Investigating GPCR:RAMP interactions using nanobodies

使用纳米抗体研究 GPCR:RAMP 相互作用

• 批准号: BB/R016615/1
• 负责人: Mark Wheatley
• 金额: $ 51.48万
• 依托单位: Coventry University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR016615%2F1
• 关键词: Investigating; GPCR; RAMP; interactions; using

The largest target for clinical drugs is a family of proteins called G-protein-coupled receptors (GPCRs). These are found on the surface of cells, where they bind molecules that cells release when they need to communicate with one another, such as hormones and neurotransmitters. On binding a hormone, the GPCR is activated, binds to another protein (often a G-protein, hence the name GPCR) and subsequently generates a chemical signal to the inside of the cell to bring about the required changes. GPCRs are thus of enormous interest, both in terms of basic biology but also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level and function at the cellular level, is fundamentally important and is one of the 'big questions' in biology today. GPCRs have been classified into several families of structurally-related receptors. Family B GPCRs share a similar structure comprising a bundle of 7 transmembrane helices (7TM) linked by loops plus a large extracellular domain (ECD) and are important therapeutic targets for treatment of debilitating conditions such as obesity, diabetes, osteoporosis and migraine. They are activated by medium sized peptides which interact with both the ECD and the 7TM bundle although detailed understanding of the activation process is still limited. The pharmacology and signalling characteristics of family B GPCRs can be profoundly altered by association with a family of accessory membrane proteins referred to as Receptor Activity Modifying Proteins or RAMPs. The activation mechanisms of family B GPCRs and their interactions with RAMPs are poorly understood, although it is beyond question that the formation of these complexes is of profound pharmacological importance. Moreover, currently we do not know where these GPCR:RAMP complexes are localised in native human tissue. The main reason for this is the current lack of tools to properly localise or identify the GPCR:RAMP complexes in native tissues, or to selectively target them.Antibodies are powerful tools that will provide mechanistic and functional insights into family B GPCRs, their interactions with RAMP accessory proteins and localise GPCR:RAMP complexes in human tissue slices. Single domain antibodies (called nanobodies) made by llamas are particularly useful for this sort of task. However, generating antibodies that recognise native GPCRs in tissues has been a difficult challenge as GPCRs are unstable when extracted from the cell membrane by detergent as a pre-requisite for their purification. Recently, we have pioneered a way to 'solubilise' GPCRs without detergent, using a molecular 'pastry cutter' to generate GPCRs still in their native state, embedded in a miniscule disc of cell membrane (referred to as a 'SMALP'), thereby preserving the native environment. In collaboration with our Industrial Partner UCB, we have already shown that GPCR-SMALP can be used to isolate GPCR nanobodies. We will generate antibodies to two main family B receptors - the 'CGRP receptor' and the calcitonin receptor (which exists both alone and bound to RAMP). In addition to nanobodies that bind to only one target, we will engineer 'designer' antibodies that target two things simultaneously, so called 'bi-specifics'. These will target/bridge GPCRs:RAMP complexes, or will target two different domains in the same receptor, such as the ECD and 7TM, similar to natural activators (see above). We already have antibodies to the ECD, and loops, of a parathyroid receptor (PTH1R) suitable for making such 'designer' bi-specifics. Our nanobodies and 'bi-specifics' will be used to probe signalling by family B GPCRs, how it is regulated by RAMPs and to localise GPCR:RAMP complexes in tissue samples.Overall, this will provide important insights into how RAMPs regulate family B GPCRs and identify where this occurs in human tissue samples thereby providing physiological insights as well as defining underpinning mechanisms.

