Figure 2 - Compound 2a is an antagonist for proximal and downstream signals from hS1P₁ receptor activation.

(a) EC₅₀ shift by 2a and 2b of 3 activation of hS1P₁ receptors. Curves show GTP-S binding as a percentage of maximal responses induced by 3 for 3 alone (closed squares) and in the presence of S1P antagonists 2a (1 M; inverted triangles) and 2b (10 M; circles). EC₅₀ values were 50 nM for 3 alone and 10 M and 600 nM, respectively, in the presence of molar excess 2a and 2b. (b) K_i values for 3 activation of hS1P₁ receptors derived from antagonist competition curves for 2a (inverted triangles) and 2b (circles) were, respectively, 18 nM and 2.89 M. (c) We serum starved CHO cells stably transfected with S1P₁ for 16 h, then we incubated them with 2a at 10 M for 1 h and stimulated them with either 1 or 3 at the indicated concentrations for 5 min. We performed western blot analyses of phospho-Akt, total Akt, phospho-ERK1/2 and total ERK1/2 levels as described in Methods (n 3). Data are representative autoradiograms of one of three independent experiments. 2a is a competitive inhibitor of Akt and ERK phosphorylation in response to S1P₁ activation by either 1 or 3.