Cellular G Protein-Coupled Receptor Kinase Levels Regulate Sensitivity of the α_2B-Adrenergic Receptor to Undergo Agonist-Induced Down-Regulation

Abstract

Chronic coactivation of ␣ 2B -and ␤ 2 -adrenoceptors (AR) was recently reported to down-regulate the ␣ 2B -AR at a lower threshold epinephrine (EPI) concentration compared with the activation of ␣ 2B -AR alone. This is the result of a modest ␤ 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 ␤ 2 -AR activation, decreases the EC 50 concentration for agonist-induced down-regulation of the ␣ 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 downregulation of the ␣ 2B -AR is 30 M. A 2-to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC 50 to 0.2 M (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 M (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8-to 10-fold, the EC 50 concentration (0.02 M EPI) for ␣ 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 ␣ 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 ␣ 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 ␣ 2B -AR and thus adds a new dimension to an already intricate signaling network.

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α2B-Adrenoceptor

“…The human ␣ 2A -AR mutant (Del 293-304) that lacks the four serine residues in the third intracellular loop thought to be phosphorylated by GRKs does not acutely desensitize but does undergo down-regulation (Jewell-Motz et al, 1997). In contrast to these observations, recent studies in BE(2)-C and BN17 cells and in GRK3-and GRK2-overexpressing NG108 cells suggest that the ␣ 2A -and ␣ 2B -AR are rendered more sensitive to agonist-induced down-regulation because of a 2-to 3-fold up-regulation of GRK3 (Bawa et al, 2003;Desai et al, 2004Desai et al, , 2005. In addition, mutation of potential GRK phosphorylation sites ( 322 SSTS 325 changed to AAVA) in the third intracellular loop of opossum ␣ 2C -AR prevents receptor down-regulation after 24-h exposure to 0.3 M NE (Deupree et al, 2002).…”

“…One explanation for these conflicting reports could be the difference in level of receptor expression between the different studies. In studies supporting the role of GRKs in down-regulation, the receptor was expressed at levels Յ300 fmol/mg protein (Deupree et al, 2002;Desai et al, 2004Desai et al, , 2005, whereas the negative results were observed in systems in which receptor expression was Ն700 fmol/mg protein (Eason et al, 1994;Jewell-Motz and Liggett, 1995;Jewell-Motz et al, 1997). Another contributing factor could be the low levels of expression of GRKs in CHO and COS cells commonly used for these studies (Menard et al, 1997).…”

“…When overexpressed in cells, it acts to sequester the G ␤␥ subunits released on receptor activation, thereby preventing the recruitment of GRK3 and GRK2 . We chose to jpet.aspetjournals.org transfect NG108 cells with GRK3ct because our previous study showed that the ␣ 2B -AR is more sensitive to modulation by GRK3 than by GRK2 (Desai et al, 2004(Desai et al, , 2005. There are a number of other inhibitors of GRKs such as heparin and Zn ϩ , but the nonspecificity of these agents is a limiting factor in their use.…”

“…We have previously shown an increase in sensitivity of the ␣ 2A -or ␣ 2B -AR to undergo agonist-induced down-regulation after an increase in GRK levels (Bawa et al, 2003;Desai et al, 2004Desai et al, , 2005. In BN17 cells, coactivation of ␣ 2B -and ␤ 2 -AR results in a modest selective up-regulation of GRK3 and increases the sensitivity of the ␣ 2B -AR to undergo agonist-induced down-regulation (Desai et al, 2004).…”

“…In BN17 cells, coactivation of ␣ 2B -and ␤ 2 -AR results in a modest selective up-regulation of GRK3 and increases the sensitivity of the ␣ 2B -AR to undergo agonist-induced down-regulation (Desai et al, 2004). Likewise, in NG108 cells, modest overexpression of GRK3 and GRK2 differentially modulates the sensitivity of ␣ 2B -AR to undergo agonist-induced down-regulation (Desai et al, 2005). However, an absolute requirement of GRK-mediated phosphorylation in the down-regulation of GPCRs in general, and ␣ 2B -AR in particular, is controversial.…”