Florey Neuroscience Institutes, University of Melbourne Melbourne, VIC, Australia.
Front Endocrinol (Lausanne). 2012 Feb 1;3:11. doi: 10.3389/fendo.2012.00011. eCollection 2012.
Insulin-like peptide 3 (INSL3) is a hormone and/or paracrine factor which is a member of the relaxin peptide family. It has key roles as a fertility regulator in both males and females. The receptor for INSL3 is the leucine rich repeat (LRR) containing G-protein coupled receptor 8 (LGR8) which is now known as relaxin family peptide receptor 2 (RXFP2). Receptor activation by INSL3 involves binding to the LRRs in the large ectodomain of RXFP2 by residues within the B-chain of INSL3 as well as an interaction with the transmembrane exoloops of the receptor. Although the binding to the LRRs is well characterized the features of the peptide and receptor involved in the exoloop interaction are currently unknown. This study was designed to determine the key INSL3 determinants for RXFP2 activation. A chimeric peptide approach was first utilized to demonstrate that the A-chain is critical for receptor activation. Replacement of the INSL3 A-chain with that from the related peptides INSL5 and INSL6 resulted in complete loss of activity despite only minor changes in binding affinity. Subsequent replacement of specific A-chain residues with those from the INSL5 peptide highlighted that the N-terminus of the A-chain of INSL3 is critical for its activity. Remarkably, replacement of the entire N-terminus with four or five alanine residues resulted in peptides with near native activity suggesting that specific residues are not necessary for activity. Additionally removal of two amino acids at the C-terminus of the A-chain and mutation of Lys-8 in the B-chain also resulted in minor decreases in peptide activity. Therefore we have demonstrated that the activity of the INSL3 peptide is driven predominantly by residues 5-9 in the A-chain, with minor additional contributions from the two C-terminal A-chain residues and Lys-8 in the B-chain. Using this new knowledge, we were able to produce a truncated INSL3 peptide structure which retained native activity, despite having 14 fewer residues than the parent peptide.
胰岛素样肽 3（INSL3）是一种激素和/或旁分泌因子，属于松弛肽家族成员。它在男性和女性的生育调节中起着关键作用。INSL3 的受体是富含亮氨酸重复序列（LRR）的 G 蛋白偶联受体 8（LGR8），现在称为松弛肽家族肽受体 2（RXFP2）。INSL3 通过其 B 链内的残基与 RXFP2 的大胞外结构域中的 LRR 结合，以及与受体的跨膜外环相互作用来激活受体。尽管与 LRR 的结合已得到很好的描述，但目前尚不清楚肽和受体中涉及外环相互作用的特征。本研究旨在确定 INSL3 决定 RXFP2 激活的关键因素。首先利用嵌合肽方法证明 A 链对于受体激活至关重要。尽管与结合亲和力只有微小变化，但用来自相关肽 INSL5 和 INSL6 的 A 链替代 INSL3 的 A 链会导致完全丧失活性。随后用 INSL5 肽中的特定 A 链残基替换特定 A 链残基突出了 INSL3 的 A 链的 N 末端对于其活性至关重要。值得注意的是，用四个或五个丙氨酸残基替换整个 N 末端会导致肽具有近乎天然的活性，这表明特定残基对于活性不是必需的。此外，A 链 C 末端的两个氨基酸残基的去除和 B 链中赖氨酸 8 的突变也导致肽活性略有降低。因此，我们已经证明，INSL3 肽的活性主要由 A 链的 5-9 个残基驱动，A 链的两个 C 末端残基和 B 链中的赖氨酸 8 有较小的额外贡献。利用这一新知识，我们能够产生一种截短的 INSL3 肽结构，尽管其残基数比亲本肽少 14 个，但仍保留天然活性。
