Nanoluciferase-based complementation assay for systematic profiling of GPCR–GRK interactions

Christie B. Palmer, Giulia D'Uonnolo (Main author), Rafael Luís (Main author), Max Meyrath, Tomasz Uchański, Andy Chevigné* (Main author), Martyna Szpakowska

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

4 Citations (Scopus)


G protein-coupled receptor kinases (GRKs) are a family of seven soluble receptor-modifying enzymes which are essential regulators of GPCR activity. Following agonist-induced receptor activation and G protein dissociation, GRKs prime the receptor for desensitization through phosphorylation of its C terminus, which subsequently allows arrestins to bind and initiate the receptor internalization process. While GRKs constitute key GPCR-interacting proteins, to date, no method has been put forward to readily and systematically determine the preference of a specific GPCR towards the seven different GRKs (GRK1-7). This chapter describes a simple and standardized approach for systematic profiling of GRK1-7–GPCR interactions relying on the complementation of the split Nanoluciferase (NanoBiT). When applied to a set of GPCRs (MOR, 5-HT1A, B2AR, CXCR3, AVPR2, CGRPR), including two intrinsically β-arrestin-biased receptors (ACKR2 and ACKR3), this methodology yields highly reproducible results highlighting different GRK recruitment profiles. Using this assay, further characterization of MOR, a crucial target in the development of analgesics, reveals not only its GRK fingerprint but also related kinetics and activity of various ligands for a single GRK.

Original languageEnglish
Pages (from-to)309-321
Number of pages13
JournalMethods in Cell Biology
Publication statusPublished - Jun 2022


  • ACKR3
  • Arrestin
  • B2AR
  • GRK2
  • GRK3
  • GRK5
  • GRK6
  • Kinase
  • MOR
  • NanoBiT
  • opioids


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