Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions.,Biochemistry

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Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions.
Biochemistry ( IF 2.9 ) Pub Date : 2020-08-31 , DOI: 10.1021/acs.biochem.0c00576
Xi-Ping Huang , Terrence P Kenakin , Shuo Gu ₁ , Brian K Shoichet ₁ , Bryan L Roth

Affiliation

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration- and pH-dependent manner. We then show that single histidine mutants have differential and varying degrees of effects on proton-sensing and metal ion modulation. Some histidine residues play dual roles in proton-sensing and metal ion modulation, while others are important in one or the other but not both. Two extracellular disulfide bonds are predicted to constrain histidine residues to be spatially close to each other. Combining histidine mutations leads to reduced proton activity and resistance to metal ion modulation, while breaking the less conserved disulfide bond results in a more severe reduction in proton-sensing over metal modulation. The small-molecule positive allosteric modulators (PAMs) ogerin and lorazepam are not affected by these mutations and remain active at mutants with severely reduced proton activity or are resistant to metal ion modulation. These results suggest GPR68 possesses two independent allosteric modulation systems, one through interaction with divalent metal ions at the extracellular surface and another through small-molecule PAMs in the transmembrane domains. A new GPR68 model is developed to accommodate the findings which could serve as a template for further studies and ligand discovery by virtual ligand docking.

中文翻译：

GPR68的胞外组氨酸残基对二价金属离子的质子传感和变构调节的不同作用。

GPR68是一种孤立的G蛋白偶联受体，可感知质子，与多种G蛋白偶联，并被二价金属离子激活或抑制。尽管尚不清楚这些组氨酸残基如何在质子传感和金属离子调节中发挥作用，但它具有七个细胞外组氨酸残基。在这里，我们证明二价金属离子是变构调节剂，可以以浓度和pH依赖的方式激活或抑制质子活性。然后，我们表明单个组氨酸突变体对质子传感和金属离子调制具有不同程度的影响。一些组氨酸残基在质子传感和金属离子调节中起着双重作用，而其他一些则在一个或另一个中起重要作用，但不是两者都重要。预测两个胞外二硫键会限制组氨酸残基在空间上彼此靠近。组氨酸突变的结合导致质子活性的降低和对金属离子调制的抗性，而破坏较不保守的二硫键会导致质子传感比金属调制更严重的降低。小分子正构构调节剂（PAM）ogerin和劳拉西m不受这些突变影响，并在质子活性严重降低或对金属离子调节具有抗性的突变体上保持活性。这些结果表明，GPR68具有两个独立的变构调节系统，一个通过与细胞外表面的二价金属离子相互作用，另一个通过跨膜结构域的小分子PAM相互作用。

更新日期：2020-09-29

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