The Human and Mouse Repertoire of the Adhesion Family of G-protein-coupled Receptors

Highlights

• •

  A tethered peptide agonist (termed the Stachel sequence) activates adhesion GPCRs

• •

  The Stachel sequence is highly specific for the given adhesion GPCR

• •

  Structural changes within the ectodomain induce active peptide conformation

• •

  The Stachel sequence is essential for receptor activation in vivo

Summary

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied grade of the GPCR superfamily. aGPCRs are involved in many developmental processes and immune and synaptic functions, only the mode of their signal transduction is unclear. Here, we evidence that a curt peptide sequence (termed the Stachel sequence) within the ectodomain of two aGPCRs (GPR126 and GPR133) functions as a tethered agonist. Upon structural changes within the receptor ectodomain, this intramolecular agonist is exposed to the vii-transmembrane helix domain, which triggers 1000 poly peptide activation. Our studies prove high specificity of a given Stachel sequence for its receptor. Finally, the function of Gpr126 is abrogated in zebrafish with a mutated Stachel sequence, and signaling is restored in hypomorphic gpr126 zebrafish mutants upon exogenous Stachel peptide application. These findings illuminate a way of aGPCR activation and may prompt the development of specific ligands for this currently untargeted GPCR family.

Graphical Abstract

Introduction

Adhesion G protein-coupled receptors (aGPCRs) are amongst the largest proteins in nature and consist of a long extracellular domain (ECD), a seven-transmembrane domain (7TM), and an intracellular C-terminal tail (ICD) (Effigy 1A;

,

). Another characteristic of this class is an autoproteolytic cleavage event that occurs at the GPCR proteolytic site (GPS), located within the GPCR autoproteolysis-inducing (Gain) domain, which cleaves aGPCRs into an Northward-terminal fragment (NTF) and a C-terminal fragment (CTF) (

Araç et al., 2012

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; Figure 1A). aGPCRs play essential roles in controlling prison cell and tissue polarity (

) and tin can modulate synaptic functions (

,

). Although increasing information about aGPCR relevance is being obtained from mutant animal models, human diseases, and variant-associated phenotypes, fiddling is known about the molecular function, activation, and signal transduction of this receptor grade (

,

).

Figure 1 Identification of a Putative Agonistic Region in GPR126 and GPR133

Prove full caption

(A) Cartoon of a prototypical aGPCR. The extracellular domain (ECD) contains a indicate peptide (SP) and the Proceeds/GPS domain. aGPCRs also possess a 7TM domain and an intracellular domain (ICD). Autoproteolysis at the GPS yields an Due north-final fragment (NTF) and a C-terminal fragment (CTF). For immunological detection, all constructs were epitope tagged with an N-last HA epitope (yellowish foursquare) and a C-terminal FLAG epitope (green trapezoid).

(B) hGPR126 and hGPR133 constructs (CTF and ΔGPS-CTF) lacking the NTF and ECD, respectively, were generated. Chimeric constructs were generated by fusing the N terminus of the human P2Y₁₂ receptor (green line) onto the GPR126 and GPR133 mutants. The red half-circle symbolizes the C-terminal portion of the GPS after its cleavage site. Run into also Table S1.

(C–Due east) camp levels from COS-7 cells transfected with WT and mutant GPR126 and GPR133.

(C) P2Y₁₂-CTF mutants have increased basal activity compared with the WT, and this is abolished in ΔGPS-CTF mutants.

(D) The constitutive action of P2Y₁₂-CTF(GPR126) is increased by deletion of Thr⁸¹³. Receptor activity is abolished when the outset iii or more aa later the cleavage site are deleted.

(E) Single positions within the C-terminal GPS sequence were mutated in GPR126 and GPR133 to alanine equally shown. See Figures S1C–S1F for expression studies of all constructs. Data are shown every bit ways ± SEM of three independent experiments, each performed in triplicate. Empty vector (eV) served as the negative command (eV; army camp level: 3.68 ± 2.54 nM). Statistics were obtained by two-way ANOVA and Bonferroni post hoc examination: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

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The first indirect functional information for G protein coupling by aGPCRs were obtained in studies of GPR56 by knockdown experiments involving M_12/thirteen/p115 RhoGEF pathway components (

Iguchi et al., 2008

• Iguchi T.
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). An intriguing observation was reported in a study of gpr126 mutant zebrafish (zf), which showroom defects in peripheral myelination (

Monk et al., 2009

• Monk K.R.
• Naylor S.G.
• Glenn T.D.
• Mercurio South.
• Perlin J.R.
• Dominguez C.
• Moens C.B.
• Talbot W.S.

