Biology subjects

Drube, J.

Publications and source records attributed to Drube, J..

4 recordsLinked to original sources

bioRxiv2023

GRK specificity and beta gamma dependency determines a GPCR`s potential in biased agonism

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent {beta}-arrestin recruitment. Recently, it was shown that GPCRs differentially depend on GRK2/3, GRK2/3/5/6 or GRK5/6 for their regulation. The four ubiquitously expressed GRKs are classified into the cytosolic GRK2/3 and the membrane-tethered GRK5/6 subfamily. In vitro studies revealed that GRK2/3 interact with the membrane-localized G protein {beta}{gamma}-subunits. Yet, the role of this interaction as crosslink between G protein activation and {beta}-arrestin binding to GPCRs remained strongly underappreciated. Here we systematically show that the G{beta}{gamma}-GRK2/3 interaction is key for these GRKs to mediate {beta}-arrestin2 binding to Gs-, Gi- and Gq-coupled GPCRs. In our GRK2/3/5/6 knockout cells, without endogenous GRK background, the utilized GRK2/3 mutants devoid of the G{beta}{gamma} interaction site significantly diminished {beta}-arrestin2 recruitment to the beta-2 adrenergic receptor (b2AR), muscarinic M2 and M5 acetylcholine receptors (M2R, M5R). This effect was overwritten by artificially tethering GRK2/3 via a CAAX motif to the plasma membrane independently of free G{beta}{gamma}. Hence, the membrane recruitment is crucial for GRK2/3-mediated {beta}-arrestin2 binding to GPCRs, which is naturally induced via the G{beta}{gamma} interaction. This connects the {beta}-arrestin interaction for GRK2/3-regulated receptors inseparably with the associated G protein activation. We outline a theoretical framework of how GRK dependence on free G{beta}{gamma} can determine a GPCRs potential in biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we propose that it will likely be mechanistically unattainable to create {beta}-arrestin-biased ligands for the subgroup of GRK2/3-regulated GPCRs, while GRK5/6-mediated receptor regulation is independent from G{beta}{gamma} availability. Accordingly, one should first determine the GRK specificity of a GPCR to ultimately assess the receptors potential for the development of biased ligands.

pharmacology and toxicology↗

bioRxiv2022

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation-state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonists and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

pharmacology and toxicology↗

bioRxiv2021

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

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This leads to the recruitment of arrestins resulting in desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffold. This multifunctionality is also reflected by their diverse roles in pathological conditions like cancer, influenza infection, malaria, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRK2, GRK3, GRK5, and GRK6 in western blot analysis. We thereby identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross reactivity. Thus, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained western blot results. Utilizing the most suitable antibodies we established the western blot-based, cost-effective, simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in five commonly used cell lines revealing differential isoform expression.

molecular biology↗

bioRxiv2021

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

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.

cell biology↗