported using the (e) LexA/lexAop, (f) GAL4/UAS and (g) QF2/QUAS binary expression systems. Top panels show Cirl-NRS activity in the eyes (double chevrons), proboscis (chevrons), and the pedicel (white arrows) and funiculus (grey arrows) of the antenna. Middle panels show Cirl-NRS activity in the leg, bottom panels show a close-up of the femorotibial joint with Cirl-NRS-positive mechanosensory neurons (white arrowheads). Scale bars = 250 µm (heads and legs), 50 µm (joints). All experiments independently repeated 3x with similar results.

Extended Data Fig. 5 Binary expression system controls.

a, Expression control of lexAop-myr-mCherry. b, Expression control of lexAop2-mCherry. c, Expression control of UAS-RFP.nls. d, Expression control of 2xhrGFP.nls. Scale bars = 50 µm. Same fly as in c expressing both reporters.

Extended Data Fig. 6 Manipulation of leg-joint movement.

a, Position of femorotibial joint in adult metathoracic leg. Adapted from ref. 67. b,c, Adult flies were glued to a support and videotaped before, during and after the leg immobilization procedure. In the photographs the fly is displayed only during the immobilization interval, when the experimental metathoracic leg is fixed in extension with a taut restraint during the leg extension (b) and flexion (c) phases. The support plate fixation point of the restraint is not depicted in the images. The contralateral leg was allowed to move freely during all intervals of the procedure. Dashed lines indicate axes of the femur and tibia, between which the angle was determined for the immobilized and mobile leg, respectively. d, The motion range of the joint (Δα) was determined by measuring the difference between the femoro-tibia axes angle during maximal extension (αe) and flexion (αf). For clarity axes of mobile leg as shown in b,c were mirrored in the illustration.

Extended Data Fig. 7 Colocalization of Cirl-NRS-LexA and Cirl proteins.

a–c, Comparisons of L3 larvae carrying wild-type (a), γ-secretase-resistant (b) or GAIN-domain cleavage-incompetent (c) Cirl-NRS-LexA variants showed that Cirl dissociation is receptor autoproteolysis-dependent in all neurons except in Kenyon cells (chevron) and a few individual neurons throughout the CNS (arrowheads). nls = nuclear localization sequence; Scale bars, 50 µm. d–f, Schematic illustrations of tagged NRS sensor variants. g–i, Single planes of central brain hemispheres from different NRS sensor variants immunostained using anti-HA (in magenta) and anti-V5 (in green) antibodies to visualize the ECR and C termini of NRS sensor variants (right panel). Scale bar = 30 µm. j–l, Insets of merged hemisphere images shown in g–i (dashed rectangles). N- and C-terminal NRS termini colocalize in the membrane in central brain hemisphere cells of third instar larvae (arrowheads). Scale bar = 10 µm. m, L3 larval brain expressing the transcriptional reporter Cirlp-GAL4 (green) and the release sensor Cirl-NRS-LexA (magenta). Scale bar = 50 µm. n–p, Immunohistochemical co-staining of RFP-Cirl (green) and different Cirl-NRS variants (magenta) show colocalization of both proteins in the membrane in central brain hemisphere cells of L3 larvae (arrowheads). Dashed rectangles indicate position of areas magnified in the insets below. Scale bar = 30 µm, inset = 10 µm. All experiments independently repeated 3x with similar results.

Extended Data Fig. 8 Loss of Tollo does not affect Cirl expression levels or localization.

