G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor–arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of β-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with β-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and β-arrestin mutants, we present evidence for differential receptor–β-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.

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.

Low intrinsic efficacy can explain the reduced side effects of apparently biased μ-opioid receptor agonists.

Opioid analgesics are powerful pain relievers; however, over time, pain control diminishes as analgesic tolerance develops. The molecular mechanisms initiating tolerance have remained unresolved to date. We have previously shown that desensitization of the μ-opioid receptor and interaction with β-arrestins is controlled by carboxyl-terminal phosphorylation. Here we created knockin mice with a series of serine- and threonine-to-alanine mutations that render the receptor increasingly unable to recruit β-arrestins. Desensitization is inhibited in locus coeruleus neurons of mutant mice. Opioid-induced analgesia is strongly enhanced and analgesic tolerance is greatly diminished. Surprisingly, respiratory depression, constipation, and opioid withdrawal signs are unchanged or exacerbated, indicating that β-arrestin recruitment does not contribute to the severity of opioid side effects and, hence, predicting that G-protein-biased µ-agonists are still likely to elicit severe adverse effects. In conclusion, our findings identify carboxyl-terminal multisite phosphorylation as key step that drives acute μ-opioid receptor desensitization and long-term tolerance.

We have cloned Rev-erbfi, a novel isoform of the Rev-erba orphan nuclear receptor. The DNA binding domains of Rev-erba and fl are highly related to each other and to the retinoic acid related orphan receptor (ROR)/RZR subfamily of nuclear receptors. Indeed, we find that all three receptors bind as monomers to the sequence AATGT-AGGTCA. Whereas RORal constitutively activates transcription through this sequence, both isoforms of Reverb are inactive. When coexpressed, both Reverb isoforms suppress the transcriptional activity of RORal. Our data define Rev-erb and ROR/RZR as a family of related receptors with opposing activities on overlapping regulatory networks. (Molecular Endocrinology 8: 1253-1281,1994)