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

Drube, J.; Haider, R. S.; Matthees, E. S. F.; Reichel, M.; Zeiner, J.; Fritzwanker, S.; Ziegler, C.; Barz, S.; Klement, L.; Kliewer, A.; Miess, E.; Kostenis, E.; Schulz, S.; Hoffmann, C.

doi:10.1101/2021.02.12.430971

bioRxiv · 10.1101/2021.02.12.430971

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

Drube, J.·Haider, R. S.·Matthees, E. S. F.·Reichel, M.·Zeiner, J.·Fritzwanker, S.·Ziegler, C.·Barz, S.·Klement, L.·Kliewer, A.·Miess, E.·Kostenis, E.·Schulz, S.·Hoffmann, C.

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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 {beta}-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which {beta}-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 {beta}-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and {beta}-arrestin mutants, we present evidence for differential AT1R-{beta}-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 {beta}-arrestin complex formation.

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Drube, J., Haider, R. S., Matthees, E. S. F., Reichel, M., Zeiner, J., Fritzwanker, S., Ziegler, C., Barz, S., Klement, L., Kliewer, A., Miess, E., Kostenis, E., Schulz, S., Hoffmann, C.. 2021-02-13. GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation. https://doi.org/10.1101/2021.02.12.430971

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