Figure 3. Cys-ClAc pair-wise crosslinking to pinpoint intermolecular pairs of proximal amino acids in ligand-CRF1R complexes.

(A) Nucleophilic substitution reaction between a cysteine (Cys) thiol and a α-chloroacetamide (ClAc) moiety. (B) Transiently transfected 293T cells expressing each Cys-CRF1R mutant were incubated with each of the ClAc-peptide ligands. The mutation site in CRF1R is indicated in the upper row. The positions of ClAc moiety in the ligands are indicated in the right column of each panel. The peptide indicated with a star (*) bears the ClAc not on the side chain of a Lys, but directly on the N-terminus (see Table 1). Whole-cell lysates were separated on 10% SDS-PAGEs and analyzed by Western blotting using an anti-CRF antibody. The non-deglycosylated ligand-CRF1R complex runs at an apparent molecular weight of ~70–100 kDa (Coin et al., 2013). The non-crosslinked ligand is not detected (MW ~3–4 kDa). Signals were considered as hits if their intensity was higher than a threshold defined as 50% of the most intense signal per ligand.

DOI: http://dx.doi.org/10.7554/eLife.27711.009

Figure 3—figure supplement 1. Photo-labeling of Cys-CRF1R mutants and photo-crosslinking of [Lys(ClAc)]-^dFXCRF(12-41) analogues.

Whole-cell lysates of transiently transfected 293T cells were separated on 10% SDS-PAGE gels and analyzed by Western blotting using an anti-ligand antibody as indicated. The non-crosslinked ligands were not detected (MW ~3–4 kDa). (A) To ensure that the Cys-CRF1R mutants are expressed on the cell surface and still retain the ability to bind CRF1R ligands, cells expressing each of the mutants were photo-labeled with 100 nM [Bpa¹²]-Ucn1. The latter is a Ucn1 analogue containing the photo-activatable amino acid p-benzoyl-Phe (Bpa), which binds to CRF1R with about the same affinity as Ucn1 and gives high labeling yields of the receptor at the cell surface (Kraetke et al., 2005). The residues noted at the top of each panel indicate the Cys-mutation site in CRF1R. The lysates were deglycosylated by PNGase F to obtain sharper bands. The detected bands refer to the covalent [Bpa¹²]-Ucn1-CRF1R complex running at an apparent MW of ~40 kDa and demonstrate that the labeled mutants are expressed at the cell surface and able to bind the ligand. Except for Y77^ECDC-CRF1R and W169^2.64C-CRF1R, all mutants gave a detectable crosslinking band. (B) To assess whether the ^dFXCRF(12-41) analogues containing ClAc moieties bind CRF1R, each of the peptide antagonists indicated at the top was applied to cells transiently expressing A119^1.39Azi-, Y195^3.36Azi- and K262^ECL2Azi-CRF1R mutants and crosslinked with UV light. The peptide indicated with a star (*) bears the ClAc not on the side chain of a Lys, but directly on the N-terminus (see Table 1). Bands corresponding to the fully glycosylated ligand-CRF1R complex at an apparent MW of ~70–100 kDa were detected with all Azi-CRF1R mutants for all nine ligands.

DOI: http://dx.doi.org/10.7554/eLife.27711.010