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

Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the α2B-adrenoceptor (AR) more sensitive to agonist-induced down-regulation ( J Pharmacol Exp Ther 312: 767–773, 2005). However, an absolute requirement of GRK3 and GRK2 for α2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous α2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the α2B-AR. Pretreatment of NG108 cells with 20 μM epinephrine (EPI) begins down-regulating the α2B-AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in α2B-AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in α2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gβγ subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the α2B-AR caused by 24-h pretreatment with 20 μM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the α2B-AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonist-induced down-regulation of the α2-AR.