GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation

Abstract

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor-arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of β-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which β-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent β-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and β-arrestin mutants, we present evidence for differential AT1R-β-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

Differential Regulation of GPCRs—Are GRK Expression Levels the Key?

“…Thus, GRK-mediated receptor phosphorylation is crucial for the formation of "core" and "hanging" GPCR-arrestin complexes. Phosphorylation is often also hypothesized to be the starting point of arrestin complex formation, however, the precise determination of succession of these binding events is still occluded, as arrestins also have an affinity for active, yet unphosphorylated GPCRs (Gurevich and Gurevich, 2006;Haider et al, 2019;Drube et al, 2021). Differential spacing of negative charges at the receptor C-terminus has been shown to induce specific conformational changes in arrestins (Lee et al, 2016;Nuber et al, 2016;Mayer et al, 2019).…”

“…Depending on the availability of kinases in a cellular system, the same GPCR could be phosphorylated by GRK2/3 or GRK5/6 only, to induce specific functions, or by all GRK isoforms to achieve the activation of all possible β-arrestin functions. (C) Certain GPCRs have been shown to be functionally phosphorylated by GRK2/3 only, or GRK2/3/5/6 ( Drube et al, 2021 ). This might constitute another layer of coupling preference at the foundation of the “barcode” hypothesis.…”

“…Recent studies which utilize the CRISPR/Cas9 technology to achieve a partial ( Moller et al, 2020 ) or complete genetic ablation ( Drube et al, 2021 ) of GRK2, 3, 5, and/or 6 suggest that GPCR-specific GRK-coupling preferences might determine which isoforms regulate a given receptor ( Figure 2C ). Using β-arrestin recruitment as a read-out for GRK-mediated receptor regulation, two subsets of GPCRs have been identified ( Drube et al, 2021 ): receptors that are functionally phosphorylated by GRK2, 3, 5, and 6 and those for which arrestin recruitment could only be mediated by GRK2 and 3. By analysis of the β2ADR, this study shows that even though GRK2 and GRK6 preferentially phosphorylate distinct C-terminal sites ( Nobles et al, 2011 ), the individual overexpression of either kinase mediates β-arrestin recruitment to the same extent.…”

Suitability of GRK antibodies for individual detection and quantification of GRK isoforms in western blots

“…Expression pcDNA3 plasmids for human GRK2 (NP_001610.2), GRK3-1 (NP_005151), GRK5 (NP_005299.1), and GRK6-1 (NP_001004106.1), as well as the kinase dead mutants GRK2-K220R, GRK5-K215R and GRK6-1-K215R were previously described (Drube 2021).…”

Viewpoints on the First Transatlantic GPCR Symposium for Early-Career Investigators

“…Natasha’s work investigated the effect of GRKs on Gq coupling and downstream signaling. She used novel Crispr/Cas9 knockout cell lines to show that GRK2 can sequester G-protein subunits to attenuate signaling through phospholipase C. This result demonstrated important effects in cell signaling unrelated to the canonical kinase functionality ascribed to the GRK family.…”