FIGURE 4.

Characterization of cross-linked CNBr-cleaved fragments of V114M, H115M and H117M CRFR1 mutants. A, scheme showing the location of the three CRFR1 methionine (M) mutations, the endogenous methionine at position 206, and the potential cross-linked site. B, the different CRFR1 mutants, expressed in COS-7 cells were cross-linked to ¹²⁵I-YQB-SVG as in Fig. 1B and then analyzed on Tricine-PAGE before (-CNBr) and after (+CNBr) CNBr cleavage. The cleaved products were lyophilized, resuspended in sample buffer, and analyzed on Tricine-PAGE followed by autoradiography. The positions of the molecular mass markers are shown on the right side of the gel. The Y116M receptor mutant was not functional, and cross-linking could not be performed.