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的最大分子群之一，因此是许多人类疾病和失调的主要药物靶点，包括抑郁症、药物成瘾和神经退行性疾病——所有这些疾病与社会的相关性都在增加，并且变得越来越普遍。然而，尽管GPCR具有核心重要性，但它们的结合伙伴（配体）如何进入通常埋在GPCR分子内的活性位点仍不清楚。重要的是，我们最近观察到一种嗅觉GPCR，它的配体除了典型的内结合位点外，还在GPCR外表面的前庭中拥有第二个结合位点，它阻断了配体通往内结合位点的通道，从而限制了受体（配体-门控）并降低了其有效结合强度。我们在初步的实验中已经表明，在两个神经递质受体和另一个嗅觉受体中存在这样的前庭。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）控制配体进入构成了一种古老而保守的机制，以微调gpcr的结合亲和力。所提议的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在一系列嗅觉和神经递质受体中的存在并检查其功能，这些受体来自几种物种，包括人类和脊椎动物模型系统斑马鱼。首先，我们将使用几种独立的方法通过计算机辅助理论预测其结构来识别选定受体中的前庭。其次，我们将引入具有预测前庭结合位点的受体的氨基酸序列突变，以消除这些结合位点。第三，我们将通过在细胞中表达野生型和突变型受体，并通过随后的信号转导级联观察受体的激活，来研究突变受体中配体的结合如何受到影响。我们预测，该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，并可能对靶向药物设计产生重大影响，这对抑郁症、药物成瘾和神经退行性记忆丧失（5 -羟色胺能、胆碱能受体和多巴胺能受体）等疾病至关重要。

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).