Cellular G Protein-Coupled Receptor Kinase Levels Regulate Sensitivity of the α2B-Adrenergic Receptor to Undergo Agonist-Induced Down-Regulation

Chronic coactivation of α 2B - and β 2 -adrenoceptors (AR) was recently reported to down-regulate the α 2B -AR at a lower threshold epinephrine (EPI) concentration compared with the activation of α 2B -AR alone. This is the result of a modest β 2 -AR-dependent up-regulation of G protein-coupled receptor kinase 3 (GRK3). In the present study, we determined that increasing GRK2 or GRK3 levels, independent of β 2 -AR activation, decreases the EC 50 concentration for agonist-induced down-regulation of the α 2B -AR using NG108 cells with or without overexpression (2- to 10-fold) of GRK2 or GRK3. In parental NG108 cells, the EC 50 concentration of EPI required for down-regulation of the α 2B -AR is 30 μM. A 2- to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC 50 to 0.2 μM (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 μM (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8- to 10-fold, the EC 50 concentration (0.02 μM EPI) for α 2B -AR down-regulation is reduced 1000-fold. These data clearly suggest that a modest (2- to 3-fold) up-regulation of GRK3 is more effective at enhancing the sensitivity of α 2B -AR to down-regulation after exposure to EPI than a modest up-regulation of GRK2, but that both GRK2 and GRK3 are equally effective at inducing α 2B -AR down-regulation when up-regulated 8- to 10-fold. To our knowledge, this is the first report to systematically demonstrate that GRKs, particularly GRK3, play a pivotal role in modulating the agonist EC 50 concentration that down-regulates the α 2B -AR and thus adds a new dimension to an already intricate signaling network.