Abstract

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 Gi/o 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 Gi/o‐ 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 Gi/o and PKCs which collaboratively regulate ERK1/2 signaling.

Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call