临床药物的最大靶点是称为G蛋白偶联受体（GPCR）的蛋白质家族。它们存在于细胞表面，当细胞需要相互交流时，它们会结合细胞释放的分子，如激素和神经递质。在结合激素时，GPCR被激活，与另一种蛋白质（通常是G蛋白，因此称为GPCR）结合，随后向细胞内部产生化学信号，以引起所需的变化。因此，无论是在基础生物学方面，还是在制药工业的商业开发方面，GPCR都引起了极大的兴趣。了解GPCR如何在分子水平上发挥作用，在细胞水平上发挥作用，是至关重要的，也是当今生物学中的“大问题”之一。GPCR已被分类为结构相关受体的几个家族。家族B GPCR具有相似的结构，包括由环连接的一束7个跨膜螺旋（7 TM）加上大的细胞外结构域（ECD），并且是用于治疗使人衰弱的病症如肥胖症、糖尿病、骨质疏松症和偏头痛的重要治疗靶标。它们被与ECD和7 TM束相互作用的中等大小的肽激活，尽管对激活过程的详细理解仍然有限。家族B GPCR的药理学和信号传导特征可以通过与称为受体活性修饰蛋白或RAMP的辅助膜蛋白家族的结合而被深刻地改变。家族B GPCR的激活机制及其与RAMP的相互作用知之甚少，尽管毫无疑问这些复合物的形成具有深刻的药理学重要性。此外，目前我们不知道这些GPCR：RAMP复合物在天然人体组织中的位置。其主要原因是目前缺乏适当定位或鉴定天然组织中的GPCR：RAMP复合物或选择性靶向它们的工具。抗体是强大的工具，将提供对B家族GPCR、它们与RAMP辅助蛋白的相互作用以及在人体组织切片中定位GPCR：RAMP复合物的机制和功能见解。由美洲驼制造的单域抗体（称为纳米抗体）对这类任务特别有用。然而，产生识别组织中天然GPCR的抗体一直是一个困难的挑战，因为当通过去污剂从细胞膜中提取GPCR作为其纯化的先决条件时，GPCR是不稳定的。最近，我们开创了一种在没有洗涤剂的情况下“溶解”GPCR的方法，使用分子“糕点切割器”来产生仍然处于天然状态的GPCR，嵌入在细胞膜的微小圆盘中（称为“SMALP”），从而保护天然环境。在与我们的工业合作伙伴UCB的合作中，我们已经证明GPCR-SMALP可以用于分离GPCR纳米抗体。我们将产生针对两个主要家族B受体-"CGRP受体“和降钙素受体（其单独存在并与RAMP结合）的抗体。除了只与一个靶点结合的纳米抗体外，我们还将设计出同时针对两个目标的“设计师”抗体，即所谓的“双特异性”抗体。这些将靶向/桥接GPCR：RAMP复合物，或者将靶向相同受体中的两个不同结构域，例如ECD和7 TM，类似于天然活化剂（参见上文）。我们已经有了针对甲状旁腺受体（PTH 1 R）的ECD和环的抗体，适用于制造这种“设计师”双特异性抗体。我们的纳米抗体和“双特异性”将用于探测B家族GPCR的信号传导，以及RAMP如何调节信号传导，并定位组织样本中的GPCR：RAMP复合物。总体而言，这将为RAMP如何调节B家族GPCR提供重要的见解，并确定这在人体组织样本中发生的位置，从而提供生理学见解并定义基础机制。

项目成果

Ligand-induced conformational changes in a SMALP-encapsulated GPCR

• DOI: 10.1016/j.bbamem.2020.183235
• 发表时间: 2020-06-01
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
• 影响因子: 3.4
• 作者: Routledge, Sarah J.;Jamshad, Mohammed;Wheatley, Mark
• 通讯作者: Wheatley, Mark

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles.

• DOI: 10.1039/d1nr02419a
• 发表时间: 2021-08-21
• 期刊: Nanoscale
• 影响因子: 6.7
• 作者: Grime RL ;Logan RT ;Nestorow SA ;Sridhar P ;Edwards PC ;Tate CG ;Klumperman B ;Dafforn TR ;Poyner DR ;Reeves PJ ;Wheatley M
• 通讯作者: Wheatley M