A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133

Abstract

Adhesion G protein-coupled receptors (aGPCRs) comprise the second largest yet least studied class of the GPCR superfamily. aGPCRs are involved in many developmental processes and immune and synaptic functions, but the mode of their signal transduction is unclear. Here, we show that a short 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 seven-transmembrane helix domain, which triggers G protein activation. Our studies show 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 mode of aGPCR activation and may prompt the development of specific ligands for this currently untargeted GPCR family.

Fig. 1. Identification of a putative agonistic region in GPR126 and GPR133

(A) Cartoon of a prototypical aGPCR. The extracellular domain (ECD) contains a signal peptide (SP) and the GAIN/GPS domain. aGPCRs also possess a 7TM domain and an intracellular domain (ICD). Autoproteolysis at the GPS yields an N-terminal fragment (NTF) and a C-terminal fragment (CTF). For immunological detection, all constructs were epitope-tagged with an N-terminal HA epitope (yellow square) and a C-terminal FLAG epitope (green trapezoid). (B) hGPR126 and hGPR133 constructs, CTF and ΔGPS-CTF, were generated which lack the NTF and the ECD, respectively. 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. See also supp. Table S1. (C–E) cAMP levels from COS-7 cells transfected with wt and mutant GPR126 and GPR133. (C) P2Y₁₂-CTF mutants have increased basal activity compared to wt, which is abolished in ΔGPS-CTF mutants. (D) Constitutive activity of P2Y₁₂-CTF(GPR126) is increased by deletion of Thr⁸¹³. Receptor activity is abolished when the first three or more aa after the cleavage site are deleted. (E) Single positions within the C-terminal GPS sequence were mutated in GPR126 and GPR133 to alanine as shown. See suppl. Fig. S1C-F for expression studies of all constructs. Data are shown as means ± SEM of three independent experiments each performed in triplicates. EV served as negative control (eV; cAMP level: 3.68 ± 2.54 nM). Statistics were performed by two-way ANOVA and Bonferoni post-hoc test: *p<0.05; **p<0.01; ***p<0.001.

Fig. 2. GPR126 agonistic peptides are derived from the C-terminal part of the GPS

(A) Application of 1 mM peptides of different lengths derived from the C-terminal part of the GPS beginning at the cleavage site of GPR126 revealed agonistic properties as measured by cAMP accumulation. The highest agonistic efficacy was detected for a peptide containing 16 amino acids (p16). Negative controls: eV, and GPR126-P2Y₁₂-ΔGPS-CTF mutant. Basal cAMP levels were3.8 ± 1.6 nM. ( B) Different p16 concentrations were tested on wt P2Y₁₂, wt GPR126, and P2Y₁₂-ΔGPS-CTF. Inset: concentration-response curve of p16 at wt GPR126 revealed an EC₅₀ value >400 μM. Basal eV levels were 3.2 ± 0.7 nM. (C) COS-7 cells endogenously express low levels of GPR126 (see suppl. Fig S2C). Endogenous and transfected GPR126 are knocked down with primate GPR126-specific siRNA as shown by abolished cAMP formation (x-fold over eV; basal cAMP: 5.5 ± 2.2 nM). This was confirmed by a dynamic mass redistribution assay (Epic Biosensor Measurements) (suppl. Fig. S2D) and reduced cell surface ELISA (see suppl. Fig S2E). (D) The specificity of p16 was confirmed on endogenous GPR126. Mutation of position 6 (Leu⁶Ala) abolished the response of p16 in EPIC measurements, as indicated by a picometer (pm) shift of the resonant wavelength caused by dynamic mass redistribution within the cell. (E) A systematic alanine-scan within the p16 peptide showed that the six amino acids downstream of Thr⁸¹³ are required for receptor activation. Basal cAMP levels were 3.8 ± 1.6 nM. (F) p16 Gly⁴Ala (1 mM) blocked activation of GPR126 by p16 (500 μM). Basal cAMP levels were 18.7 ± 9.4 nM. Data are shown as means ± SEM of three independent experiments each performed in triplicates. Statistics were performed by two-way ANOVA and Bonferoni post-hoc test: *p<0.05; **p<0.01; ***p<0.001.

Fig. 3. Tethered agonistic peptides are receptor-specific

(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 properties as measured by cAMP accumulation. The highest agonistic efficacy was detected for a peptide containing 13 amino acids (p13). Negative controls: eV, and GPR126-P2Y₁₂-ΔGPS-CTF mutant. Basal cAMP levels were 5.2 ± 2.0 nM. (B) Concentration-response curve of the p13 peptide revealed an EC₅₀ > 400 μM. Basal eV levels were 2.9 ± 0.2 nM. (C) Specificity of the p16 (GPR126) and the p13 (GPR133) peptides were verified using wt P2Y₁₂, wt GPR126 and wt GPR133 as controls. p16 peptide activated wt GPR126 and P2Y₁₂-ΔGPS-CTF(GPR126) whereas it exhibited unspecific activity in control receptors due to endogenous expression of GPR126 in COS-7 cells (Fig. 2). 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 triplicates. Statistics were performed by two-way ANOVA and Bonferoni post-hoc test: *p<0.05; **p<0.01; ***p<0.001.

Fig. 4. Tethered agonistic peptides function in vivo

(A) Sequences of wild-type (wt) and stl215 alleles. stl215 is characterized by a 6 base pair (bp) in-frame deletion which results in the removal of amino acids Gly831 and Ile832. The BtsCI restriction enzyme site targeted by the TALEN is underlined. (B) Schematic representation of Gpr126 showing the stl215 allele compared to st49 and st63 alleles. (C) Dorsal view of 4 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 mbp 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 central nervous system (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). (E) Quantification of 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 CNS (arrowhead) and PLLn (arrow). (F) Scoring rubric for PLLn mbp expression, enlarged panels show PLLn only key. “Strong” = strong and consistent mbp expression, “some” = weak but consistent mbp expression, “weak” = weak and patchy mbp expression, “none” = no mbp expression. wt larvae treated with DMSO (G) or 100 μM p16 (H) have strong PLLn mbp expression. DMSO-treated gpr126^st63/st63 mutants have reduced PLLn mbp expression (I), which is significantly rescued with peptide treatment (J). (K) Quantification of WISH experiments. Bars indicate proportion of larvae with each PLLn mbp expression phenotype (as defined in F). **p<0.0001, combined gpr126^st63/st63 mutants with “some” and “strong” vs. combined gpr126^st63/st63 mutants with “weak” and “none”, Fisher’s Exact Test. wt = gpr126^+/+ and gpr126^+/st63 siblings of gpr126^st63/st63 mutants. N=3 technical replicates, n=105 wt (51 DMSO-treated, 54 peptide-treated), n=53 gpr126^st63/st63 (21 DMSO-treated, 32 peptide-treated), n=8 gpr126^st49/st49 (4 DMSO-treated, 4 peptide-treated).