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

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 W6.53 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.

黏附性G蛋白偶联受体ADGRG2，也称为GPR64，是雄性生育力的关键调节因子，可维持离子/ pH稳态以及与囊性纤维化跨膜传导调节因子（CFTR）的偶联。人们对ADGRG2功能的分子基础了解甚少，部分原因是尚未有关于ADGRG2内源性配体的报道，这限制了用于探究ADGRG2活性的工具。已有研究表明，ADGRG2可被一种名为p15的肽激活，该肽源自其自身N末端区域，即所谓的斯塔赫尔（Stachel）序列。然而，p15的低亲和力限制了其在ADGRG2特性研究中的应用。在本研究中，我们利用丙氨酸扫描诱变来检测对p15诱导的ADGRG2活性起关键作用的残基。接下来，我们设计了系统的策略，通过天然和非天然氨基酸替换来优化ADGRG2的肽激动剂。我们获得了一种优化的ADGRG2斯塔赫尔肽T1V/F3Phe(4 - Me)（VPM - p15），它激活ADGRG2的亲和力显著提高（>2个数量级）。然后，我们对ADGRG2中在VPM - p15结合时对ADGRG2激活起重要作用的残基进行了表征，发现切换开关W6.53以及ADGRG2的胞外环2（ECL2）区域的残基是VPM - p15相互作用以及VPM - p15诱导的Gs或抑制素信号传导的关键决定因素。我们的研究不仅为探究ADGRG2的功能提供了一种有用的工具，还为进一步优化斯塔赫尔肽以激活黏附性G蛋白偶联受体成员提供了新的见解。