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CRISPR/Cas9-mediated knockout of G proteins and ß-arrestins define pathway specific contributions to

Ashley Nilson

University of Texas Medical Branch


Dopamine D1 receptors (D1R) are G protein-coupled receptors that regulate important neurobiological processes including movement, memory and responses to rewarding stimuli. The D1R is also a drug target for treating motor deficits in Parkinson’s disease and cognitive and motivational deficits in neuropsychiatric diseases. The D1R canonically activates heterotrimeric Gαs and Gαolf G proteins that, in turn, activate adenylyl cyclase to increase cAMP/PKA signaling; however, the D1R also engages β-arrestin proteins that may transduce an independent wave of intracellular signal transduction. The relative importance of G proteins versus β-arrestins for controlling the efficacy and duration of D1R signaling remains largely undefined. Here, we delineate transducer-specific contributions involved in D1R signaling using CRISPR/Cas9 genome editing to stably knockout Gαs and Gαolf G proteins (Gαs/olf KO) or β-arrestin1/2 (Arr1/2 KO) in HEK293 cells. Western blots confirmed an absence of Gαs/olf proteins or β-arrestin1/2 proteins in knockout cells versus parent cells. By measuring live-cell cAMP production using the GloSensor and dose responses of selective agonists for the D1R (SKF-81297), endogenous β2AR (isoproterenol), Gαs/olf (cholera toxin) or adenylyl cyclase (forskolin), we confirmed no cAMP was generated in the Gαs/olf KO cells. Re-expression of Gαs or Gαolf demonstrated receptor-specific differences in coupling to the G proteins to activate cAMP signaling. The D1R had significantly higher cAMP efficacy when coupling to Gαs compared to Gαolf. In contrast, β2AR-mediated cAMP signaling occurred via Gαs but not Gαolf. Using antibody feeding followed by confocal imaging of an HA-tagged D1R or a cell surface ELISA assay, we determined agonist-mediated HA-D1R endocytosis was blocked in Arr1/2 KO cells, but that Gαs/olf KO had no effect on endocytosis. Re-expression of β-arrestin1 or 2 in Arr1/2 KO cells restored receptor endocytosis. In addition, Arr1/2 KO cells showed impaired agonist-mediated D1R cAMP desensitization. Taken together, this study provides fundamental insights into mechanisms of D1R signal transduction suggesting β-arrestins are essential for D1R endocytosis and desensitization and that Gαs coupling primarily activates cAMP signaling. Using this robust CRISPR/Cas9 KO model, future studies will examine downstream signaling events using proteomics and biased agonists to define the role of both β-arrestins and Gαs/olf for D1R pharmacology and signaling.

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