A One thousand protein-coupled receptor is essential for Schwann cells to initiate myelination.

• Crossref
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). This phenotype was reversible through forskolin-induced circadian AMP (cAMP) peak, suggesting G_s-protein coupling. Other studies provided direct evidence of G_s-protein coupling by measuring intracellular cAMP levels induced past basal activeness of the aGPCRs GPR133 (

) and GPR126 (

Mogha et al., 2013

• Mogha A.
• Benesh A.East.
• Patra C.
• Engel F.B.
• Schöneberg T.
• Liebscher I.
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Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation.

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). Furthermore, experiments with chimeric 1000 proteins, stoichiometric titrations of the Gα_s subunit and receptor, and Gα_s subunit knockdown experiments (

) strongly support Grand protein coupling for GPR133.

Although it is now clear that aGPCRs couple to 1000 proteins, it remains unclear whether endogenous binding partners can induce activation of aGPCRs. Interestingly, several studies have described increased receptor activity when an N-terminal deletion mutant was expressed (

,

,

,

; see Figure 1A). These observations led to the assumption that the ectodomain functions as an inverse agonist, although at least two scenarios for aGPCR activation take been proposed (

): (1) the ectodomain contains an inverse agonist that inhibits 7TM signaling; and (two) ligand binding at the ECD or NTF removal changes the conformation of an aGPCR and exposes a tethered agonist (Figures S1A and S1B).

To test these two models, nosotros used the human being (h) GPR126 and GPR133 to analyze the contribution of the ECD to receptor basal activity, because G_south-protein coupling has been experimentally suggested for these aGPCRs (

,

,

). Systematic mutagenesis studies revealed tethered peptide sequences inside the most C-final part of the ECD, which specifically activates Grand poly peptide signaling via 7TM interactions in vitro. Finally, nosotros performed loss-of-function and rescue experiments in zfgpr126 mutants to confirm the in vivo and evolutionarily conserved significance of the tethered agonist. Together, our results define a machinery of aGPCR activation.

Results

ECD Deletion Activates GPR126 and GPR133

First, nosotros deleted the ECDs of hGPR126 and hGPR133 at their natural GPS cleavage sites and tested the mutants in camp assays. In these constructs, termed CTF(GPR126) and CTF(GPR133), the NTF between the signal peptide and the GPS cleavage site was removed, but the ECD role located C-terminally to the GPS cleavage site remained attached to the 7TM (CTF in Effigy 1B and Table S1). All mutants defective the ECD displayed significantly increased basal activities in campsite assays (Figure 1C), consistent with results from other NTF-deficient aGPCRs. Both mutants were poorly detected at the prison cell surface via hemagglutinin (HA)-tag staining (Figure S1C). Appropriately, total ELISA and confocal imaging revealed an absence of the HA tag in CTF(GPR126) constructs. However, confocal imaging of the C-last FLAG tag showed specific membrane fluorescence (Effigy S1D). We therefore speculate that the HA tags in the CTF mutant constructs are processed during intracellular protein maturation, thereby precluding detection. Because the North termini of rhodopsin-similar receptors tin can improve cell-surface expression and detection of other GPCRs (

,

), we added an HA-tagged P2Y₁₂ N terminus to the balance ECD of the CTF mutants. This generated chimeric P2Y₁₂-CTF(GPR126) and P2Y₁₂-CTF(GPR133) receptors (Figure 1B), which enabled proper plasma membrane detection via HA-tag visualization (Figure S1C). As observed for the CTF constructs, P2Y₁₂-CTF(GPR126) and P2Y₁₂-CTF(GPR133) displayed high constitutive activeness (Figure 1C). These results demonstrate that deletion of the NTF activates hGPR126 and hGPR133.

