Figure 7. 5-HT_1B-mediated phosphorylation of ERK1/2 is dependent on β-arrestins in N2A-1B cells.

N2A-1B control cells, β-arrestin 1 KO cells (β-Arr1 KO), and β-arrestin 2 KO cells (β-Arr2 KO) received treatment with the 5-HT_1B agonist CP-94253 (100 nM) alone or pretreatment with the 5-HT_1B antagonist SB-224289 (1 μM) 1 hour prior to agonist treatment and compared to unstimulated control cells treated with vehicle (PBS). (a) Levels of phospho-ERK1 significantly differed by treatment (F_2,36 = 21.4, p < 0.0001) and cell type (F_2,36 = 4.79, p = 0.014), with a significant interaction between treatment and cell type (F_4,36 = 7.017, p < 0.001). 5-HT_1B receptor stimulation with agonist significantly increased levels of phospho-ERK1 in control cells (p < 0.0001) but not in β-Arr1 KO (p = 0.918) or β-Arr2 KO cells (p = 0.997). (b) Levels of phospho-ERK2 also significantly differed by treatment (F_2,36 = 19.3, p < 0.0001) and cell type (F_2,36 = 4.362, p = 0.020), with a significant interaction between treatment and cell type (F_4,36 = 4.469, p = 0.049). 5-HT_1B receptor stimulation significantly increased levels of phospho-ERK2 in control cells (p = 0.0003) but not in β-Arr1 KO (p = 0.917) or β-Arr2 KO cells (p = 0.680). Furthermore, SB-224289 blocks agonist-induced ERK1/2 activation, reducing phospho-ERK1 levels in control (p = 0.045) and β-Arr2 KO cells (p = 0.043) (a) and reducing phospho-ERK2 levels in control (p = 0.048) and β-Arr1 KO cells (p = 0.014) (b). Data are expressed as the percent change in pERK signal compared to the no agonist control from each independent biological replicate. Error bars represent SEM and data are averages of 5 independent biological replicates for all groups (two-way ANOVA with Dunnett’s post hoc tests; ***p < 0.001, *p < 0.05).