Abstract 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.
Abstract 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.
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 βarrestins (βarrs). 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 βarrs, but can recruit and form stable complexes with βarrs. We found that GLP-1R recruits clathrin adaptor protein-2 for agonist-induced internalization in both βarr-dependent and -independent manners. These results provide a valuable resource for GPCR signaling and reveal the plasticity of different GPCRs to employ or not βarrs in the clathrin-mediated internalization.
// Julia Drube 1 , Thomas Ernst 2 , Markus Pfirrmann 3 , Benadict Vincent Albert 1 , Sebastian Drube 4 , Daniela Reich 1 , Anne Kresinsky 1 , Kathrin Halfter 3 , Claudio Sorio 5 , Christian Fabisch 2 , Andreas Hochhaus 2 and Frank-D. Böhmer 1 1 Institut für Molekulare Zellbiologie, CMB, Universitätsklinikum Jena, Jena, Germany 2 Abteilung Hämatologie und Internistische Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany 3 Institut für Medizinische Informationsverarbeitung, Biometrie und Epidemiologie (IBE), Ludwig-Maximilians Universität, Munich, Germany 4 Institut für Immunologie, Universitätsklinikum Jena, Jena, Germany 5 Department of Medicine, University of Verona, Verona, Italy Correspondence to: Frank-D. Böhmer, email: boehmer@med.uni-jena.de Keywords: chronic myeloid leukemia; protein-tyrosine phosphatases; PTPRC; CD45; PTPRG Received: November 30, 2017 Accepted: December 08, 2017 Published: January 15, 2018 ABSTRACT The introduction of second-generation tyrosine kinase inhibitors (TKIs) targeting the protein-tyrosine kinase (PTK) BCR-ABL1 has improved treatment response in chronic myeloid leukemia (CML). However, in some patients response still remains suboptimal. Protein-tyrosine phosphatases (PTPs) are natural counter-actors of PTK activity and can affect TKI sensitivity, but the impact of PTPs on treatment response to second-generation TKIs is unknown. We assessed the mRNA expression level of 38 PTPs in 66 newly diagnosed CML patients and analyzed the potential relation with treatment outcome after 9 months of nilotinib medication. A significantly positive association with response was observed for higher PTPN13, PTPRA, PTPRC (also known as CD45), PTPRG, and PTPRM expression. Selected PTPs were then subjected to a functional analysis in CML cell line models using PTP gene knockout by CRISPR/Cas9 technology or PTP overexpression. These analyses revealed PTPRG positively and PTPRC negatively modulating nilotinib response. Consistently, PTPRG negatively and PTPRC positively affected BCR-ABL1 dependent transformation. We identified BCR-ABL1 signaling events, which were affected by modulating PTP levels or nilotinib treatment in the same direction. In conclusion, the PTP status of CML cells is important for the response to second generation TKIs and may help in optimizing therapeutic strategies.
Glioblastoma (GBM) is the most common and aggressive primary brain malignancy. Adhesion G protein-coupled receptors (aGPCRs) have attracted interest for their potential as treatment targets. Here, we show that CD97 (ADGRE5) is the most promising aGPCR target in GBM, by virtue of its de novo expression compared to healthy brain tissue. CD97 knockdown or knockout significantly reduces the tumor initiation capacity of patient-derived GBM cultures (PDGCs) in vitro and in vivo. We find that CD97 promotes glycolytic metabolism via the mitogen-activated protein kinase (MAPK) pathway, which depends on phosphorylation of its C terminus and recruitment of β-arrestin. We also demonstrate that THY1/CD90 is a likely CD97 ligand in GBM. Lastly, we show that an anti-CD97 antibody-drug conjugate selectively kills tumor cells in vitro. Our studies identify CD97 as a regulator of tumor metabolism, elucidate mechanisms of receptor activation and signaling, and provide strong scientific rationale for developing biologics to target it therapeutically in GBM.
Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β1-adrenoceptors, arrestin-biased signalling via β2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol's cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system.
Human G protein-coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis.
Abstract ID 98801Poster Board 159 Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors. They are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs couple to Gs and Golf to increase cAMP levels, while the D2-like DARs couple to Gi/o to decrease cAMP levels. All DARs also recruit β-arrestin which activates distinct signaling cascades and can also initiate receptor desensitization and internalization. There are seven GRK isoforms (GRKs1-7), with GRK2, GRK3, GRK5, and GRK6 being widely expressed. Using systematic mutational analyses, our lab has previously mapped the GRK-mediated phosphorylation sites on both the D1R and D2R, with phosphorylation occurring exclusively on ICL3 for the D2R, and both ICL3 and the C-terminus for the D1R. GRK-mediated phosphorylation of the D1R was found to be required for subsequent β-arrestin recruitment. In contrast, while GRKs play a role in β-arrestin recruitment to the D2R, GRK-mediated D2R phosphorylation is dispensable for this process. We have now sought to determine which GRK isoform(s) are involved in regulating β-arrestin recruitment to these receptors using cell lines in which the expression of specific GRK isoforms were selectively eliminated via CRISPR, as well as by utilizing isoform-selective GRK inhibitors. β-arrestin recruitment to both the D1R and D2R was severely impaired in cells lacking all GRKs (total KO), whereas selective expression of GRK isoforms 2, 3, 5 and 6 in the total KO cells was able to rescue β-arrestin recruitment. The kinase activity of each GRK was required for this recue for the D1R. Interestingly, the GRK2/3 and GRK5/6 isoforms differed in their requirement of kinase activity for the rescue of β-arrestin recruitment to the D2R. Individual and double GRK KO cells revealed that the GRK5/6 subfamily is more important for β-arrestin recruitment to the D1R, while the GRK2/3 subfamily is more important for the D2R. Treatment with GRK2/3-selective inhibitors recapitulated these findings with the GRK KO cells in that they impaired β-arrestin recruitment to the D2R, but had limited effect on this process for the D1R. Intriguingly, in cells with endogenous levels of GRK expression, β-arrestin recruitment to both the D1R and D2R were unaffected by inhibitors of GRK5/6. However, these inhibitors could block the rescue of β-arrestin recruitment to the D2R observed with GRK5 or GRK6 overexpression but had little effect on the rescue of β-arrestin recruitment to the D1R or the Gs-coupled β2 adrenergic receptor. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that DAR regulation, and GPCR regulation in general, by specific GRK isoforms can add layers of regulatory fine-tuning through differentially directing signaling or trafficking outcomes. National Institute of Neurological Disorders and Stroke Intramural Research Program, National Institutes of Health (ZIA NS002263)
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