Figure 5

Constitutive activity of HA-S1P ₄ and HA-S1P ₄ -Gα _i1 (C³⁵¹I). Comparison of basal, vehicle and stimulated (10 μM S1P) GTPγS binding for membranes prepared from CHO cells transfected to express HA-S1P₄ or HA-S1P₄ fused to PTx insensitive Gα_i1. Where indicated, cells were treated with 100ng/ml PTx for 24 hours prior to harvesting. PTx treatment of HA-S1P₄-transfected membranes prevented activation by S1P and also caused a dramatic reduction in basal signalling, indicative of constitutive activity. In contrast, when the HA-S1P₄-Gα_i1 fusion-expressing membranes were treated with PTx, there was only a slight reduction in basal signalling and the receptor still responded to exogenous S1P, indicating that the receptor signalled via the tethered, PTx-insensitive G protein. Basal signalling in PTx-treated HA-S1P₄-Gα_i1-expressing membranes exceeded that seen with membranes from untransfected CHO cells.