Howard Florey Institute, University of Melbourne, Victoria 3010, Australia.
Peptides. 2010 Sep;31(9):1730-6. doi: 10.1016/j.peptides.2010.05.021. Epub 2010 Jun 4.
INSL3 is a member of the insulin-IGF-relaxin superfamily and plays a key role in male fetal development and in adult germ cell maturation. It is the cognate ligand for RXFP2, a leucine-rich repeat containing G-protein coupled receptor. To date, and in contrast to our current knowledge of the key structural features that are required for the binding of INSL3 to RXFP2, comparatively little is known about the key residues that are required to elicit receptor activation and downstream cell signaling. Early evidence suggests that these are contained principally within the A-chain. To further explore this hypothesis, we have undertaken an examination of the functional role of the intra-A-chain disulfide bond. Using solid-phase peptide synthesis together with regioselective disulfide bond formation, two analogs of human INSL3 were prepared in which the intra-chain disulfide bond was replaced, one in which the corresponding Cys residues were substituted with the isosteric Ser and the other in which the Cys were removed altogether. Both of these peptides retained nearly full RXFP2 receptor binding but were devoid of cAMP activity (receptor activation), indicating that the intra-A-chain disulfide bond makes a significant contribution to the ability of INSL3 to act as an RXFP2 agonist. Replacement of the disulfide bond with a metabolically stable dicarba bond yielded two isomers of INSL3 that each exhibited bioactivity similar to native INSL3. This study highlights the critical structural role played by the intra-A-chain disulfide bond of INSL3 in mediating agonist actions through the RXFP2 receptor.
胰岛素样肽 3（INSL3）是胰岛素-IGF-松弛素超家族的成员，在男性胎儿发育和成年生殖细胞成熟中发挥关键作用。它是 RXFP2 的同源配体，RXFP2 是一种富含亮氨酸重复的 G 蛋白偶联受体。迄今为止，与我们目前对 INSL3 与 RXFP2 结合所需的关键结构特征的了解相比，人们对引发受体激活和下游细胞信号转导所需的关键残基知之甚少。早期证据表明，这些主要包含在 A 链中。为了进一步探索这一假设，我们研究了 A 链内二硫键的功能作用。我们使用固相肽合成技术和区域选择性二硫键形成技术，制备了两种人 INSL3 的类似物，其中一个类似物中的 A 链内二硫键被取代，相应的 Cys 残基被等电子的 Ser 取代，另一个类似物中的 Cys 残基被完全去除。这两种肽都保留了几乎全部的 RXFP2 受体结合能力，但没有 cAMP 活性（受体激活），表明 A 链内二硫键对 INSL3 作为 RXFP2 激动剂的能力有重要贡献。用代谢稳定的二碳键取代二硫键得到了两种 INSL3 的异构体，它们都表现出与天然 INSL3 相似的生物活性。这项研究强调了 INSL3 的 A 链内二硫键在通过 RXFP2 受体介导激动剂作用中的关键结构作用。