a, Schematic illustration of the experimental set-up for affinity-immunoprecipitation of Cirl ligands. b, Tollo-GAL4 and Cirl-NRS-LexA co-labelling shows co-expression of Cirl-NRS-LexA+ (magenta) and Tollo-GAL4+ (green) in specific areas of the brain hemispheres and VNC (inset). Strong Cirl-NRS-LexA>lexAop-myr-mCherry activity in the central brain is found in the mushroom body (asterisk) and in a reticular pattern in the cortex (arrows). Scale bar, 25 µm. Inset: some cells in the VNC display Tollo-GAL4+/Cirl-NRS-LexA+ co-labelling (closed arrowheads) while others are either Tollo-GAL4+ or Cirl-NRS-LexA+ (open arrowheads). Scale bar, 10 µm. Experiment independently repeated 6x with similar results. c, Principle of synaptic interaction screen between Tollo-GAL4+ and Cirl-NRS-LexA+ cells through t-GRASP. d,e, t-GRASP signals in L3 brain hemispheres enhanced by an anti-GFP immunostaining; neuroblasts visualized using anti-Mir antibody. Scale bar = 50 µm. Representative t-GRASP signals upon co-expression by  Tollo-GAL4 and Cirl-NRS-LexA are abundant (e), but hardly detectable in control flies lacking the drivers (d). t-GRASP signals appear to line cell boundaries (arrowheads). Scale bar, 10 µm. f, Quantification of t-GRASP signals in the brain indicates that Tollo-GAL4+ and Cirl-NRS-LexA+ cells are contacting each other. (i) and (ii) relate to images in d and e, respectively. pre-t-GRASP/+; post-t-GRASP/+ (n = 4 independent flies), Tollo-GAL4>pre-t-GRASP; Cirl-NRS-LexA>post-t-GRASP (n = 5 independent flies). Data are presented in a box-whisker plot (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Data were compared with a two-tailed unpaired t-test (confidence interval = 95 %). See also Source Data. g, Illustration of C-terminally V5-tagged Cirl. h, Western blot analysis showing similar Cirl expression levels in the presence and absence of Tollo. α-tubulin served as loading control. Experiment independently repeated 2x with similar results. For gel source data, see Supplementary Fig. 1h. i, Confocal images of Cirl expression in larval brains appears unaltered in TolloKO. Scale bar 100 µm. Experiment independently repeated 3x with similar results.

Source data

Extended Data Fig. 9 Cirl-NTF release occurs in glial cells and is sufficient for maintaining the pool of neuroblasts.

a, Single confocal plane showing sparse co-labelling of Cirl-NRS-LexA and pan-glial repo-GAL4 marker in the L3 CNS (arrowhead). Boxed region magnified in inset. Asterisk indicates mushroom body. Scale bar, 50 µm; scale bar inset, 10 µm. b, Schematic of Cirl-T2A-LexA reporter allele. c, Cirl-NRS-LexA is sufficient for neuroblast pool size maintenance. Quantification of Mir+ neuroblasts in L3 central brain (n = 8 independent flies per genotype). Data are presented in a box-whisker plot (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Following Shapiro–Wilk normality testing data were analysed with ordinary one-way ANOVA with Tukey’s test (confidence interval = 95 %). P values are displayed above data. See also Source Data. d, Cirl is only required in CG cells but not neuroblasts or GMCs to maintain a normal neuroblast pool size. Filled circle indicates presence of transgene. Quantification of Mir+ neuroblasts in L3 central brain of independent flies with the genotype UAS-CirlRNAi (n = 8 independent flies), Cirlp-GAL4 (n = 11 independent flies), Cirlp-GAL4>UAS-CirlRNAi (n = 9 independent flies), 55B12-GAL4 (n = 8 independent flies), 55B12-GAL4 (n = 7 independent flies), Tollo-GAL4 (n = 9 independent flies) and Tollo-GAL4>UAS-CirlRNAi (n = 8 independent flies). Data are presented in a box-whisker plot (all data points plotted; horizontal line represents median, boxes the 25th and 75th percentiles, whiskers minimum and maximum values). Data were analysed with ordinary one-way ANOVA with Tukey’s test (confidence interval = 95 %). P values are displayed above data. See also Source Data.

Source data

Extended Data Fig. 10 The aGPCR family in Drosophila melanogaster.

a, Structural layout of all known aGPCRs of Drosophila melanogaster. Domain abbreviations: 7TM, heptahelical transmembrane; CA, cadherin; GAIN, GPCR autoproteolysis-inducing; HRM, hormone-receptor motif; IG, immunoglobulin; LAM, laminin; EGF, epidermal growth factor; LRR, leucine rich repeat. b, Phylogenetic comparison of GAIN domain amino acid sequences using the Jukes-Cantor algorithm. Human PKD1 GAIN domain was used as an outgroup. c, Amino acid sequence alignment of the GPS of all fly aGPCRs shows conservation of the GPS site in four of the five receptors. Dashed vertical line indicates the site of GAIN-domain-mediated self-cleavage. NTF side boxed in blue. d, Structure of Mayo-NRS and Ketchup-NRS.

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Scholz, N., Dahse, AK., Kemkemer, M. et al. Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation. Nature 615, 945–953 (2023). https://doi.org/10.1038/s41586-023-05802-5

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