The ECDs of GPR126 and GPR133 Comprise Agonistic Domains

We generated GPR126 and GPR133 mutants in which the unabridged ECD, including the entire GPS motif, was deleted or replaced past the N terminus of P2Y₁₂ (ΔGPS-CTF; P2Y₁₂-ΔGPS-CTF; Figure 1B). None of the constructs displayed constitutive activity (Effigy 1C), although these chimeras were expressed at the jail cell surface (Effigy S1C). These results argue against the inverse agonist model of aGPCR activation considering constitutive activeness caused past the release of an inverse agonist would not depend on the presence of the residue GPS motif. These results betoken toward an activation model that requires the residual GPS motif, and we hypothesized that the GPS sequence downstream of the cleavage site contains determinants required for receptor activation.

To identify this potential tethered agonist, we sequentially deleted amino acids (aa) C-terminal to the GPS cleavage site in GPR126. Functional analysis showed that while the most North-terminal aa (Thr⁸¹³; Figure 1B) was not essential for receptor activation (Figure 1D), deletion of the first two, besides as larger deletions that removed aa following Thr⁸¹³, abolished basal receptor action. This abolishment was not due to expression changes, since full and cell-surface expression levels were not significantly different betwixt the constructs (Figure S1E). To maintain right aa length C concluding to the cleavage site, we exchanged several positions with alanine. Again, mutants with an substitution of position 813 retained constitutive activity, whereas the commutation of positions 815, 818, and 819 abolished activity in P2Y₁₂-CTF(GPR126) (Figure 1E), but expression levels were not afflicted (Figure S1F). Mutagenesis studies at respective positions in P2Y₁₂ -CTF(GPR133) revealed almost identical results (Figures 1E and S1F). These experiments support the being of a defined agonistic region C-terminal to the GPS.

A Tethered Peptide Activates GPR126

To demonstrate that the aa sequence C-last to the GPS cleavage site has agonistic backdrop, we tested peptides derived from this domain on P2Y₁₂-ΔGPS-CTF(GPR126). Excitingly, systematic truncation of the peptide's C terminus revealed several agonistic peptides (Figure 2A). The most efficient peptide, p16 (sixteen aa long), was used for further construction-function studies. Northward-concluding deletion of the commencement two aa abolished the agonistic abilities of p16 (p16-i and p16-2; Figure 2A). This does non contradict the results of Figures 1D and 1E, because in the original CTF mutants, the starting time aa were replaced past the P2Y₁₂ Due north terminus or by alanine. Thus, these changes are tolerated, whereas the deletions in p16 are non. N-terminal extension beyond the cleavage site past one (p16+1) or 2 to iv (p16+2 to p16+4) aa showed reduced or no agonistic activity of p16, respectively (Effigy 2A). This indicates that noncleaved aa upstream of Thr⁸¹³ are non part of the agonistic structure. In concentration-response curves, p16 displayed low potency (EC₅₀ > 400 μM) on both P2Y₁₂-ΔGPS-CTF(GPR126) and wild-type (WT) GPR126 (Effigy 2B), which tin can be explained by the natural ane:ane stoichiometry of the covalently bound agonist in its natural conformation. The higher cell-surface expression of WT GPR126 compared with P2Y₁₂-ΔGPS-CTF(GPR126; Figure S1C) explains the increased efficacy of p16 on WT GPR126 activation. Fourth dimension-course analyses of army camp accumulation (Figure S2A) and GTPγS binding assays (Effigy S2B) in response to p16 supported p16-induced G protein coupling in GPR126-transfected cells. Note that eV-transfected cells showed residual cAMP accumulation (Figure 2C) and GTPγS bounden, indicating endogenous expression of GPR126 in COS-7 cells. This was confirmed by RT-PCR (Figure S2C), cAMP assays (Effigy 2C), and kinetic dynamic mass redistribution (DMR) measurements (Epic; Corning Life Sciences) with small interfering RNA (siRNA)-mediated knockdown of the endogenous GPR126 (Figures S2D and S2E).

Figure 2 GPR126 Agonistic Peptides Are Derived from the C-Concluding Part of the GPS

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(A) Application of 1 mM peptides of dissimilar lengths derived from the C-terminal office of the GPS, get-go at the cleavage site of GPR126, revealed agonistic properties every bit measured by military camp aggregating. The highest agonistic efficacy was detected for a peptide containing xvi aa (p16). Negative controls: eV and GPR126-P2Y₁₂-ΔGPS-CTF mutant. Basal cAMP levels were 3.viii ± 1.half dozen nM.

