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. 2017 Aug 3;6:e27711. doi: 10.7554/eLife.27711

Figure 1. Photo-crosslinking mapping of CRF1R to determine footprints of agonists and antagonists.

(A) Sequence alignment of CRF1R peptide ligands. The classification into N-terminus, C-terminus and helical connector is adopted from Beyermann et al. (2000). Antagonists derived from CRF are well characterized in the literature (Rivier and Rivier, 2014), while Ucn1(8-40) was introduced in this work for the first time for direct comparison of agonist/antagonist pairs. In dFXCRF(12-41), substitutions of the two native Met residues to Nle and the N-terminal D-amino acid to enhance its stability and potency (Rivier and Rivier, 2014). (B–D) Activation of CRF1R by CRF and Ucn1 in the presence and absence of competitive antagonists. The assay measures cAMP accumulation in live HEK293 cells stimulated with serial dilutions of each ligand via a luciferase reporter. Plots are representative mean ± s.e.m. of at least three independent experiments, each run in triplicates. (B) CRF1R activation elicited by each of the ligands used in this study. Sub-nanomolar EC50 values for the two agonists CRF and Ucn1 agree with literature data (Rivier and Rivier, 2014). As expected, dFXCRF(12-41) did not elicit any significant receptor activation up to the maximal tested concentration of 100 nM, while CRF(9-41) and Ucn1(8-40) showed minimal residual activity (<10%). (C, D) CRF1R activation by either CRF or Ucn1, in the presence of a constant concentration of each of the antagonists used in this study. (E) Photo-crosslinking experiments. Transiently transfected 293T cells expressing each Azi-CRF1R mutant (residues replaced by Azi are indicated in the upper row) were incubated with each of the five peptide ligands indicated in the right column, followed by UV irradiation (365 nm). Whole-cell lysates were separated on 10% SDS-PAGE and analyzed by Western blotting using either anti-CRF or anti-Ucn1 antibodies as indicated. To obtain sharper bands, samples were deglycosylated by PNGaseF. The subdivision of transmembrane helices and ECLs is based on the crystal structure of the CRF1R TMD (Hollenstein et al., 2013). The deglycosylated ligand-CRF1R complex runs at an apparent MW of ~40 kDa (Coin et al., 2013). The non-crosslinked ligand is not detected (MW ~3–4 kDa).

DOI: http://dx.doi.org/10.7554/eLife.27711.002

Figure 1—source data 1. Numerical values of cAMP accumulation assay.
The assay measures cAMP accumulation in live HEK293 cells stimulated with serial dilutions of each ligand via a luciferase reporter. Raw luminescence data of the firefly reporter luciferase were normalized to the Renilla control and to 100% activation of agonists. At least three independent experiments were performed per ligand, and each experiment was run in triplicate. Values in the table represent a single measurement. Mean ± s.e.m. of triplicates for one representative experiment are reported in Figure 1B–D.
DOI: http://dx.doi.org/10.7554/eLife.27711.003
elife-27711-fig1-data1.xlsx (12.3KB, xlsx)
DOI: 10.7554/eLife.27711.003
Figure 1—source data 2. Numerical values of whole-cell ELISA of HA-Azi-CRF1R mutants.
Related to Figure 1—figure supplement 1. The absorption was measured at 492 nm. Each data point was obtained as mean of three independent experiments, each performed in triplicate. Mean ± s.e.m. of the means of the triplicates are plotted in Figure 1—figure supplement 1B.
DOI: http://dx.doi.org/10.7554/eLife.27711.004
elife-27711-fig1-data2.xlsx (14.1KB, xlsx)
DOI: 10.7554/eLife.27711.004

Figure 1.

Figure 1—figure supplement 1. Expression profiles of a subset of Azi-CRF1R mutants.

Figure 1—figure supplement 1.

Western blot analysis of expression levels of the whole set of mutants used in this study has been reported previously (Coin et al., 2013). Based on those results, we selected here a representative mutant for each domain of CRF1R (top row). (A) Whole-cell lysates of 293T cells transiently expressing each Azi-CRF1R mutant were resolved on 10% SDS-PAGE and analyzed by Western blotting using an M2 anti-FLAG-HRP antibody. The Azi mutation site is indicated in the top row. The fully glycosylated mature CRF1R runs at an apparent MW of ~70–100 kDa. Bands at a lower MW represent non-mature forms (Coin et al., 2013). (B) Whole-cell ELISA of HA-Azi-CRF1R mutants. The absorptions at 492 nm (OD492) are shown as mean ± s.e.m. obtained from three independent experiments each performed in triplicates and normalized to wild type (wt). The positions replaced by Azi are identical to (A).
DOI: http://dx.doi.org/10.7554/eLife.27711.005