FIGURE 1.

Properties of the Bpa-substituted sauvagine, [Tyr⁰, Gln¹, Bpa¹⁷]SVG (YQB-SVG). A, binding and signaling properties. Intact LLCPK-1 cells expressing mCRFR1 were incubated with ¹²⁵I-YQ-SVG (∼100,000 cpm) in the presence of increasing concentrations of YQB-SVG or YQ-SVG for 2 h at 18 C; the cells were rinsed (three times) with binding buffer; the cells were lysed with 1 m NaOH (0.25 ml × 3), and the amount of bound radioligand was determined by counting in a γ-counter. The data (B/B0%) are calculated from the means ± S.D. of triplicates. Stimulation of cAMP accumulation by YQB-SVG and YQ-SVG was performed in LLCPK-1 cells expressing CRFR1. The cells were challenged with increasing concentration of the agonist for 20 min in presence of 2 mm 3-isobutyl-1-methylxanthene, supernatant was removed, and intracellular cAMP was extracted by lysing the cells with 100 mm HCl and measured by specific radioimmunoassay. The data are the means ± S.D. of triplicates. B, photoaffinity cross-linking of ¹²⁵I-YQ-SVG to LLCPK-1 cells stably expressing mCRFR1. The cells were incubated with ¹²⁵I-YQB-SVG (200,000 cpm/well) in presence of increasing concentrations of YQB-SVG for 2 h at 18 C; the cells were then rinsed with binding buffer and exposed to UV light. The receptor-ligand complexes were extracted with SDS sample buffer and loaded on 5–20% SDS-PAGE, followed by autoradiography.