(B) Unlike p16 concentrations were tested on WT P2Y₁₂, WT GPR126, and P2Y₁₂-ΔGPS-CTF. Inset: the concentration-response curve of p16 at WT GPR126 revealed an EC_fifty > 400 μM. Basal eV levels were three.2 ± 0.vii nM.

(C) COS-7 cells endogenously express low levels of GPR126 (encounter Figure S2C). Endogenous and transfected GPR126 were knocked downwardly with primate GPR126-specific siRNA every bit shown by abolished cAMP germination (x-fold over eV; basal cAMP: 5.5 ± 2.2 nM). This was confirmed by a DMR assay (Epic biosensor measurements; Effigy S2D) and reduced cell-surface ELISA (run across Figure S2E).

(D) The specificity of p16 was confirmed on endogenous GPR126. Mutation of position six (Leu⁶Ala) abolished the response of p16 in Epic measurements, every bit indicated by a picometer (pm) shift of the resonant wavelength caused by DMR within the cell.

(East) A systematic alanine scan within the p16 peptide showed that the six aa downstream of Thr⁸¹³ are required for receptor activation. Basal military camp levels were 3.eight ± ane.6 nM.

(F) p16 Gly^ivAla (1 mM) blocked activation of GPR126 by p16 (500 μM). Basal cAMP levels were xviii.7 ± ix.4 nM. Data are shown as ways ± SEM of three independent experiments, each performed in triplicate. Statistics were obtained by two-manner ANOVA and Bonferroni post hoc test: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

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The endogenous expression of GPR126 and the high sensitivity of the DMR technology enabled u.s. to test p16 apart from heterologous overexpression systems. As shown in Figure 2D, p16 induced concentration-dependent cellular responses very similar to those found with isoprenaline and β-adrenergic receptor endogenously expressed in COS-7 cells. Mutation of position 6 (Leu^{half dozen}Ala) in p16 abolished the response (Figure 2nd), confirming specificity. To identify functionally relevant positions in the peptide, nosotros performed a systematic alanine scan (Figure 2E). Equally expected from our receptor mutagenesis data (Figures 1D and 1E), the more Due north-concluding aa (positions +2 to +7) were required for agonistic activity, whereas positions +8, +ten, +12, and +14 to +xvi could be replaced with Ala and still testify agonistic properties. These data are in line with a loftier evolutionary conservation of the N-final portion of this peptide sequence (Figure S2F). Interestingly, the peptide p16 Gly⁴Ala blocked p16-induced GPR126 activation at double concentration (Effigy 2F), indicating that p16 Gly⁴Ala can compete with the p16 binding site. Together, these data support the notion that the tethered peptide p16 activates GPR126.

A Tethered Peptide Activates GPR133

To determine whether activation past a tethered peptide is common for aGPCRs, we performed like studies with GPR133. The P2Y₁₂-ΔGPS-CTF(GPR133) could be activated by a peptide derived from the thirteen aa (p13) downstream of the putative cleavage site (Figure 3A). Concentration-response measurements of p13 revealed specific activity on P2Y₁₂-ΔGPS-CTF(GPR133) and WT receptor (EC₅₀ > 400 μM; Effigy 3B). The derived peptides were highly specific for the aGPCR from which they originated: GPR133 p13 did not activate GPR126, and GPR126 p16 did not activate GPR133 (Figure 3C). Because the importance of GPS cleavage for aGPCR expression and activeness has been the subject of controversy (

), we tested two cleavage-scarce mutants: GPR126T⁸⁴¹A (

Moriguchi et al., 2004

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) and GPR133H⁵⁴⁰R (

). Both mutants were expressed and activated past their corresponding peptides (Figures S2G–S2I), indicating that cleavage at the GPS is not required for aGPCR activation by the tethered agonistic peptides. These information demonstrate that the tethered peptide p13 activates GPR133. Together with our analysis of GPR126, these studies suggest that tethered peptide activation is a mutual signaling modality for the aGPCR form.

Figure 3 Tethered Agonistic Peptides Are Receptor Specific

Bear witness full caption

(A) Application of 1 mM peptides of different lengths derived from the C-terminal part of the GPS, beginning at the cleavage site of GPR133, revealed agonistic backdrop as measured past army camp accumulation. The highest agonistic efficacy was detected for a peptide containing 13 aa (p13). Negative controls: eV and GPR126-P2Y₁₂-ΔGPS-CTF mutant. Basal cAMP levels were 5.2 ± ii.0 nM.

