Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Research Center for Translational Medicine at East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China.
Arch Biochem Biophys. 2021 Jun 15;704:108872. doi: 10.1016/j.abb.2021.108872. Epub 2021 Apr 16.
The gastric peptide ghrelin has important functions in energy metabolism and cellular homeostasis by activating growth hormone secretagogue receptor type 1a (GHSR1a). The N-terminal residues of ghrelin orthologs from all vertebrates are quite conserved; however, in orthologs from Cavia porcellus and Phyllostomus discolor, Ser2 and Leu5 are replaced by a smaller Ala and a positively charged Arg, respectively. In the present study, we first demonstrated that the hydrophobic Leu5 is essential for the function of human ghrelin, because Ala replacement caused an approximately 100-fold decrease in activity. However, replacement of Leu5 by an Arg residue caused much less disruption; further replacement of Ser2 by Ala almost restored full activity, although the [S2A] mutation itself showed slight detriments, implying that the positively charged Arg5 in the [S2A,L5R] mutant might form alternative interactions with certain receptor residues to compensate for the loss of the essential Leu5. To identify the responsible receptor residues, we screened GHSR1a mutants in which all conserved negatively charged residues in the extracellular regions and all aromatic residues in the ligand-binding pocket were mutated separately. According to the decrease in selectivity of the mutant receptors towards [S2A,L5R]ghrelin, we deduced that the positively charged Arg5 of the ghrelin mutant primarily interacts with the essential aromatic Phe286 at the extracellular end of the sixth transmembrane domain of GHSR1a by forming cation-π and π-π interactions. The present study provided new insights into the binding mechanism of ghrelin with its receptor, and thus would facilitate the design of novel ligands for GHSR1a.
胃肽 ghrelin 通过激活生长激素促分泌素受体 1a（GHSR1a）在能量代谢和细胞稳态中发挥重要作用。所有脊椎动物 ghrelin 同源物的 N 端残基非常保守；然而，在来自豚鼠和巨嘴蝠的同源物中，Ser2 和 Leu5 分别被较小的 Ala 和带正电荷的 Arg 取代。在本研究中，我们首先证明了 ghrelin 的疏水性 Leu5 对于其功能至关重要，因为 Ala 取代导致活性降低约 100 倍。然而，Leu5 被 Arg 残基取代引起的破坏要小得多；进一步用 Ala 取代 Ser2 几乎完全恢复了全部活性，尽管[S2A]突变本身显示出轻微的不利影响，这意味着[S2A,L5R]突变体中的带正电荷的 Arg5 可能与某些受体残基形成替代相互作用，以弥补必需的 Leu5 的缺失。为了确定负责的受体残基，我们筛选了 GHSR1a 突变体，其中细胞外区域的所有保守负电荷残基和配体结合口袋中的所有芳香族残基分别被突变。根据突变体受体对[S2A,L5R]ghrelin的选择性降低，我们推断 ghrelin 突变体中的带正电荷的 Arg5 主要通过形成阳离子-π 和 π-π 相互作用与 GHSR1a 第六跨膜域细胞外端的必需芳香族 Phe286 相互作用。本研究为 ghrelin 与其受体的结合机制提供了新的见解，从而有助于设计新型 GHSR1a 配体。
