Fig. 4.
Tripartite antagonism of CLR. ERK activity was assessed in individual HEK-HA-CLR/myc-RAMP1 cells expressing FRET biosensors for cytosolic ERK (cytoEKAR, A, C, and E) or nuclear (nucEKAR, B, D, and F) ERK. Cells were preincubated with vehicle, CGRP8–17, or CGRP8–37–Chol for 30 min and washed. CGRP-stimulated ERK activity was assessed immediately after washing (A and B, 30 min preincubation) or 4 h after washing (C and D, 4 h prepulse). (A–D) Kinetics. (E and F) Area under curve (AUC). (G and H) Effects of graded concentrations of CGRP8–37–Chol on cytosolic (G) and nuclear (H) ERK signaling in populations of HEK-HA-CLR/myc-RAMP1 cells. (A–F) n = 159–417 cells, n = 3 experiments; (G and H) n = 4–9 experiments. ***P < 0.001 to vehicle; ∧∧∧P < 0.001 to antagonist vehicle control. ANOVA, Tukey’s test.