CRISPR/Cas9‐mediated knockout of G proteins and β‐arrestins delineates their distinct but interdependent roles in dopamine D1 receptor signaling

The dopamine D1 receptor (D1R) is a G protein‐coupled neurotransmitter receptor which controls key brain functions including voluntary movement, memory, attention and reward. Selective agonist activation of the D1R may also provide novel neurotherapeutics for neurodegenerative and neuropsychiatric disorders. The D1R canonically activates the heterotrimeric G proteins Gα s , and possibly Gα olf , which activate adenylyl cyclase (AC) to increase cAMP/PKA signaling. The D1R also engages β‐arrestin proteins which may transduce β‐arrestin dependent signaling by scaffolding and activating kinases. However, the distinct roles of Gα s , Gα olf versus β‐arrestins 1 and 2 for D1R signaling remains largely undefined. Here we utilize CRISPR/Cas9 engineered HEK293 cells in which Gα s , Gα olf , β‐arrestin1 and β‐arrestin2 are stably knocked out (KO) to delineate D1R pharmacology and signaling mechanisms. Western blots confirmed a complete loss of Gα s , Gα olf , β‐arrestin1 or β‐arrestin2 proteins in KO cells versus parental wildtype cells. Dose responses with the D1R agonist SKF‐81297 or β2AR agonist isoproterenol induced robust cAMP production in wildtype cells that was eliminated in Gα s/olf KO cells. Re‐expression of Gα s or Gα olf in KO cells rescued D1R‐mediated cAMP signaling similar to wildtype cells. Notably, the D1R had significantly higher cAMP signaling when coupling via Gα s as compared to Gα olf , while the β2AR only coupled with Gα s to stimulate cAMP signaling. Western blotting indicated Gα s/olf KO completely blocked agonist‐induced CREB phosphorylation by both D1R and β2AR agonists, indicating a strict dependence on Gα s/olf and cAMP/PKA signaling to activate CREB. In major contrast, β‐arrestin1/2 KO significantly increased the intensity and duration of cAMP signaling and CREB phosphorylation by the β2AR. β‐arrestin1/2 KO also entirely blocked agonist‐induced D1R internalization and impaired D1R desensitization while Gα s/olf KO had no effect on D1R internalization. Key controls of re‐expressing Gα s , Gα olf, β‐arrestin1 or β‐arrestin2 in KO cells completely restored signaling or internalization similar to wildtype cells, indicating CRISPR/Cas9 targeting did not result in non‐specific or compensatory effects in the pathways under study. Notably, β‐arrestin1/2 KO also potentiated and prolonged D1R or β2AR‐induced ERK1/2 phosphorylation. D1R‐induced ERK1/2 phosphorylation in β‐arrestin1/2 KO cells was eliminated by the SRC inhibitor SU6656 or the MEK inhibitor U0126, suggesting that D1R signaling via a Gα s /SRC/MEK pathway is essential for ERK1/2 activation. Taken together, our studies indicate that Gα s is the dominant transducer for D1R cAMP, CREB and ERK1/2 signaling and that β‐arrestins provide distinctly interdependent roles with G proteins in modulating the duration and intensity of D1R and β2AR signaling cascades. Using CRISPR/Cas9 KO model cells, future studies will examine the distinct contributions of β‐arrestins and Gα s/olf for D1R pharmacology, which will be useful in defining and validating novel D1R biased agonists. Support or Funding Information NIH DA047643 (JAA); Rising STAR award from the University of Texas System (JAA); Institutional funding from UTMB (JAA)