Inverse Agonist-induced Up-regulation of the Human Beta2-adrenoceptor in Transfected Neuroblastoma X Glioma Hybrid Cells

Neuroblastoma X glioma hybrid NG108-15 cells were transfected to express stably either the wild-type human beta2-adrenoceptor or a constitutively active mutant (CAM) version of this receptor. Basal adenylyl cyclase activity in cells expressing the CAM beta2-adrenoceptor correlated well with the level of expression of the receptor and was substantially greater than that in cells expressing the wild-type beta2-adrenoceptor. The CAM beta2-adrenoceptor displayed higher affinity for the agonist isoprenaline than the wild-type receptor but not for the antagonist alprenolol or the inverse agonist betaxolol. Pretreatment of cells harboring the CAM beta2-adrenoceptor with betaxolol resulted in a large (4-7-fold within 24 hr) up-regulation in levels of this receptor. This was not observed after exposure of the CAM beta2-adrenoceptor-expressing cells to alprenolol, and a much smaller effect of betaxolol was produced in cells expressing the wild-type receptor. Betaxolol-mediated up-regulation of the CAM beta2-adrenoceptor was both time and concentration dependent. However, this up-regulation did not result in a substantial alteration in the cellular distribution profile of the receptor. Half-maximal up-regulation of the CAM beta2-adrenoceptor required concentrations of betaxolol similar to those needed to cause half-maximal inhibition of basal adenylyl cyclase activity, indicating the receptor up-regulation is associated with the inverse agonist properties of this compound. Despite the large up-regulation of CAM beta2-adrenoceptor levels, treatment with betaxolol did not significantly alter levels of the G protein that couples to this receptor (G(Salpha)). After sustained treatment with betaxolol, Northern analyses did not demonstrate up-regulation of either CAM beta2-adrenoceptor or G(Salpha) mRNA, and up-regulation of the receptor was prevented by cotreatment of the cells with cycloheximide. These data indicate that the up-regulation of the receptor by betaxolol is likely to reflect an increase in translational efficiency of existing mRNA and/or stabilization of the receptor polypeptide from proteolytic degradation and indicate that such effects can be produced by inverse agonists but not by neutral antagonists.