(a) Competition binding assays of [Y]6-AII analogue
to AT1R, AT2R wild type, and mutants: AT1R, open circle; wild type
AT2R, black circles; AT2R-Y189A, blue diamonds; AT2R-Y189N, green
triangle; AT2R-F272(6.51)A, red square; AT2R-F272(6.51)H, orange triangle. Kd and Ki values
are given in Supplementary Table S3. (b,c)
Plots of fold selectivity values for the two AII receptor subtypes
(IC50(AT1R)/IC50(AT2R)]) versus % of cis (b) and the value of Hammet substituent constants σpara (c) for the different AII analogues (see also Supplementary Table S5). (d) PC12W cells, either
transduced with the Ad-AT2R or untransduced, were used for the evaluation
of the AT2R agonistic effect of [Y]6-AII in the presence
of either 1 nM AII or [Y]6-AII. Agonist-induced neurite
outgrowth by AII or [Y]6-AII for 24 h stimulation was quantified
by counting neurite-positive cell numbers in five randomly selected
photos/well. The neurite outgrowth-positive cells were defined as
the cells with neurite length longer than their cell diameters. This
experiment was carried out in triplicate and repeated twice.