(B) The concentration-response bend of the p13 peptide revealed an EC₅₀ > 400 μM. Basal eV levels were two.9 ± 0.2 nM.

(C) Specificity of the p16 (GPR126) and the p13 (GPR133) peptides were verified using WT P2Y₁₂, WT GPR126, and WT GPR133 equally controls. p16 peptide activated WT GPR126 and P2Y₁₂-ΔGPS-CTF(GPR126), whereas information technology exhibited unspecific activeness in control receptors due to endogenous expression of GPR126 in COS-7 cells (Figure S2C). The p13 peptide specifically activated WT GPR133 and P2Y₁₂-ΔGPS-CTF(GPR133). Basal camp levels were 3.0 ± 0.8 nM. Data are shown as means ± SEM of three independent experiments, each performed in triplicate. Statistics were obtained by two-way ANOVA and Bonferroni mail hoc test: ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

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Tethered Peptide Activation of Gpr126 In Vivo

Nosotros next sought to test the in vivo relevance of aGPCR tethered peptide activation. For these studies, nosotros used zf considering previous mutant analyses demonstrated that Gpr126 is essential for Schwann cell myelination and ear development and that these physiological functions require campsite elevation (

Geng et al., 2013

• Geng F.Due south.
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,

). Although several zf gpr126 mutant alleles have been recovered in genetic screens (

Pogoda et al., 2006

• Pogoda H.G.
• Sternheim N.
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), none specifically bear upon the tethered agonist sequence. Therefore, nosotros utilized transcription-activator-similar effector nucleases (TALENs) to target this region (Figures S3A and S3B). We isolated a mutant, gpr126 ^stl215, which lacks simply two codons (Gly⁸³¹-Ile⁸³²) within the tethered agonist sequence (Figures 4A, 4B , and S3C). The gpr126 ^stl215 mutants were grossly normal compared with WT animals (Effigy S3D), but developed swollen ears (Figure 4C), failed to express myelin bones protein (mbp, a marker of mature Schwann cells) forth the posterior lateral line nerve (PLLn) (Figures 4D and 4E), and did not myelinate peripheral axons (Figures S3E–S3H). These defects completely phenocopy the previously published gpr126 ^st49 mutant, which has an early finish codon in the Gain domain upstream of the GPS motif (Figure 4B;

Monk et al., 2009

• Monk K.R.
• Naylor S.G.
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). Chiefly, the Gly⁸³¹-Ile⁸³² deletion introduced by the gpr126 ^stl215 mutation did non modify the prison cell-surface expression of the receptor (Figures S4A and S4B). Therefore, we conclude that the phenotypes observed in gpr126 ^stl215 mutants are caused by loss of a functional tethered agonist.

Figure 4 Tethered Agonistic Peptides Function In Vivo

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(A) Sequences of WT and stl215 alleles. stl215 is characterized by a vi bp in-frame deletion, which results in the removal of aa Gly831 and Ile832. The BtsCI brake enzyme site targeted by the TALEN is underlined.

(B) Schematic representation of Gpr126 showing the stl215 allele compared with st49 and st63 alleles.

(C) Dorsal view of 4 days postfertilization (dpf) larvae. Arrowheads indicate normal ear morphology in the gpr126 ^+/+ larva (WT) and swollen ears in the gpr126 ^{stl215/stl215} larva (stl215).

(D) Lateral view of whole-mount in situ hybridization (WISH) of zf larvae at 4 dpf. The posterior lateral line nerve (PLLn) is marked with an arrow; mbp expression in the CNS is indicated with an arrowhead. mbp expression can be observed in the CNS, but not in the PLLn of gpr126 ^stl215/215 mutant larvae (st215).

(Due east) Quantification of the swollen ear phenotype and PLLn mbp expression out of the total number of larvae scored per genotype (WT = gpr126 ^+/+ and gpr126 ^stl215/+).

(F–J) WISH of 5 dpf larvae showing mbp expression in the CNS (arrowhead) and PLLn (arrow).

(F) Scoring rubric for PLLn mbp expression; enlarged panels show the PLLn-just cardinal. "Strong" = strong and consistent mbp expression, "some" = weak just consequent mbp expression, "weak" = weak and patchy mbp expression, "none" = no mbp expression.

