CRISPR/Cas9‐mediated Knockout of G Proteins and β‐arrestins Determines Transducer Specific Contributions to Dopamine D1 Receptor Signaling
Authors: Jain Manish K. , Nilson Ashley N. , Felsing Daniel E. , Inoue Asuka , Allen John A. 
Published: June 2021

Dopamine D1 receptors (D1R), a subclass of G protein‐coupled receptors (GPCRs), regulate important neurobiological processes including movement, memory and responses to rewarding stimuli. D1Rs are also drug targets for treating motor deficits in Parkinson's disease and cognitive and motivational deficits in neuropsychiatric diseases. The D1R canonically activates heterotrimeric Gs and Golf G‐proteins which, in turn, activate adenylyl cyclase to increase cAMP/PKA signaling; however, the D1R also engages β‐arrestin proteins which may transduce an independent wave of intracellular signal transduction by scaffolding and activating kinases. The relative importance of G proteins versus 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 (KO) Gs, Golf, multiple G‐proteins (Gs, Golf, Gq, G11 and G12) and β‐arrestin1/2 in HEK293 cells. Western blots confirmed a complete loss of the various G proteins or β‐arrestin1/2 proteins in KO cells versus wildtype cells. By measuring kinetic live‐cell cAMP production using the GloSensor and dose responses of selective agonists for the D1R (A‐77636, SKF‐81297), β2AR (isoproterenol) or Gs/olf (cholera toxin), we confirmed no cAMP was generated in the Gs/olf and multiple G protein KO cells. In contrast, β‐arrestin 1/2 KO greatly elevated and prolonged the duration of cAMP production after time course treatments with agonists for the D1R or the β2AR. Using antibody feeding followed by live‐cell confocal imaging of an HA tagged D1R, or a cell surface ELISA assay, we determined agonist‐mediated HA‐D1R endocytosis was entirely blocked in β‐arrestin 1/2 KO cells, but that Gs/olf KO had no effect on endocytosis. β‐arrestin1/2 KO also showed impaired agonist‐mediated D1R desensitization. Re‐expression of β‐arrestin 1 or 2 or both isoforms together in KO cells, restored receptor endocytosis and reduced the elevated D1R cAMP responses to that of wildtype cells. Lastly, knockout of Gs/olf or β‐arrestin1/2 differentially influenced the activation level and duration of ERK phosphorylation (p‐ERK). Western blotting indicated Gs/olf knockout completely blocked agonist‐induced p‐ERK induction by both D1R and β2AR agonists over a time course of 40 minutes. However, β‐arrestin1/2 knockout greatly increased and prolonged the duration of D1R and β2AR‐mediated p‐ERK. Taken together, this study provides fundamental insights into mechanisms of D1R signal transduction suggesting β‐arrestins are essential for D1R endocytosis and they primarily limit the intensity and duration of Gs/olf‐mediated D1R signaling. Gs/olf was essential for activating both cAMP signaling and ERK phosphorylation. Using this robust CRISPR/Cas9 KO model, future studies will examine downstream signaling events using proteomics and biased agonists to further define the role of both β‐arrestins and Gs/Golf for D1R pharmacology and signaling.

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UTMB Center for Addiction Research and Dept. of Pharmacology and Toxicology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.