Optimization of a peptide ligand for the adhesion GPCR ADGRG2 provides a potent tool to explore receptor biology
| Autor: | Guige Hou, Dongfang He, Chuanshun Ma, Xiao Yu, Zhao Yang, Peng Xiao, Ke Yu, Shengchao Guo, Yujing Sun, Jun-Yan Wang, Hui Lin, Daolai Zhang, Jin-Peng Sun, Wen-Tao An, You-Chen Song, Ming-Liang Ma |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
ADGRG2 Models Molecular Protein Conformation alpha-Helical Regulator Gene Expression Peptide Ligands Protein Engineering Biochemistry Receptors G-Protein-Coupled Mice Stachel peptide agonist Transgenes aGPCR adhesion G protein–coupled receptor Receptor chemistry.chemical_classification Peptide chemical synthesis Alanine scanning adhesion G protein–coupled receptor (aGPCR) Recombinant Proteins Cell biology signal transduction Research Article CTF C-terminal fragment Protein Binding TBST Tris Buffer Saline Tween20 Biology 03 medical and health sciences Structure-Activity Relationship Arrestin Animals Humans Protein Interaction Domains and Motifs BRET bioluminescence resonance energy transfer Molecular Biology G protein-coupled receptor Binding Sites 030102 biochemistry & molecular biology G protein–coupled receptor (GPCR) Cell Biology ECD extracellular domain Kinetics 030104 developmental biology NTF N-terminal fragment HEK293 Cells chemistry Amino Acid Substitution Mutagenesis Site-Directed Peptides Function (biology) |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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