(Grand–J) The WT larvae treated with DMSO (G) or 100 μM p16 (H) accept stiff PLLn mbp expression. DMSO-treated gpr126 ^st63/st63 mutants accept reduced PLLn mbp expression (I), which is significantly rescued with peptide treatment (J).

(Thou) Quantification of WISH experiments. Bars indicate the proportion of larvae with each PLLn mbp expression phenotype (as divers in F). ^∗∗p < 0.0001, combined gpr126 ^st63/st63 mutants with "some" and "strong" versus combined gpr126 ^st63/st63 mutants with "weak" and "none"; Fisher's exact exam. WT = gpr126 ^+/+ and gpr126 ^+/st63 siblings of gpr126 ^st63/st63 mutants. due north = 3 technical replicates, n = 105 WT (51 DMSO-treated, 54 peptide-treated), north = 53 gpr126 ^st63/st63 (21 DMSO-treated, 32 peptide-treated), and n = viii gpr126 ^st49/st49 (iv DMSO-treated, 4 peptide-treated).

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Finally, we tested whether p16 serves as an agonist for endogenous Gpr126 in vivo, using zf PLLn mbp expression as an assay. The gpr126 ^st63 allele contains a point mutation in the first extracellular loop of the 7TM that converts a conserved cysteine residue to tyrosine (C⁹¹⁷Y; Figure 4B;

Monk et al., 2009

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). This mutant receptor shows reduced cell-surface expression compared with the WT (∼60% of WT levels; Figure S4A) and a concomitant reduction in basal activeness (Figure S4B). In vivo, mbp expressed is reduced, but not absent, along the PLLn (

Pogoda et al., 2006

• Pogoda H.G.
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). In contrast, mbp expression is completely absent along the PLLn of the strong loss-of-function gpr126 ^st49 and gpr126 ^stl215 mutants (Figures 4D and 4E). Therefore, we predicted that the gpr126 ^st63 allele produces a hypomorphic Gpr126 protein with reduced signaling capability. Accordingly, our ultrastructural analysis revealed that gpr126 ^st63 mutants can myelinate axons in the PLLn, although fewer axons are myelinated than in the WT (Figures S4C and S4D) (Due south.C.P. and K.R.M., unpublished data).

Because nosotros can infer that gpr126 ^st63 mutants possess a partially functional 7TM, we hypothesized that exogenous addition of p16 could increase the signaling of endogenous hypomorphic Gpr126. This analysis is feasible given that pocket-sized molecules, including peptides, can freely diffuse into the developing larva in the presence of a carrier (

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), and because the functionally important positions in p16 are almost 100% identical between Danio rerio and Human sapiens (Figure S2F). Indeed, p16 was able to activate WT zf Gpr126 in in vitro military camp assays (Figure S4B). Therefore, we treated gpr126 ^st63 mutants with 100 μM p16 in DMSO from 50–55 hr postfertilization, which constitutes a temporal window in which army camp pinnacle by forskolin administration can rescue myelination in gpr126 ^st49 mutants (

,

). We and then qualitatively scored mbp expression in the PLLn (Figure 4A). As a negative control, nosotros treated siblings with DMSO and observed normal PLLn mbp expression in the WT (gpr126 ^+/+ or gpr126 ^st63/+) and reduced or absent mbp in hypomorphic gpr126 ^st63/st63 mutants (Figures 4F–4K). Treatment with 100 μM p16 caused no meaning modify in WT larvae, merely significantly rescued mbp expression in gpr126 ^st63/st63 hypomorphs (0% "strong" or "some" in gpr126 ^st63/st63 + DMSO versus 44% "strong" or "some" in gpr126 ^st63/st63 + p16; Figures 4H, 4J, and 4K). To exam whether this upshot is specific to Gpr126 signaling, we besides assayed strong loss-of-function gpr126 ^st49 mutants, which presumably do non express a 7TM (

Patra et al., 2013

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). Exogenous treatment of gpr126 ^st49 mutants with 100 μM p16 did not rescue mbp expression in the PLLn (Figure 4K), indicating that p16 signals through the 7TM. Together, these loss- and gain-of-function experiments in zf demonstrate the in vivo relevance of tethered peptide activation of aGPCRs.

