Fig. 3: Switching RAMP expression in HUVECs produces unique signalling bias patterns for CGRP family of agonists.

a Expression of CALCR, CALCRL, RAMP1, RAMP2, and RAMP3 genes in RAMP1 expressing HUVECs. Data normalised to GAPDH expression. n = 3 independent experiments. b–f Dose–response curves were constructed for RAMP1 expressing HUVECs stimulated with CGRP, AM or AM2 and the cAMP levels quantified relative to forskolin (100 μM) (n = 4) (b), mobilisation of Ca²⁺_i relative to ionomycin (10 μM) (n = 6) (c), total NO production relative to acetylcholine (10 μM) (n = 3) (d), intracellular ERK_1/2 phosphorylation relative to PMA (10 μM) (n = 3) (e), and extent of cell proliferation (after 72 h) relative to vector treated control and VEGF (n = 4) (f). Data are analysed using a three-parameter non-linear regression curve or the operational model of receptor agonism²⁷. g–h Signalling bias plots were calculated as ∆Log(τ/K_A) (g) or ∆∆Log(τ/K_A) (h) for each agonist and for each signalling pathway. Determination of values requires normalisation to a reference agonist (AM) alone in (g), while for (h) values were normalised to both a reference agonist (AM) and a reference pathway (cAMP). All data represent mean ± SEM for n repeats.