Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli

• Published in: PloS one 2014-01, Vol.9 (1), p.e84013-e84013
• Main Authors: Jappelli, Roberto, Perrin, Marilyn H, Lewis, Kathy A, Vaughan, Joan M, Tzitzilonis, Christos, Rivier, Jean E, Vale, Wylie W, Riek, Roland
• Format: Article
• Language: English
• Subjects: Amphibian Proteins - metabolism; Animals; Bacteria; Binding; Biology; Blotting, Western; Cell culture; Cell Membrane - drug effects; Cell Membrane - metabolism; Chemical bonds; Corticotropin; Corticotropin-releasing hormone; Corticotropin-Releasing Hormone - metabolism; Culture media; Culture Media - pharmacology; Detergents - pharmacology; E coli; Escherichia coli; Escherichia coli - metabolism; Fractionation; G protein-coupled receptors; Gene fusion; Genetic Vectors; Histidine; Humans; Kinetics; Laboratories; Ligands; Localization; Mammals; Mammals - metabolism; Media (culture); Membranes; Mice; Nervous system; Peptide Fragments - metabolism; Peptide Hormones - metabolism; Peptides; Pharmacology; PhoA gene; Physical chemistry; Physiology; Protein binding; Protein Binding - drug effects; Protein Sorting Signals; Protein Structure, Tertiary; Proteins; Receptors; Receptors, Corticotropin-Releasing Hormone - chemistry; Receptors, Corticotropin-Releasing Hormone - metabolism; Recombinant Fusion Proteins - metabolism; Releasing; Rodents; Sieve analysis; Solubility; Studies; Topology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0084013

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.