FIGURE 5.

A, competition binding with [Gln¹, Bpa¹⁷, Tyr²³]SVG (QBY-SVG) against ¹²⁵I-QBY-SVG and stimulation of cAMP accumulation in LLCPK-1 cells stably expressing CRFR1; the experimental conditions are similar to those in Fig. 1A. The data are the means ± S.D. of triplicates. B, photoaffinity cross-linking of ¹²⁵I-QBY-SVG to COS-7 cells expressing the Met¹¹⁴ CRFR1 mutant in presence and absence of excess ovine CRF (1 μm). CNBr cleavage of the photo-cross-linked materials were resolved on Tricine-PAGE. C, scheme showing the relative position of Tyr²³ and Bpa¹⁷ in the QBY-SVG analog and the cross-linked site His¹¹⁷ in CRFR1. D, radiosequencing of cross-linked receptor-ligand fragment purified from the 16-kDa band depicted in B and sequenced using Edman degradation; at each cycle the radioactivity released was quantified in a γ-counter for 5 min; ∼60% of radioactivity submitted for sequencing was recovered in the fractions.