Multiple roles of beta-Arrestin2 in initiating and sustaining D3R-mediated ERK1/2 phosphorylation by G protein-biased and unbiased D3R agonists

β-arrestins play key roles in both terminating G protein signaling and activating G protein-independent signaling across many G protein coupled receptors (GPCRs); however, the roles of β-arrestins in dopamine D3 receptor (D3R)-mediated ERK phosphorylation/signaling are poorly characterized. Our previous studies showed that novel compounds SK609 and its analog SK608 are selective D3R agonists and display G biased signaling without significant recruitment of β-arrestin2 (βarr2) compared with dopamine which can potently activate G proteins and recruit β-arrestins. We hypothesize that βarr2 plays multiple roles in initiating and sustaining D3R-mediated ERK1/2 phosphorylation by G protein-biased and unbiased D3R agonists. We tested this hypothesis using neuron-like SH-SY5Y cells stably expressing D3R (SH-SY5Y-D3R cells) with βarr2 knockdown (βarr2KD) in vitro. Our results show: SK609 and SK608 or pramipexole (PRX) time-dependently elicited both the early and late phases of mono phosphorylation of ERK1/2; SK608 or SK609 only elicited the early phase of dual phosphorylation, whereas PRX induced both the early and late phases and sustained for 1-4 h; βarr2KD partially reduced mono ERK1/2 phosphorylation by PRX but not SK608 or SK609; βarr2KD significantly enhanced both the early and late phases of dual ERK1/2 phosphorylation by SK608 or SK609 whereas it partially reduced the early phase by PRX and completely abolished the late phase; Gi/o inhibitor PTX abolished PRX- or SK608 and SK609-induced mono ERK1/2 phosphorylation and SK608- and SK609-induced the early phase of dual ERK1/2 phosphorylation, whereas the early phase of dual phosphorylation by PRX was abolished but the late phase was not affected; Gbg inhibitor Galllein had no effect on PRX- induced dual phosphorylation of ERK1/2 but inhibited SK609- or SK608-indued dual phosphorylation; PKC inhibitor Go6983 greatly reduced or completely abolished PRX- or SK608- and SK609-induced the mono phosphorylation, and blocked SK608- or SK609-induced the dual phosphorylation in SH-SY5Y-D3R cells. In contrast, Go6983 abolished PRX-induced late phase of dual phosphorylation, it only partially reduced PRX-induced early phase; PTX plus Go6983 completely abolished PRX- or SK608- and SK609-induced mono or dual ERK1/2 phosphorylation in SH-SY5Y-D3R/βarr2KD cells. In summary, βarr2 not only participates in inhibiting G protein-biased D3R agonist-induced early and late phases of dual ERK1/2 phosphorylation and activating unbiased D3R agonists-induced the early and late phases of the dual phosphorylation, but also is required for G - or PKCs-dependent the early phase of the mono and dual ERK1/2 phosphorylation. These findings support multiple roles in which βarr2 acts as both signal terminator for G protein biased agonist-induced ERK1/2 signaling and signal transducer for unbiased D3R agonist-induced signaling interactions among βarr2 and G and PKCs which collaboratively regulate ERK1/2 signaling.