Optimization of a peptide ligand for the adhesion GPCR ADGRG2 provides a potent tool to explore receptor biology

Abstract

The adhesion GPCR ADGRG2, also known as GPR64, is a critical regulator of male fertility that maintains ion/pH homeostasis and CFTR coupling. The molecular basis of ADGRG2 function is poorly understood, in part because no endogenous ligands for ADGRG2 have been reported, thus limiting the tools available to interrogate ADGRG2 activity. It has been shown that ADGRG2 can be activated by a peptide, termed p15, derived from its own N-terminal region known as the Stachel sequence. However, the low affinity of p15 limits its utility for ADGRG2 characterization. In the current study, we used alanine scanning mutagenesis to examine the critical residues responsible for p15-induced ADGRG2 activity. We next designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural and unnatural amino acid substitutions. We obtained an optimized ADGRG2 Stachel peptide T1V/F3Phe(4-Me) (VPM-p15) that activated ADGRG2 with significantly improved (>2 orders of magnitude) affinity. We then characterized the residues in ADGRG2 that were important for ADGRG2 activation in response to VPM-p15 engagement, finding that the toggle switch W^6.53 and residues of the ECL2 region of ADGRG2 are key determinants for VPM-p15 interactions and VPM-p15-induced Gs or arrestin signaling. Our study not only provides a useful tool to investigate the function of ADGRG2 but also offers new insights to guide further optimization of Stachel peptides to activate adhesion GPCR members.

Keywords: ADGRG2; G protein–coupled receptor (GPCR); Stachel peptide agonist; adhesion G protein–coupled receptor (aGPCR); signal transduction.

Figure 1

Critical residues contributed to ADGRG2 tethered peptide p15 binding.A, a schematic diagram of the activation of ADGRG2 by autocleavage and the structural rearrangement in the N terminus. Autocatalytic cleavage at the GPS of ADGRG2 results in the removal of theα subunit, followed by the folding of the Stachel sequence into the 7TM domain to activate ADGRG2. B, activities of truncated ADGRG2 Stachel peptides. HEK293 cells were transfected by ADGRG2-full length (ADGRG2-FL) plasmid and stimulated by 0, 0.5, 5, 50, 500 μM ADGRG2 Stachel peptide p15, N terminus truncated peptide p13 and both N and C termini truncated peptide p7, respectively. The cAMP levels were monitored by the Glosensor assay. ∗∗p < 0.01, ∗∗∗p < 0.001, ADGRG2 Stachel peptide p15 concentration 50 μM, 500 μM compared with 0 μM. &&p < 0.01, ADGRG2 Stachel peptide p13 500 μM compared with 0 μM. #p < 0.05, ###p < 0.001, ADGRG2 Stachel peptide p13, p7 compared with p15. Each experiment was repeated 12 times. C, a systematic alanine scanning of ADGRG2 Stachel peptide p15. HEK293 cells were transfected with ADGRG2-FL, cells transfected with pcDNA3.1 were used as control. Transfected cells were stimulated by 100 μM p15(p15-WT) or its different mutants (p15-Muts), and cAMP levels were detected by the Glosensor assay. n.s. p > 0.05, ∗p < 0.05, ∗∗ p < 0.01, ∗∗∗p < 0.001, p15-Muts were compared with p15-WT. Each experiment was repeated six times.

Figure 2

Optimization of ADGRG2 Stachel peptide p15 for higher potency.A, a schematic diagram of designed mutants of ADGRG2 Stachel peptide p15 according to Stachel sequences. B–D, activities of ADGRG2 Stachel peptide p15 mutants at Thr1 (B), Phe3 (C), Ser10, Thr12, or Pro15 (D). HEK293 cells transfected with ADGRG2-ΔGPS-β were stimulated by 100 μM p15 or p15 mutants; PBS solution containing no peptide was used as control vehicle. The cAMP levels were monitored by the Glosensor assay. Each experiment was repeated 12 times. n.s. p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001, signaling activities of ADGRG2 Stachel peptide p15 mutants were compared with p15. E–F, dose–curves of p15, p15 mutants T1V, F3Tyr(Me), F3Phe(4-Me), F3(1-Nal), T1V/F3(1-Nal), and T1V/F3Phe(4-Me). HEK293 cells transfected with ADGRG2-ΔGPS-β were stimulated by increasing concentrations of p15 or p15 mutants. Each experiment was repeated six times.