Discussion

We define a mutual intramolecular agonistic domain for the aGPCRs GPR126 and GPR133 that comprises a sequence between the GPS cleavage site and TM1. Because of its activating nature and its position at the very C terminus of the ECD, nosotros refer to this agonistic sequence as the "Stachel sequence" (Stachel is the German word for stinger). Our analysis of gpr126 ^stl215 suggests that Stachel-mediated activation of Gpr126 is essential for Schwann cell myelination in zf (Figures 4C–4E and S3E–S3G); notwithstanding, the in vivo mechanisms that unmask this tethered agonistic domain are unknown. Gain domain crystal structures revealed that the Stachel sequence lies buried between two β sheets (

Araç et al., 2012

• Araç D.
• Boucard A.A.
• Bolliger M.F.
• Nguyen J.
• Soltis S.M.
• Südhof T.C.
• Brunger A.T.

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.

• Crossref
• PubMed
• Scopus (227)
• Google Scholar

). We and others have shown that CTF-only truncation mutant aGPCRs possess increased basal activity (Figures 1C–1E;

,

,

,

). In all of these studies, the disquisitional Gain domain β sheets were deleted forth with the residue of the NTF, presumably exposing the Stachel sequence. Therefore, structural changes in vivo due to extracellular molecules interacting with the ECD (

,

) or even mechanical removal of the NTF may expose the Stachel sequence to activate the 7TM. The low analogousness of the Stachel sequence to the 7TM suggests a fast on/off ligand-receptor interaction and supports activation by mechanical signals (

Karpus et al., 2013

• Karpus O.N.
• Veninga H.
• Hoek R.M.
• Flierman D.
• van Buul J.D.
• Vandenakker C.C.
• vanBavel E.
• Medof Thou.East.
• van Lier R.A.
• Reedquist K.A.
• Hamann J.

Shear stress-dependent downregulation of the adhesion-Thou protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

• Crossref
• PubMed
• Scopus (39)
• Google Scholar

).

Peptide agonists usually bind to their cognate receptor in a sequential two-step mechanism (

Monteclaro and Charo, 1996

• Monteclaro F.S.
• Charo I.F.

The amino-last extracellular domain of the MCP-ane receptor, but not the RANTES/MIP-1alpha receptor, confers chemokine selectivity. Testify for a two-step mechanism for MCP-1 receptor activation.

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). The kickoff step requires loftier-affinity interactions with extracellular loop regions, whereas the second stride is mediated past depression-affinity interactions with the helix package, promoting receptor activation. Based on our findings, the first stride is non required for aGPCRs, considering the activating peptide is part of the receptor'due south own ECD and therefore covalently bound to the 7TM. In the second footstep of our model of aGPCR activation, the Stachel sequence is predicted to collaborate with extracellular loops and upper helix bundles equally in other peptide/peptide-GPCR pairs (

Thompson et al., 2012

• Thompson A.A.
• Liu W.
• Chun E.
• Katritch 5.
• Wu H.
• Vardy E.
• Huang X.P.
• Trapella C.
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• Crossref
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), which requires a depression affinity. This model is also consistent with protease-activated receptors in which thrombin cleaves the receptor's Northward terminus and exposes an activating tethered agonist (

).

Large ECDs are not unique to the aGPCR family unit. The ectodomains of glycoprotein hormone receptors (the rhodopsin-like GPCR form) are as well composed of several hundred aa forming leucine-rich repeat domains. In glycoprotein hormone receptors, a conserved module termed the swivel region (

) connects the ECD to the 7TM in a manner like to that observed for the GPS domain in aGPCRs. Although the interspaced swivel region does not share the predicted 3D structural identity with the GPS motif, both the hinge region and the GPS motif possess multiple disulfide bonds that class at least ii loops of the polypeptide chain (

Araç et al., 2012

• Araç D.
• Boucard A.A.
• Bolliger M.F.
• Nguyen J.
• Soltis Due south.Thou.
• Südhof T.C.
• Brunger A.T.

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.

• Crossref
• PubMed
• Scopus (227)
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). Interestingly, hinge-region mutations of glycoprotein hormone receptors can activate these rhodopsin-like GPCRs, suggesting an "intramolecular agonistic unit" (

Krause et al., 2012

• Krause G.
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• Kleinau G.

Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

• Crossref
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). Similarly, mutations in Cys⁷⁷⁵, Cys⁷⁹⁴, Cys⁸⁰⁷, and Cys⁸⁰⁹ of GPR126, which unremarkably form disulfide bridges in the GAIN domain, displayed constitutive activity in army camp assays (Figures S4E and S4F). These data provide farther testify that structural changes in the GPS region promote activation via the Stachel sequence.

Our results are compatible with an activation scenario of aGPCRs in which an intramolecular agonistic domain (the Stachel sequence) is unmasked upon structural changes of the ECD, which subsequently triggers 7TM-mediated activation of G protein-signaling cascades (Figures S1B [cis signaling] and S4G). Recent show indicates that the ECD of GPR126 and other aGPCRs tin mediate biological functions independently of the 7TM (trans signaling) (

,

). Our discovery may facilitate attempts to distinguish betwixt trans- and cis-dependent functions; for example, phenotypic perturbations in model organisms through peptide agonists could be attributed to cis signaling of the receptor (eastward.grand., Figures 4F–4K). Our study defines a signaling modality for aGPCRs and lays the foundation for rational ligand design to promote a deeper agreement of the physiology and therapeutic usefulness of this emerging class of GPCRs.

Experimental Procedures

aGPCR Constructs and Functional Assays

Epitope-tagged, full-length human being aGPCR sequences were inserted into pcDps, and mutant aGPCRs were generated by PCR (Table S1). For functional assays, transfected COS-vii cells were divide into 48-well plates and cAMP concentrations were adamant with the Alpha Screen cAMP assay kit (PerkinElmer Life Sciences) according to the manufacturer's protocol. To measure characterization-free receptor activation, a DMR assay (Epic biosensor measurements; Corning Life Sciences) with COS-vii cells endogenously expressing GPR126 was performed as previously described (

Schröder et al., 2010

• Schröder R.
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• Crossref
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). To estimate cell-surface and total expression of receptors carrying N-final HA and C-final FLAG tags, ELISA was used (

Schöneberg et al., 1998

• Schöneberg T.
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• Grüters A.
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). Assay data were analyzed with GraphPad Prism version 6.0 for Windows (GraphPad Software) and statistical details are given in each figure fable.

Peptide Synthesis

Solid-phase peptide synthesis of the peptides was performed on an automatic peptide synthesizer (MultiPep; Intavis AG) using standard Fmoc chemistry.

Zebrafish Studies

Adult zebrafish were maintained in the Washington Academy Zebrafish Consortium facility in accordance with institutional animal protocols (http://zebrafish.wustl.edu/husbandry.htm). Embryos were nerveless from heterozygous gpr126 mutant adults, and mutant larvae were compared with WT siblings for all assays. See the Supplemental Experimental Procedures for details on TALEN mutagenesis, in situ hybridization, transmission electron microscopy, and peptide treatment.

Additional details regarding the methods used in this work are available in the Supplemental Experimental Procedures.

Writer Contributions

I.L., J.South., L.F., 1000.-U.S., L.One thousand.D., and T.S. performed the in vitro experiments. S.C.P., Northward.A., A.Chiliad., and Grand.R.M. performed the zebrafish experiments and S.C.P. and North.A. analyzed the results. I.L., J.Southward., L.F., and T.S. analyzed the information. S.R. synthesized the peptides. I.L. and T.S. designed the study and wrote the newspaper with contributions from all of the authors.

Acknowledgments

We give thanks Xianhua Piao and Thue W. Schwartz for their very helpful comments and discussion of the paper. Nosotros thank Marilyn Levy and Robyn Roth for aid with transmission electron microscopy. This work was supported by the Deutsche Forschungsgemeinschaft (Sfb 610 to T.South. and FOR 2149 to I.50. and T.S.), the BMBF (IFB Adiposity Diseases Leipzig to J.South. and I.L.), the NIH/NINDS (F32NS087786 to S.C.P. and R01NS079445 to 1000.R.M.), the Boehringer Ingelheim Fonds (MD stipend to N.A.), and the University of Leipzig (Formel one startup grant to I.L.).

Supplemental Information

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Article Info

Publication History

Published: December 18, 2014

Accepted: Nov 22, 2014

Received in revised form: October 10, 2014

Received: June 30, 2014

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This is an open access commodity under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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DOI: https://doi.org/10.1016/j.celrep.2014.eleven.036

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