Addressing the architecture, dynamics and activation mechanism of the CGRP receptor

G蛋白偶联受体（gpcr）是人类基因组中最大的蛋白家族，也是治疗药物的最大靶点；因此，它们具有巨大的科学和实际意义。他们被分成许多科。其中，a家族是了解得最好的，但b家族包含的受体可能在许多疾病状态中都很重要，因此了解这些受体的功能非常重要，这不仅有助于我们进一步了解基础生物学知识，也有助于新药的设计。降钙素基因相关肽（CGRP）存在于整个神经系统中，在调节心血管系统（心脏和血管）、免疫系统和炎症方面尤为重要。CGRP的受体具有特殊的科学意义，因为它涉及一种名为CLR的GPCR和另一种名为RAMP1的蛋白质。RAMP1是一个蛋白家族的成员，它可以调节许多gpcr，其中最典型的是CLR。CGRP在心血管疾病和任何涉及炎症的疾病中也可能很重要。这种肽是偏头痛的主要原因，阻断CGRP受体的药物在临床试验中显示出很大的希望；然而，由于毒性问题，到目前为止还不可能在临床上使用这些药物。因此，迫切需要开发能够作用于CGRP受体的新药。CGRP受体由两部分组成。细胞膜上有一个叫做跨膜结构域的部分。它与细胞外区域相连，细胞外区域位于细胞外部。CGRP与该结构的两个部分相互作用，导致跨膜结构域改变形状。这导致受体与其他蛋白质相互作用，导致细胞活化。我们有CGRP受体部分的晶体结构它在细胞外部。不幸的是，我们不知道CGRP如何与之结合，也不知道它如何与跨膜结构域结合。这严重限制了我们对受体的理解和设计针对它的药物的能力。我们之前使用了一种称为定点诱变的技术的实验数据来构建CGRP受体跨膜结构域的计算机模型。这个跨膜结构域与我们的计算机模型产生后结晶的两个b家族gpcr的跨膜结构域非常相似。这给了我们信心，我们结合实验和计算方法的方法是有价值的。在这个项目中，我们打算扩展这种方法来研究CGRP如何结合到受体的两个结构域以及这是如何导致受体被激活的。我们将使用诱变和其他方法，其中我们物理交联CGRP到受体，以确定接触点。然后我们将使用这些来构建计算模型，我们可以通过进一步的实验来完善。利用计算机，我们可以预测当CGRP与受体结合时，受体形状将如何变化，从而确定受体激活的机制。这一知识将有利于设计新药，既可以阻断受体，也可以促进其激活。

G protein coupled receptors (GPCRs) are the largest family of proteins in the human genome and also the largest target for therapeutic drugs; thus they are of enormous scientific and practical interest. They are divided into a number of families. Of these, family-A is the best understood, but family-B includes receptors which are likely to be important in many disease states and so it is important to understand how these function, both to further our knowledge of fundamental biology and also for the design of new drugs.Calcitonin gene-related peptide (CGRP) is found throughout the nervous system and is particularly important in regulating both the cardiovascular system (the heart and blood vessels) and also the immune system and inflammation. The receptor for CGRP is of special scientific interest as it involves a GPCR called CLR and also a second protein called RAMP1. RAMP1 is a member of a protein family that modulates a number of GPCRs of which the best characterised is CLR. CGRP is also likely to be important both in cardiovascular disorders and any disease that involves inflammation. The peptide is a major cause of migraine and drugs which block CGRP receptors have shown great promise in clinical trials; however, so far it has not been possible to use these clinically because of toxicity problems. Thus, there is an urgent need to develop new drugs that could act on CGRP receptors.The CGRP receptor is made up of two parts. A portion called the transmembrane domain is found in the membranes of cells. This is connected to the extracellular domain, which is on the outside of cells. CGRP interacts with both parts of this structure and causes the transmembrane domain to change shape. This causes the receptor to interact with other proteins, leading to cell activation. We have a crystal structure of the part of the CGRP receptor that is on the outside of cells. Unfortunately, we do not know how CGRP binds to this, nor do we know how it binds to the transmembrane domain. This severely limits our understanding of the receptor and our ability to design drugs that could target it.We have previously used experimental data from a technique known as site-directed mutagenesis to construct a computer model of the transmembrane domain of the CGRP receptor. This transmembrane domain is very similar to the transmembrane domains of two family-B GPCRs which were crystallised after our computer model was produced. This gives us confidence that our approach of combining experimental and computational methods is valuable. In this project, we intend to extend the approach to study how CGRP binds to both domains of the receptor and how this causes the receptor to become activated. We will use mutagenesis and also methods where we physically cross-link CGRP to the receptor to identify contact points. We will then use these to construct computational models, which we can refine through further experimentation. Using a computer, we can predict how the receptor shape will change when CGRP binds to it, so identifying the mechanism for receptor activation. This knowledge will be benefitial in the design of new drugs which can either block the receptor or promote its activation.