Involvement of G protein-coupled receptor kinase (GRK) 3 and GRK2 in down-regulation of the alpha2B-adrenoceptor.

Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the alpha2B-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 alpha2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous alpha2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the alpha2B-AR. Pretreatment of NG108 cells with 20 microM epinephrine (EPI) begins down-regulating the alpha2B-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 alpha2B-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 alpha2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gbetagamma 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 alpha2B-AR caused by 24-h pretreatment with 20 microM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the alpha2B-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 alpha2-AR.

Two new behavioral QTLs, Emo4 and Reb1, map to mouse Chromosome 1: Congenic strains and candidate gene identification studies.

By use of newly developed subcongenic strains of mice from a parental B6.129-Il10-/- knockout/congenic strain, we have narrowed the critical region for a new behavioral QTL, called Emo4, for open-field activity to a segment of Chromosome 1 between Erbb4 (68.4Mb) and B3gnt7 (86.2 Mb). We have also uncovered an additional QTL governing repetitive beam breaks in the open field. This QTL, called Reb1, maps to the interval between Asb1 (91.4 Mb) and NM_172851 (100.0 Mb) and is one of the first QTLs mapped for this type of behavior. Genome-wide microarray expression analyses were then undertaken to help to identify candidate genes that may be the cause of these genetic differences in open-field performance. In this effort, we analyzed global gene expression differences in the amygdalae by use of Affymetrix GeneChips between B6, B6.129-Il10-/-, and B6.129R4. Several probe sets representing target Chr 1 genes were found that showed significantly differential expression in the subcongenic and congenic strains. Several candidate genes have been identified. One of these regions coincides with an homologous region in humans that has been associated with autism, a disease whose symptoms include repetitive actions. This study illustrates that the use of congenic strains combined with global gene expression analyses can produce a list of viable candidates. It further shows that caution should be observed when analyzing the effects of knockout/congenic strains because many of the gene expression differences in these comparisons could not be attributable to the ablated Il10 gene but rather to passenger gene effects.

Cellular G protein-coupled receptor kinase levels regulate sensitivity of the {alpha}2b-adrenergic receptor to undergo agonist-induced down-regulation.

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

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.