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.

Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane region

“…Several studies analyzing the role of the amino acids composing the tethered agonist are in accordance with our identification of phenylalanine at position 3, as the most N-terminal residue playing a key role in aGPCR activation (GPR64/ADGRG2, Azimzadeh et al, 2019 andSun et al, 2020;GPR126, Liebscher et al, 2014;Lphn3/ADGRL3, Mathiasen et al, 2020). Similarly, amino acids at positions 6 and 7, albeit not strictly conserved as leucine and methionine residues in all aGPCR tethered ligands, Leucine 998 and Methionine 999 are key for GPR116 activation.…”

“…It will be interesting to identify the position of ECL2 in the peptide-activated state. As an extreme example, in GPR52, a class A receptor, it was recently shown that ECL2 can act as a tethered ligand and activates the receptor by interacting with the TM barrel (Lin et al, 2020). This is yet another potential mechanism of aGPCR activation and cannot be excluded for other members of the aGPCR family.…”

“…With regard to the tethered agonist peptide, a beta strand conformation has been suggested, followed by a turn element that may be key in receptor activation (detailed in Vizurraga et al, 2020). However, only recently a study explored the modalities of the tethered agonist-mediated activation (Sun et al, 2020). We set out to investigate in-depth the tethered agonist-induced activation of aGPCRs, by NTF removal or exogenous GAP addition, using GPR116/ADGRF5 as an example.…”

Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2

“…While finalizing this manuscript, we noticed that a separate group reported that several residues, including the tryptophan and isoleucine of the ECL2, participate in the binding of Stachel peptide to ADGRG2 37 . Although this study corroborates some of our findings, differences exist between the two studies.…”

Conserved residues in the extracellular loop 2 regulateStachel-mediated activation of ADGRG2

“…Whether the ECL2 of other aGPCRs play similar roles in receptor stability and activation by Stachel peptides warrants further studies. While finalizing this manuscript, we noticed that a separate group reported that several residues, including the tryptophan and isoleucine of the ECL2, participate in the binding of Stachel peptide to ADGRG2 37 . Although this study corroborates some of our findings, differences exist between the two studies.…”