Biology subjects

Drube, J.

Publications and source records attributed to Drube, J..

11 recordsLinked to original sources

bioRxiv2026

Chemokine and opioid peptide scavenging through constitutive and ligand-induced release of ACKR3-bearing extracellular vesicles

Atypical chemokine receptors (ACKRs) are non-signaling GPCRs that regulate ligand availability, with ACKR3 functioning as a dual scavenger of chemokines and opioid peptides. Here, we demonstrate that following ligand stimulation, besides the canonical internalization, ACKR3 is released on extracellular vesicles (EVs). ACKR3 was also found on EVs released under basal conditions, although to a lesser extent. These observations were confirmed across multiple cellular contexts, including endogenous systems. Mechanistically, basal and ligand-induced EV release are independent of GRKs and {beta}-arrestin but each relies on distinct trafficking routes and C-terminal determinants. Ligand-induced EV release is associated with plasma membrane localization and receptor recycling pathways. In contrast, basal EV release is governed by intracellular sorting processes and influenced by receptor ubiquitination and RAMP3. Functionally, EV-associated ACKR3 retains high-affinity ligand binding, enabling sequestration of CXCL12 and opioid peptides and thereby attenuating their signaling through CXCR4 and MOR. We also show that the release on EVs, in particular under basal conditions, is observed for other receptors such as KOR, CXCR4 and several ACKRs. Collectively, these findings establish EVs as regulators in chemokine and opioid systems and as a previously underappreciated dimension of ACKR3 and more broadly GPCR biology.

Cell Biology↗

bioRxiv2025

Cytoplasmic tail composition modulates the G protein and arrestin-3 signaling bias of the adhesion GPCR LPHN2

The class B2 Adhesion GPCRs (aGPCRs) combine cell adhesion with GPCR signaling to control diverse developmental and physiological processes. How aGPCRs interact with and integrate distinct groups of effectors including G proteins, arrestins, and G protein receptor kinases (GRKs) remains unclear. Here, we find that diversity in the aGPCR C-terminal intracellular tail modulates G protein activation, arrestin-3 recruitment, and utilization of selective GRKs in LPHN2, a postsynaptic aGPCR essential for synapse formation. The C-terminal tail of LPHN2 is required for G protein activation and arrestin-3 recruitment. LPHN2 with an intact tail recruits arrestin-3 in the absence of G protein activation, suggesting constitutive arrestin-3-biased signaling. Alternative splicing of the LPHN2 tail modulates G protein activation and arrestin-3 binding independently, supporting that it controls G protein vs arrestin-3 bias. GRKs are important but not essential for arrestin-3 recruitment to LPHN2. Moreover, GRK2 increases arrestin-3 recruitment only in a subset of LPHN2 variants. Collectively, these results show that the mechanisms of the interactions of class B2 aGPCRs and arrestin are distinct from those of class A GPCRs and that splicing of the LPHN2 C-terminal tail determines G protein vs arrestin bias.

molecular biology↗

bioRxiv2025

Non-canonical internalization mechanisms of mGlu receptors

Cell surface density of G protein-coupled receptors (GPCRs) is tightly regulated through constitutive and agonist-induced internalization. Whereas the mechanisms of constitutive internalization remain elusive, agonist-induced internalization is accepted to involve receptor phosphorylation by GPCR kinases (GRKs), {beta}-arrestin binding and AP2 recruitment, targeting receptors to clathrin-coated pits. Dimeric class C metabotropic glutamate (mGlu1 to 8) receptors regulate synaptic transmission but their internalization process is ambiguous. Here, we used diffusion-enhanced energy transfer (DERET) to decipher their internalization kinetics. We showed that all mGlu receptors are constitutively internalized. However, only mGlu1, 5 and 3 homodimers are agonist-induced internalized, that require neither GRKs, nor {beta}-arrestins. In contrast, the constitutive internalization involves only {beta}-arrestins. This systematic study further illustrates how different class C receptors are relative to most other GPCRs, revealing non-canonical internalization mechanisms. These insights in mGlu receptor dynamics will help promoting the therapeutic action of drugs targeting mGlu receptors.

