Expression and Functional Characterization of Membrane-Integrated Mammalian Corticotropin Releasing Factor Receptors 1 and 2 in Escherichia coli

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β -PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2β expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2β product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2β were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2β membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

促肾上腺皮质激素释放因子受体（Corticotropin-Releasing Factor Receptors, CRFRs）属于B1类G蛋白偶联受体，可结合促肾上腺皮质激素释放因子家族的肽类物质，是应激反应中的关键介导因子。为解析该类受体的结合特异性并开展结构研究，本研究在大肠杆菌（E. coli）中过量表达了全长人源CRFR1α、小鼠源CRFR2β，以及缺失N端胞外结构域的受体片段。在细菌中表达的不同CRFR2β-PhoA基因融合产物的特性，与七螺旋膜拓扑模型的预测结果一致。本研究通过细胞分级分离与蛋白质印迹（Western blot）分析，评估了重组组氨酸标签（histidine-tagged）CRFR1α和CRFR2β的表达水平及细菌亚细胞定位情况；同时考察了大肠杆菌宿主菌株、培养基类型，以及PelB或DsbA信号肽对蛋白表达的影响。总体而言，绝大多数受体蛋白可整合至细菌细胞膜中。在所有实验条件下，CRFR2β的产物产量均显著高于CRFR1α。经去污剂筛选分析后，含有CRFR1α和CRFR2β的细菌细胞膜最适合用两性离子去污剂FC-14进行增溶。不同肽配体与CRFR1α、CRFR2β膜组分的结合特性，在一定程度上与真核细胞中观察到的复杂药理学特征相似。本研究认为，所构建的可产功能性CRFRs的大肠杆菌表达系统，可用于该类受体的大规模表达以开展结构研究。