G protein-coupled receptors: Evolution of ligand-mediated gating as regulator of binding affinity

G蛋白偶联受体（GPCR）是将信号转导到神经细胞中的最大分子组之一，因此是许多人类疾病和病症的主要药物靶标，包括抑郁症，药物成瘾和神经退行性疾病-所有这些都与社会相关并变得更加普遍。然而，尽管GPCR具有核心重要性，但仍不清楚它们的结合伴侣（配体）如何进入通常埋在GPCR分子内的活性位点。重要的是，我们最近观察到嗅觉GPCR，其配体具有-除了典型的内部结合位点-在GPCR的外表面上的前庭中的第二结合位点，其阻断配体朝向内部结合位点的通道，从而门控受体（配体门控）并降低其有效结合强度。我们在初步实验中已经证明，这种前庭存在于两种神经递质受体和另一种嗅觉受体中。从这些结果中，我们得出了我们的中心假设：通过外部结合位点（前庭）门控配体进入构成了微调GPCR结合亲和力的古老且保守的机制。该项目的最终目标是严格检验这一假设。为此，我们现在将确定前庭的存在并检查前庭在一系列嗅觉和神经递质受体中的功能，这些受体选自包括人类和脊椎动物模型系统斑马鱼在内的几个物种。首先，我们将确定前庭在选定的受体通过计算机辅助理论预测其结构，使用几个独立的方法。第二，我们将在具有预测的前庭结合位点的受体的氨基酸序列中引入突变以消除这些结合位点。第三，我们将研究如何结合的配体是通过表达野生型和突变体受体在细胞中的影响，并通过随后的信号转导级联可视化受体的激活。我们预测，拟议项目的成功完成将对GPCR激活的基本机制产生深刻的新见解，也可能对靶向药物设计产生强烈的影响，这在抑郁症，药物成瘾和神经退行性记忆丧失（多巴胺能，胆碱能受体和多巴胺能受体）等疾病中至关重要。

G protein-coupled receptors (GPCRs) are one of the largest groups of molecules transducing signals into nerve cells, and as such are the major pharmaceutical target for many human diseases and disorders, including depression, drug addiction, and neurodegenerative conditions - all of which increase in relevance for society and become more prevalent. However, despite the central importance of GPCRs, it is still unclear how their binding partners (ligands) access the active site which is often buried inside the GPCR molecules. Importantly, we have recently observed for an olfactory GPCR that its ligand possesses - in addition to the canonical inner binding site - a second binding site in a vestibule on the external surface of the GPCR, which blocks the passage of the ligand towards the inner binding site, thereby gating the receptor (ligand-gating) and decreasing its effective binding strength. We have in preliminary experiments already shown the presence of such vestibules in two neurotransmitter receptors and one other olfactory receptor. From these results we derive our central hypothesis: gating of ligand access via an external binding site (vestibule) constitutes an ancient and conserved mechanism to fine- tune binding affinity of GPCRs. The ultimate goal of the proposed project is to stringently examine this hypothesis. To that end we will now determine the presence and examine the function of a vestibule in a series of olfactory and neurotransmitter receptors chosen from several species including humans and the vertebrate model system zebrafish. First, we will identify vestibules in the selected receptors by computer-aided theoretical prediction of their structure using several independent methods. Second, we will introduce mutations in the amino acid sequence of receptors with predicted vestibular binding sites to eliminate these binding sites. Third, we will examine how binding of ligands is influenced in mutant receptors by expressing wild-type and mutant receptors in cells and visualizing the activation of the receptors through the subsequent signal transduction cascade. We predict that a successful completion of the proposed project will yield profound new insights into the basic mechanism of GPCR activation and could also have strong implications for targeted drug design, which is of critical importance in diseases such as depression, drug addiction, and neurodegenerative memory loss (serotonergic, cholinergic receptors, and dopaminergic receptors).