Targeted Elimination of G Proteins and Arrestins Defines Their Specific Contributions to Both Intensity and Duration of G Protein-coupled Receptor Signaling

Abstract

G protein-coupled receptors (GPCRs) can initiate intracellular signaling cascades by coupling to an array of heterotrimeric G proteins and arrestin adaptor proteins. Understanding the contribution of each of these coupling options to GPCR signaling has been hampered by a paucity of tools to selectively perturb receptor function. Here we employ CRISPR/Cas9 genome editing to eliminate selected G proteins (Gαq and Gα11) or arrestin2 and arrestin3 from HEK293 cells together with the elimination of receptor phosphorylation sites to define the relative contribution of G proteins, arrestins, and receptor phosphorylation to the signaling outcomes of the free fatty acid receptor 4 (FFA4). A lack of FFA4-mediated elevation of intracellular Ca2+ in Gαq/Gα11-null cells and agonist-mediated receptor internalization in arrestin2/3-null cells confirmed previously reported canonical signaling features of this receptor, thereby validating the genome-edited HEK293 cells. FFA4-mediated ERK1/2 activation was totally dependent on Gq/11 but intriguingly was substantially enhanced for FFA4 receptors lacking sites of regulated phosphorylation. This was not due to a simple lack of desensitization of Gq/11 signaling because the Gq/11-dependent calcium response was desensitized by both receptor phosphorylation and arrestin-dependent mechanisms, whereas a substantially enhanced ERK1/2 response was only observed for receptors lacking phosphorylation sites and not in arrestin2/3-null cells. In conclusion, we validate CRISPR/Cas9 engineered HEK293 cells lacking Gq/11 or arrestin2/3 as systems for GPCR signaling research and employ these cells to reveal a previously unappreciated interplay of signaling pathways where receptor phosphorylation can impact on ERK1/2 signaling through a mechanism that is likely independent of arrestins.

Genetic evidence that β-arrestins are dispensable for the initiation of β 2 -adrenergic receptor signaling to ERK

“…The use of β-arrestin-KO cells suggests that, at least for V2R, β-arrestins are not strictly required for ERK signaling by this class B GPCR. A recent study on the free fatty acid receptor 4 (FFA4), which couples with Gq, demonstrates that FFA4-induced ERK phosphorylation is dependent on Gq but not β-arrestins (32). Thus, dispensable role of β-arrestin in GPCR-induced ERK activation is likely to be generalizable, and is supported by several additional studies (33, 34).…”

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

“…The dye was washed out with Ringer's solution containing 250 μM EDT for 5 min, and the cells were placed in the incubator for at least 1 h before the experiments. The ERK KTR vector (pLentiCMV Puro DEST ERKKTRClover), originally made by the Covert laboratory (29), was obtained from Addgene (#59150). TTP 22 and TBB were purchased from Tocris, and cmpd101 (GRK2/3 blocker) was from HelloBio.…”

“…For example, stable arrestin binding is considered to require prior binding of a receptor kinase and phosphorylation of the receptor. Because the biased receptor does not interact productively with G proteins, both the receptor kinase-binding steps and the arrestin-binding steps could be facilitated by an absence of competition from G proteins (29).…”

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation

“…Performed essentially as described in (29) with a difference in the alkaline phosphatase substrate and the use of Gq/11 CRISPR-knockout cells (44). We used the fluorescent substrate, 4-Methylumbelliferyl phosphate (4-MUP), instead of p-Nitrophenyl Phosphate (p-NPP).…”

“…For the TGFa shedding assay, HEK293 cells that lack Gq/11, which were previously modified via CRISPR/Cas9 and characterized (44) in order to reduce non-specific shedding activity.…”