In Vitro Expressed GPCR Inserted in Polymersome Membranes for Ligand-Binding Studies
In Vitro Expressed GPCR Inserted in Polymersome Membranes for Ligand-Binding Studies
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DOI：
10.1002/anie.201204645
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Sinner, Eva-Kathrin
中科院分区：
文献类型：
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作者：
May, Sylvia;Andreasson-Ochsner, Mirjam;Sinner, Eva-Kathrin
G蛋白偶联受体(GPCRs)是人类基因组中最大的基因家族(约占所有基因的2%)，在细胞通讯、细胞黏附和信号转导中发挥着不可或缺的作用。[1]由于它们在不同的生理过程中发挥核心作用，这些七个跨膜结构域的蛋白与许多疾病密切相关就不足为奇了，事实上，大约40%的上市药物或那些正在开发的靶向GPCRs的药物。[2]对于大多数这些受体，结构与功能的关系仍然难以捉摸。此外，还有相当数量的孤儿受体具有未知的内源性配体。[3]因此，大量的工作被用于开发配体结合分析来识别内源性配体或筛选新的药物先导。[4-9]筛选分析成功开发的一个关键决定因素是将GPCRs稳定地固定在活性构象上，最好是固定在表面上，以允许可靠的基于微阵列的筛选形式。一旦筛选成功，这些配体就可以在功能分析中进行进一步的测试。生产GPCRs的传统方法涉及在宿主细胞中过表达。这种方法通常会对细胞的新陈代谢施加压力，造成压力甚至毒性。由于蛋白质聚集和错误折叠，蛋白质产量也往往较低。另一种选择是从细胞膜中提取GPCRs，纯化后将其重组为基于脂质的膜系统。然而，这种方法往往会导致GPCR功能完整性的丧失。无细胞蛋白质合成绕过了这些缺陷，但没有解决固有的脆弱脂质双层的有限适用性。[10]为了克服与基于脂质的系统相关的稳定性问题，基于嵌段共聚物的仿生膜已经开发出来，由于它们的两亲性，形成具有类脂特性但具有增强稳定性的双层膜。[11-14]我们先前描述了使用嵌段共聚物膜作为插入平台来体外合成膜蛋白。[15，16]在此，我们首次展示了体外合成的多巴胺受体D2(DRD2；长链)，GPCR，形成嵌段共聚物小泡，导致所谓的蛋白质多聚体(方案1)。我们使用流式细胞术来证明DRD2的表达，并通过抗体和配体结合来研究构象的完整性。通过在表面固定的多聚体上体外表达DRD2，以及未标记的多巴胺将结合的荧光配体从受体上置换，证明了配体结合的特异性。这种方法引起了广泛的兴趣，因为原则上，我们的系统可以适应任何类型的膜蛋白，并可能为以微阵列为基础的配基结合分析的发展开辟新的途径。方案1.a)蛋白质多聚体的合成：蛋白质多聚体是通过体外表达膜蛋白并自发插入到聚合物膜中而产生的。将编码该蛋白的互补DNA和聚合体直接加入到体外表达混合物中。B)与蛋白多聚体结合的抗体和配体。上盒：通过结合特异性抗体和荧光标记的二抗检测纯化的蛋白多聚体中的膜蛋白。下框：为了确定膜蛋白是否以活性构象结合到聚合体中，通过孵育纯化的…与蛋白聚合体进行配体结合。
G-protein-coupled receptors (GPCRs) constitute the largest gene family in the human genome (ca. 2% of all genes) and play an indispensable role in cell communication, cell adhesion, and signal transduction.[1] Given their central role in diverse physiological processes, it comes as no surprise that these seven-transmembrane domain proteins are significantly involved in many diseases and, indeed, about 40% of all marketed drugs or those in development target GPCRs.[2] For the majority of these receptors, however, the structure–function relationships remain elusive. Furthermore, there is a considerable number of orphan receptors with unknown endogenous ligands.[3] As such, substantial effort is directed towards the development of ligand-binding assays to identify either endogenous ligands or to screen for new drug leads.[4–9] A key determinant for the successful development of screening assays is the stable immobilization of GPCRs in an active conformation, preferably onto surfaces, to allow for a reliable micro-array-based screening format. Upon a screening hit such ligands can then be further tested in functional assays. Conventional methods of producing GPCRs involve overexpression in host cells. This approach typically exerts a strain on the cells metabolism, causing stress or even toxicity. Protein yields also tend to be low, owing to protein aggregation and mis-folding. An alternative is to extract GPCRs from the cell membrane, purify and then reconstitute them into lipid-based membrane systems. However, this approach often leads to the loss of functional integrity of the GPCR. Cell-free protein synthesis circumvents these pitfalls, but does not address the limited applicability of inherently fragile lipid bilayers.[10] To overcome the stability issues associated with lipid based systems, biomimetic membranes based on block-copolymers have been developed, which, owing to their amphiphilic nature, form bilayer membranes with lipid-like characteristics but with an enhanced stability.[11–14] We previously described the in vitro synthesis of membrane proteins using block-copolymer membranes as an insertion platform.[15, 16] Herein we show for the first time the incorporation of in vitro synthesized dopamine receptor D2 (DRD2; long form), a GPCR, into block copolymer vesicles, leading to so-called proteopolymersomes (Scheme 1). We use flow cytometry to demonstrate DRD2 expression, and investigate conformational integrity by antibody and ligand binding. The specificity of ligand binding is demonstrated by in vitro expression of DRD2 onto surfaceimmobilized polymersomes and displacement of bound fluorescent ligand from the receptor by unlabeled dopamine. This approach is of broad interest because, in principle, our system can be adapted to any type of membrane protein and may open new avenues for the development of ligand-binding assays in micro-array based formats.Scheme 1. a) Synthesis of proteopolymersomes: The proteopolymersomes were produced by in vitro expression of membrane proteins and spontaneous insertion into polymer membranes. Complementary DNA (cDNA) encoding the protein and the polymersomes were directly added to the in vitro expression mixture. b) Antibody and ligand binding to proteopolymersomes. Upper box: The membrane proteins were detected in the purified proteopolymersomes through binding of a specific antibody and detection with a fluorescently labeled secondary antibody. Lower box: To determine if the membrane protein was incorporated into the polymersomes in an active conformation, ligand binding to the proteopolymersomes was carried out by incubating purified …