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

<div><p>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 <i>E. coli</i>. 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 <i>E. coli</i> 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 <i>E. coli</i> expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.</p></div

Alkaline phosphatase fusion protein analysis in <i>E. coli</i> of mCRFR2β.

<p>(<b>A</b>) Specifically designed C-terminally truncated versions of mCRFR2β fused to bacterial membrane topology reporter alkaline phosphatase (PhoA) confer different phenotypes at the level of colony color. PhoA activity was assessed qualitatively by visual inspection of the colonies. (<b>B</b>) The bacterial colony colors conferred by these different protein fusions are in agreement with the hepta-helical transmembrane model of mCRFR2β. The aa numeration refers to the native receptors pre-protein sequence.</p

Influence of signal peptides on the expression of CRFRs.

<p>Comparative analysis of hCRFR1α (A) and mCRFR2β (B) constructs encoding no signal peptide (No SP), PelB signal peptide or DsbA signal peptide. Expressions were carried out in TB medium with Rosetta2(DE3) strain and the samples were analyzed by Western blot with His₆-tag antibody. For each expression vector tested, 1 µl of IB and M fractions were loaded on the gel; the dilution factor of each sample is indicated.</p

Detergent screen for solubilization of CRFRs.

<p>The efficacy of 12 different detergents, or detergent mixes, in solubilizing PelB-hCRFR1α (<b>A</b>) and PelB-mCRFR2β (<b>B</b>) from bacterial membranes was evaluated, after overnight incubation, by phase separation via ultracentrifugation. The resultant soluble fractions were subjected to His₆-tag antibody Western blot analysis. As a control, an equivalent aliquot of the original membrane fraction (M), which had not been subjected to solubilization, was loaded on the gel. The irregular spot present in the lower part of the B panel is due to a non-specific contamination. For abbreviations of detergent molecules see main text.</p

Saturation binding of labeled astressin.

<p>Binding of increasing concentrations of labeled astressin bound to (A) hCRFR1α or (B) mCRFR2β expressed in <i>E. coli</i> membranes. (◼) total binding; (♦) non-specific binding; (▲)specific binding.</p

Native Western blot analysis of bacterial membrane fractions.

<p>Native PAGE Western blot analysis with anti-His₆-tag antibody of detergent-free bacterial membrane fractions, which derived from the expression of PelB-hCRFR1α (R1) and PelB-mCRFR2β (R2) carried out in Rosetta2(DE3) strain and TB medium. Vector pET-26b (-) was used as negative control. The 1,048 kDa band corresponds to the smallest protein (IgM pentamer) present in the NativeMark Unstained Protein Standard (Life Technologies), which is detected by the antibody.</p

Inhibitory binding constants (K_i, nM) for CRF ligands bound to CRFRs expressed in <i>E.coli</i> membranes as measured by competitive displacement of bound labeled astressin.

<p>K_i values determined by analysis using competitive displacement of labeled astressin as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084013#s3" target="_blank">Materials and Methods</a>. r: rat; m: mouse; h: human. N.D.: No displacement.</p>*<p>Inhibitory binding constants (K_i, nM) for PD-sauvagine bound to CRFRs expressed in <i>E. coli</i> membranes were measured by competitive displacement of bound labeled PD-sauvagine.</p

PD-sauvagine binding.

<p>Displacement by PD-sauvagine (♦) or antalarmin (●) of labeled PD-sauvagine bound to hCRFR1α expressed in <i>E. coli</i> membranes.</p

Comparison of the expression level of recombinant CRFRs.

<p>Expression of PelB-hCRFR1α (R1) and PelB-mCRFR2β (R2) was carried out either in LB (A) or in TB (B) medium in Rosetta2(DE3) strain. TB derived cultures were diluted twenty times before electrophoresis, while LB derived fractions were not diluted. With either medium, equivalent volumes of both the bacterial inclusion bodies and membrane fractions were analyzed by Western blot with His₆-tag antibody. IPTG induced bacteria Rosetta2(DE3) transformed with the parental vector pET-26b (-) were used as a negative control.</p

Schematic diagram of the fusion proteins used for the expression of CRFRs.

<p>The constructs differ by the presence of a bacterial signal peptide (PelB, DsbA, or none) and do not include the native signal peptide. They encode either full-length receptors or variants lacking the ECD-1 domain. All the proteins are produced with a His₆-tag at the C-terminus. The aa numeration refers to the native receptors pre-protein sequences.</p