Wilde Caroline, Fischer Liane, Lede Vera, Kirchberger Jürgen, Rothemund Sven, Schöneberg Torsten, Liebscher Ines
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany.
*Institute of Biochemistry and Core Unit Peptide Technologies, Medical Faculty, University of Leipzig, Leipzig, Germany
FASEB J. 2016 Feb;30(2):666-73. doi: 10.1096/fj.15-276220. Epub 2015 Oct 23.
Adhesion GPCRs (aGPCRs) form the second largest, yet most enigmatic class of the GPCR superfamily. Although the physiologic importance of aGPCRs was demonstrated in several studies, the majority of these receptors is still orphan with respect to their agonists and signal transduction. Recent studies reported that aGPCRs are activated through a tethered peptide agonist, coined the Stachel sequence. The Stachel sequence is the most C-terminal part of the highly conserved GPCR autoproteolysis-inducing domain. Here, we used cell culture-based assays to investigate 2 natural splice variants within the Stachel sequence of the orphan Gs coupling aGPCR GPR114/ADGRG5. There is 1 variant constitutively active in cAMP assays (∼25-fold over empty vector) and sensitive to mechano-activation. The other variant has low basal activity in cAMP assays (6-fold over empty vector) and is insensitive to mechano-activation. In-depth mutagenesis studies of these functional differences revealed that the N-terminal half of the Stachel sequence confers the agonistic activity, whereas the C-terminal part orientates the agonistic core sequence to the transmembrane domain. Sequence comparison and functional testing suggest that the proposed mechanism of Stachel-mediated activation is relevant not only to GPR114 but to aGPCRs in general.
粘附性G蛋白偶联受体（aGPCRs）构成了G蛋白偶联受体超家族中第二大但也是最神秘的一类。尽管多项研究证明了aGPCRs的生理重要性，但这些受体中的大多数在激动剂和信号转导方面仍然是孤儿受体。最近的研究报道，aGPCRs通过一种被称为“Stachel序列”的拴系肽激动剂被激活。Stachel序列是高度保守的GPCR自蛋白水解诱导结构域的最C末端部分。在这里，我们使用基于细胞培养的试验来研究孤儿Gs偶联aGPCR GPR114/ADGRG5的Stachel序列内的2种天然剪接变体。其中一种变体在cAMP试验中具有组成性活性（比空载体高约25倍），并且对机械激活敏感。另一种变体在cAMP试验中具有低基础活性（比空载体高6倍），并且对机械激活不敏感。对这些功能差异的深入诱变研究表明，Stachel序列的N末端一半赋予激动活性，而C末端部分将激动核心序列定向到跨膜结构域。序列比较和功能测试表明，所提出的Stachel介导的激活机制不仅与GPR114相关，而且总体上与aGPCRs相关。
