β‐arrestins mediate rapid 5‐HT2A receptor endocytosis to regulate intensity and duration of signaling
Authors: Felsing Daniel E , Nilson Ashley , Jain Manish , Raval Sweta , Inoue Asuka , Allen John 
Published: June 2021

Serotonin 5‐HT2A receptors (5‐HT2AR) regulate important physiological processes including platelet aggregation, smooth muscle contraction, mood and perception. 5‐HT2AR are the site of action for most hallucinogens and are principle molecular targets for atypical antipsychotic drugs, as well as targets of antidepressants and anxiolytics. The 5‐HT2AR canonically activates heterotrimeric Gaq G‐proteins which activate phospholipase C (PLC) and formation of inositol phosphates to stimulate intracellular release of Ca2+. The 5‐HT2AR also interact with β‐arrestin proteins; however, the importance of β‐arrestins for controlling the efficacy and duration of 5‐HT2AR signaling remains largely undefined. Here we delineate contributions of β‐arrestin isoforms in 5‐HT2AR signaling and trafficking using CRISPR/Cas9 genome editing to stably knockout (KO) β‐arrestins. We first examined if agonist activation of 5‐HT2AR receptors caused plasma membrane recruitment of β‐arrestin using confocal imaging. Agonist activation of HA‐5‐HT2AR with 10 uM 5‐HT, or the selective agonist DOI, induced robust and rapid (within 30 sec) translocation of β‐arrestin2‐GFP from cytoplasm to the plasma membrane, where it strongly colocalized with HA‐5‐HT2AR. Live cell confocal imaging of HA‐5‐HT2AR determined rapid endocytosis within 3 mins of agonist stimulation. To determine if β‐arrestins control this rapid receptor endocytosis to impact signaling, we studied HEK293 cells lacking β‐arrestins. Western blots confirmed a complete loss of the β‐arrestin 1 and 2 in KO cells vs parent cells. Using a receptor cell surface ELISA assay, we confirmed 5 min agonist treatments resulted in rapid (~35%) loss of receptors from the cell surface; however, endocytosis (3 min to 45 min) was significantly reduced in β‐arrestin 1/2 KO cells. By measuring kinetic live‐cell Ca2+ release using the FLIPR assay and dose responses of selective 5‐HT2AR agonists, we determined prolonged duration of Ca2+ release in β‐arrestin 1/2 KO cells. The maximal 5‐HT2AR calcium signaling was significantly elevated by 45% (5‐HT) and 46 % (DOI) in KO cells vs. WT cells; however, agonist potency was unchanged. Re‐expression of β‐arrestin 1 or 2 in KO cells, reduced the elevated 5‐HT2AR Ca2+ responses to that of parent cells, indicating a specific effect of the genetic knockout. In addition, knockout of β‐arrestin1/2 increased and prolonged the duration of 5‐HT2AR‐mediated ERK phosphorylation. Taken together, this study indicates β‐arrestins rapidly interact with 5‐HT2AR receptors and profoundly limit both intensity and duration of Gaq‐mediated signal transduction. Moreover, these results indicate rapid 5‐HT2AR endocytosis during agonist activation is dependent on β‐arrestins, which likely limits receptor and agonist signaling. Using the robust CRISPR/Cas9 KO model, future studies will examine downstream signaling events to further define the role of β‐arrestins in regulating 5‐HT2AR agonist and antagonist signaling and pharmacology.Support or Funding InformationRising Star Award, UT System (JAA); UTMB institutional funding (JAA); T32 DA07287 (DEF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.