PT  - JOURNAL ARTICLE
AU  - J. Drube
AU  - R.S. Haider
AU  - E.S.F. Matthees
AU  - M. Reichel
AU  - J. Zeiner
AU  - S. Fritzwanker
AU  - C. Ziegler
AU  - S. Barz
AU  - L. Klement
AU  - A. Kliewer
AU  - E. Miess
AU  - E. Kostenis
AU  - S. Schulz
AU  - C. Hoffmann
TI  - GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation
AID  - 10.1101/2021.02.12.430971
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.12.430971
4099  - http://biorxiv.org/content/early/2021/02/13/2021.02.12.430971.short
4100  - http://biorxiv.org/content/early/2021/02/13/2021.02.12.430971.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.AChacetylcholineAngIIAngiotensin IIANOVAanalysis of varianceAT1RAngiotensin 1 receptorAUarbitrary unitsAUCarea under the curveAVP[Arg8]-vasopressinβarr1β-arrestin1βarr2β-arrestin2β2ADRβ2 adrenergic receptorβ2V2β2ADR with an exchanged C-terminus of the vasopressin type 2 receptorBRETbioluminescence resonance engergy transferC5aR1complement 5a receptor 1CFPcyan fluorescent proteincmpd101GRK2/3 inhibitorDAMGO[D-Ala2, N-MePhe4, Gly-ol]-enkephalinΔGRK2GRK2 knockout in HEK293ΔGRK2/3GRK2 and 3 double knockout in HEK293ΔGRK2/3/5GRK2, 3 and 5 triple knockout in HEK293ΔGRK2/3/6GRK2, 3 and 6 triple knockout in HEK293ΔGRK2/5/6GRK2, 5 and 6 triple knockout in HEK293ΔGRK3GRK3 knockout in HEK293ΔGRK3/5/6GRK3, 5 and 6 triple knockout in HEK293ΔGRK5GRK5 knockout in HEK293ΔGRK5/6GRK5 and 6 double knockout in HEK293ΔGRK6GRK6 knockout in HEK293ΔQ-GRKGRK2, 3, 5, 6 quadruple knockout in HEK293dFLRdeleted finger loop regionDMEMDulbecco’s modified eagle mediumDTTDithiothreitolEDT1,2-ethane dithiolELISAenzyme-linked immunosorbent assayEVempty vectorFCSfetal calf serumFlAsHfluorescine arsenical hairpin-binderFLRfinger loop regionGö6983PKC inhibitorGPCRG protein coupled receptorGRKGPCR kinaseHAhaemagglutininHEKhuman embrionic kidney cellIsoisoproterenolKDkinase deadKOknockoutM1R, M2R, M3R, M4R, M5Rhuman muscarinic 1,2,3,4, and 5 acetylcholine receptorsMOPμ-opioid receptorNanoLucNano luciferaseORFopen reading framePBSphosphate buffered salinePKCprotein kinase CPMAPhorbol 12-myristate 13-acetatePTH(1-34)shortened parathyroid-hormonePTH1Rparathyroid hormone 1 receptorRFUrelative fluorescence unitsSDstandard deviationSDSSodium dodecyl sulfateSEMstandard error of the meanSquasshsegmentation and quantification of subcellular shapeV2Rvasopressin type 2 receptorYFPyellow fluorescent protein