Simultaneous α_2B- and β₂-Adrenoceptor Activation Sensitizes the α_2B-Adrenoceptor for Agonist-Induced Down-Regulation

Abstract

We recently reported that ␣ 2A -adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 M) in BE(2)-C cells that express both ␣ 2 -and ␤ 2 -ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by ␤ 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 ␣ 2A -versus ␣ 2B -ARs have been reported, the present study examines the effects of simultaneous activation of ␣ 2B -and ␤ 2 -ARs on ␣ 2B -AR number and signaling. We studied NG108 cells that naturally express ␣ 2B -ARs, and BN17 cells, NG108 cells transfected to express the human ␤ 2 -AR. In NG108 cells, ␣ 2B -AR desensitization and down-regulation require treatment with 20 M EPI or NE; GRK expression was not changed. In BN17 cells expressing ␤ 2 -ARs, the threshold EPI concentration for ␣ 2B -AR desensitization and down-regulation was reduced to 0.3 M; 10 M 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 ␤-AR antagonist alprenolol (1 M) and GRK3 antisense (but not sense) DNA blocked 0.3 M EPI-and 10 M NE-induced desensitization and down-regulation of the ␣ 2B -AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of ␣ 2B -and ␤ 2 -ARs results in a 67-fold decrease in the threshold concentration of EPI required for ␣ 2B -AR down-regulation. This lower threshold for down-regulation is associated with ␣ 2B -and ␤ 2 -AR dependent up-regulation of GRK3 expression.Over the last decade, considerable attention has been given to the short-and long-term regulation of ␣ 2 -adrenoceptor signaling. Primarily using transfected receptors in cell lines that do not endogenously express the ␣ 2 -adrenoceptor, Liggett and coworkers have conducted a series of experiments examining the short-term and long-term desensitization of the three ␣ 2 -adrenoceptor subtypes (␣ 2A/D,2B and 2C ) (Liggett, 1998). Through these studies, structural components of the ␣ 2 -adrenoceptor have been identified as sites for GRK phosphorylation that are important for the short-term desensitization of these receptors. For example, four serines in the third intracellular loop are reported to be critical for shortterm desensitization of the ␣ 2A -adrenoceptor (Eason et al., 1995). However, there are structural differences within the ␣ 2 -adrenoceptor family that may lead to heterogeneity in the regulation of the three ␣ 2 -subtypes (Eason and Liggett, 1992). For example, significantly higher agonist concentrations are reportedly required for desensitization of ␣ 2A compared with ␣ 2B -adrenoceptors (Heck and Bylund, 1997). Moreover, studies have suggested that phosphorylation sites critical to short-term ␣ 2 -adrenoceptor desensitization are not important for long-term desensitization and down-regulation (Jewell-Motz and Liggett, 19...

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

“…1B). There is no change in the cellular levels of either GRK2 or GRK3 after pretreatment of NG108 cells with 20 M EPI (Desai et al, 2004). The ␣ 2A -AR undergoes desensitization in response to protein kinase C (PKC) activation (Liang et al, 1998).…”

“…In NG108 cells, 20 M EPI pretreatment down-regulates the ␣ 2B -AR with no change in the cellular levels of GRK2 or GRK3 (Desai et al, 2004). Both GRK3 and GRK2 are primarily cytosolic proteins and have to be translocated to the membrane to be able to phosphorylate their receptor substrates.…”

“…After EPI pretreatment, media containing the drugs were aspirated, and the cells were harvested by pipetting fresh drug-free medium against the cells. Intact cells were harvested and assayed for cAMP accumulation as described previously (Desai et al, 2004). Briefly, intact cells were first incubated for 10 min at 37°C in Hanks' balanced salt solution buffer.…”

“…The resultant cell lysate was diluted with 2ϫ Laemmli buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.1 mg/ml bromphenol blue) and resolved on SDS-polyacrylamide gel electrophoresis (10% gel). The resolved proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (GE Heathcare BioSciences), and the GRK2 or GRK3 and GRK3ct expression levels were determined by immunoblotting using anti-GRK2 or -GRK3, respectively (1:1000; Santa Cruz Biotechnology), in 2.5% nonfat milk as described previously (Desai et al, 2004). The blots were stripped and reprobed for GAPDH as a loading control (for cytosolic and whole cell samples) using mouse anti-rabbit GAPDH antibody (1:8000; RDI, Flanders, NJ).…”

“…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). 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).…”