Department of Pediatrics, Perinatal Institute, Section of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, United States.
Novartis Institutes for Biomedical Research, Basel, Switzerland.
Elife. 2022 Sep 8;11:e69061. doi: 10.7554/eLife.69061.
The mechanistic details of the tethered agonist mode of activation for the adhesion GPCR ADGRF5/GPR116 have not been completely deciphered. We set out to investigate the physiological importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species-swapping approaches, we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane 7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.
黏附 GPCR ADGRF5/GPR116 的束缚激动剂激活模式的机械细节尚未完全破译。我们着手研究激动肽序列上游的自动催化裂解在生理上的重要性，这是 NTF 位移和随后受体激活所必需的事件。为了检验这一假说，我们在体外和体内研究了束缚激动剂介导的 GPR116 激活。表达不可裂解 GPR116 突变体的敲入小鼠模拟了 GPR116 敲除小鼠的肺表型，证明了束缚激动剂介导的受体激活对于体内功能是必不可少的。通过定点突变和种间交换方法，我们确定了在束缚激动剂序列和细胞外环 2/3（ECL2/3）中用于 GPR116 激活的关键保守氨基酸。我们进一步强调了跨膜 7（TM7）中介导在小鼠中比在人 GPR116 中更强信号转导的残基，并在支持 GPR116 激活的模型中重现了这些发现：用于 GPR116 激活的束缚激动剂：ECL2 相互作用。