Figure 3

Signaling properties of ADGRG2 activated by optimized ADGRG2 Stachel peptide p15 T1V/F3Phe(4-Me) (VPM-p15).A–H, signaling properties of ADGRG2-FL or ADGRG2-ΔGPS-β activated by optimized ADGRG2 Stachel peptide VPM-p15. HEK293 cells transfected with ADGRG2-FL (A, C, E, G) or ADGRG2-ΔGPS-β (B, D, F, H) were stimulated by increasing the concentration of ADGRG2 Stachel peptide p15 or VPM-p15. cAMP levels were detected by the Glosensor assay (A–B), Ca²⁺ signaling activities were detected by the CalfluxVTN Ca²⁺ assay (C–D), β-arrestin1 or β-arrestin2 recruitment was detected by BRET (E–H). n.s. p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001, signaling activities of VPM-p15 were compared with p15. A–H, each experiment was repeated six times.

Figure 4

Key residues of ADGRG2 for VPM-p15 binding and signal transduction.A, a cartoon presentation of ADGRG2-ΔGPS-β highlighting the existence of the possible interactions between the VPM-p15 ligand and the binding site. The ADGRG2 structure was modeled by using PTH1R (Protein Data Bank: 6NBI) as a template. The extracellular loops are colored yellow and the ligand-binding residues are shown as side chain types and colored pink. B, a schematic serpentine representation of the ADGRG2 7TM domain residues highlighting its mutation sites. Extracellular and intracellular loops (ECL and ICL) are indicated (B). C, binding capacities of ADGRG2-ΔGPS-β WT or its mutants for VPM-p15 monitored by BRET experiments. HEK293 cells were transfected with Rluc-ADGRG2-ΔGPS-β or mutants. Transfected cells were stimulated by increasing the concentration of FITC-VPM-p15. Binding capacities were determined by BRET. Kd values of ADGRG2-ΔGPS-β WT and its mutants for binding VPM-p15 were calculated by GraphPad. n.s. p > 0.05, ∗p < 0.05, ∗∗p < 0.01, Binding capacities of ADGRG2-ΔGPS-β mutants were compared with ADGRG2-ΔGPS-β WT. D, effects of ADGRG2-ΔGPS-β mutants on VPM-p15-induced cAMP accumulation. HEK293 cells transfected with ADGRG2-ΔGPS-β or its mutants were stimulated by 100 μM VPM-p15. cAMP levels were detected by the Glosensor assay. Data were normalized by paralleling experiments with ADGRG2-ΔGPS-β WT. n.s. p > 0.05; ∗p < 0.05; ∗∗∗p < 0.001; ADGRG2-ΔGPS-β mutants were compared with ADGRG2-ΔGPS-β WT. C–D, each experiment was repeated six times.

Figure 5

Schematic diagram depicting the optimization strategies of Stachel peptides and the activation of ADGRG2. ADGRG2 could be activated by the Stachel peptide p15 derived from the Stachel sequence. However, the Stachel peptide p15 has a significantly low affinity toward ADGRG2. We then designed systematic strategies to optimize the peptide agonist of ADGRG2, using natural or unnatural amino acid substitutions. Subsequently, we obtained an optimized ADGRG2 Stachel peptide VPM-p15 that activates ADGRG2 more potently compared with the wildtype p15 peptide. Then, the binding results indicate that TM6 and ECL2 regions are important positions to constitute ADGRG2 ligand binding, which recognizes VPM-p15.