Molecular Mechanisms Of D₂R G Protein Or β-Arrestin-Dependent Pathway Stimulated ERKs Activation

Dopamine D2 receptor（D₂R）have been shown to activate extracellular signal-regulated kinases（ERKs）via distinct pathways dependent on either G-proteins orβ-arrestins.However,there has not been a systematic study of the regulatory process of D₂R-mediated ERKs activation by G protein versusβ-arrestin-dependent signaling since D₂R stimulation of ERKs reflects the simultaneous action of both pathways.Therefore,this paper intends to use a variety of molecular biological experimental means to further explore the molecular mechanism of D₂R inducing ERKs activation through G protein andβ-arrestin bias signaling pathway.In this paper,we first used D₂R with innate biased signal transduction ability,namely^[Gprot]D₂R（innate biased G protein pathway）and^[βarr]D₂R（innate biasedβ-arrestin pathway）,respectively,to explore the molecular mechanism of D₂R inducing ERKs activation through G protein andβ-arrestin signaling pathway.Secondly,wild-type D₂R was used as the research model,and biased ligand MLS1547 of specific excitatory G protein signaling pathway and biased ligand UNC9994 of specific excitatoryβ-arrestin signaling pathway were used to further verify the experimental conclusions.Our results showed that G protein-dependent ERKs activation was rapid,reaching maximal level around 2 min,was quite transient,and importantly the activated ERKs were entirely confined to the cytoplasm.In contrast,β-arrestin-dependent ERKs activation was slower,reaching maximal level around 10 min,but more sustained,and phosphorylated ERKs translocated into the nucleus.Src was commonly involved in the G protein andβ-arrestin-dependent pathways mediated ERKs activation.Secondly,Gαi2,Gβγ,EGFR,PKCβI/βII and PI3K all blocked G protein-dependent ERKs activations.In contrast,GRK2 andβ-arrestin2 played a main role inβ-arrestin-dependent ERKs activation.Endocytosis of the receptor had little effect on ERKs activation which mediated by two pathways.Furthermore,the formation of a complex composed of p-ERKs,β-arrestin2 and Importinβ1 promoted the nuclear translocation of activated ERKs.The differential regulation of different cellular components,temporal and spatial patterns of ERKs activation via these two pathways implies the existence of distinct physiological outcomes.