neuroscience↗

bioRxiv2024

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

biochemistry↗

bioRxiv2024

Relating GPCR domains with functionality: receptor helix-bundle and C-terminus differentially influence GRK-specific functions and β-arrestin-mediated regulation

G protein-coupled receptors (GPCRs) orchestrate diverse physiological responses via intracellular signaling through G proteins, GPCR kinases (GRKs), and arrestins. While the role of G proteins in receptor signaling is well-established, the contributions of GRKs and arrestins remain incompletely understood. Here, we investigate the influence of arrestin-interacting GPCR domains (helix-bundle/C-terminus) on {beta}-arrestin conformations and functions using refined biosensors and advanced cellular knockout systems. By focusing on prototypical class A (b2AR) and B (V2R) receptors and their chimeras (b2V2/V2b2), we can now characterize differential {beta}-arrestin conformational changes as primarily mediated by the receptor C-terminus or helix-bundle. Moreover, we demonstrate that some {beta}-arrestin-supported processes are governed by distinct receptor domains, such as ERK1/2 activation (helix-bundle) and arrestin co-internalization (C-terminus), while others depend on the overall GPCR configuration, such as receptor internalization. Our findings elucidate how individual GPCR domains dictate downstream signaling events, shedding light on the structural basis of receptor-specific signaling and regulation.

pharmacology and toxicology↗

bioRxiv2024

Multi-faceted roles of β-arrestins in G protein-coupled receptors endocytosis

Internalization plays a crucial role in regulating the density of cell surface receptors and has been demonstrated to regulate intracellular signaling. Dysregulation of this process has been implicated in various diseases. The vast majority of GPCRs were considered to adopt one way for internalization. We challenged this conventional view by showing that multiple pathways converge to regulate the internalization of a specific receptor, based on an unparalleled characterization of 60 GPCR internalization profiles, both in the absence and presence of individual {beta}-arrestins ({beta}arrs). Furthermore, we revealed the internalization mechanism of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR pivotal in promoting insulin secretion from pancreatic beta cells to maintain glucose homeostasis. GLP-1R can undergo agonist-induced internalization without {beta}arrs, but can recruit and form stable complexes with {beta}arrs. We found that GLP-1R recruits clathrin adaptor protein-2 for agonist-induced internalization in both {beta}arr-dependent and -independent manners. These results provide a valuable resource for GPCR signaling and reveal the plasticity of different GPCRs to employ or not {beta}arrs in the clathrin-mediated internalization.

cell biology↗

bioRxiv2023

Key phosphorylation sites for robust β-arrestin2 binding at the mu-opioid receptor revisited

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=40 SRC="FIGDIR/small/570073v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@198d7f5org.highwire.dtl.DTLVardef@18a3d9forg.highwire.dtl.DTLVardef@d3c9e8org.highwire.dtl.DTLVardef@6ca5f2_HPS_FORMAT_FIGEXP M_FIG C_FIG Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, we and others have shown that phosphorylation is important for {beta}-arrestin recruitment to the receptor, and that ligands of different efficacies induce distinct patterns, or barcodes, of receptor phosphorylation. Within the MOR C-tail, the 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser acts as a primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr residues can get subsequently phosphorylated. Here we used HEK293 GRK KO cells, in combination with phosphosite specific antibodies and site-directed mutagenesis of the MOR, to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and {beta}-arrestin2 recruitment. We show that both GRK subfamilies (GRK2/3 and GRK5/6) promote phosphorylation of Thr370 and Ser375. However, only GRK2/3 induce phosphorylation of Thr376 and Thr379, which is required to promote robust {beta}-arrestin recruitment to the receptor. Moreover, while DAMGO and fentanyl can engage all kinases to promote Thr370 and Ser375 phosphorylation, under endogenous GRK expression conditions, morphine-induced phosphorylation of these residues is specifically mediated by GRK5/6. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of the different GRK subfamilies plays an important role in determining the tissue responses of distinct opioid agonists.

pharmacology and toxicology↗

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↗