Localization of CrzR-V5 (red) and GFP-Krz (green, wild type or indicated mutants) in transfected S2 cells, stained with anti-V5 and anti-GFP antibodies. (A-A’’) Without agonist (corazonin, Crz), CrzR and Krz and diffusely cytoplasmic. (B-E’’). Localization of CrzR and GFP-Krz 10 min after addition of corazonin. Krz-KK/A and Krz-KKVL/A did not induce CrzR relocalization into endosomes. Arrows indicate representative endosomes in wild type Krz and Krz-VL/A transfections. (F) Quantification of cells containing endosomes. A cell was counted as positive if it contained 3 or more endosomes. ****, p<0.0001 in chi-squared test. (G) Interactions between wild type (WT) Krz-mVenus (or an indicated mutant) and CrzR-Rluc8 analyzed by a BRET assay. Net BRET is shown as a function of time upon stimulation of cells with 1 μM corazonin at 25 °C. The data represent the mean ±SEM of three independent experiments.