| 1. |
- Tebroke, Julia, et al.
(author)
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Wnt-3a Induces Cytokine Release in Human Mast Cells
- 2019
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In: Cells. - : MDPI. - 2073-4409. ; 8:11
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Journal article (peer-reviewed)abstract
- Mast cells are well known for their detrimental effects in allergies and asthma, and Wnt signaling has recently been implicated in asthma and other airway diseases. However, it is not known if or how Wnts affect human mast cells. Since Wnt expression is elevated in individuals with asthma and is linked to a Th2 profile, we hypothesized that mast cells could be affected by Wnts in the context of asthma. We therefore sought to investigate the role of Wnt signaling in human mast cell development and activation. We first examined the expression of the 10 main Wnt receptors, Frizzled 1-10 (FZD(1-10)), and found expression of several FZDs in human mast cells. Treatment with purified recombinant Wnt-3a or Wnt-5a did not affect the proliferation or maturation of CD34(+) progenitors into mast cells, as indicated by cellular expression of CD117 and Fc epsilon RI, activation by Fc epsilon RI crosslinking, and histamine and tryptase release. Furthermore, Wnt treatment did not change the phenotype from MCT to MCTC, since MrgX2 expression, compound 48/80-mediated activation, and carboxypeptidase A3 content were not affected. However, Wnt-3a activated WNT/beta-catenin signaling in mature human mast cells, as revealed by stabilization of beta-catenin, upregulation of IL-8 and CCL8 mRNA expression, and release of IL-8 protein. Thus, our data suggest that Wnt-3a activation of mast cells could contribute to the recruitment of immune cells in conditions associated with increased Wnt-3a expression, such as asthma.
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| 2. |
- Alexander, Stephen P. H., et al.
(author)
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The Concise Guide to PHARMACOLOGY 2023/24: G protein-coupled receptors
- 2023
-
In: BRITISH JOURNAL OF PHARMACOLOGY. - : British pharmacological society. - 0007-1188 .- 1476-5381. ; 180
-
Journal article (peer-reviewed)abstract
- The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at . G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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| 3. |
- Breznau, Nate, et al.
(author)
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Observing many researchers using the same data and hypothesis reveals a hidden universe of uncertainty
- 2022
-
In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:44
-
Journal article (peer-reviewed)abstract
- This study explores how researchers analytical choices affect the reliability of scientific findings. Most discussions of reliability problems in science focus on systematic biases. We broaden the lens to emphasize the idiosyncrasy of conscious and unconscious decisions that researchers make during data analysis. We coordinated 161 researchers in 73 research teams and observed their research decisions as they used the same data to independently test the same prominent social science hypothesis: that greater immigration reduces support for social policies among the public. In this typical case of social science research, research teams reported both widely diverging numerical findings and substantive conclusions despite identical start conditions. Researchers expertise, prior beliefs, and expectations barely predict the wide variation in research outcomes. More than 95% of the total variance in numerical results remains unexplained even after qualitative coding of all identifiable decisions in each teams workflow. This reveals a universe of uncertainty that remains hidden when considering a single study in isolation. The idiosyncratic nature of how researchers results and conclusions varied is a previously underappreciated explanation for why many scientific hypotheses remain contested. These results call for greater epistemic humility and clarity in reporting scientific findings.
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| 4. |
- Christopoulos, Arthur, et al.
(author)
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THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: G protein-coupled receptors.
- 2021
-
In: British journal of pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 178 Suppl 1
-
Research review (peer-reviewed)abstract
- The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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| 5. |
- Dijksterhuis, Jacomijn P., et al.
(author)
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High levels of WNT-5A in human glioma correlate with increased presence of tumor-associated microglia/monocytes
- 2015
-
In: Experimental Cell Research. - : Elsevier. - 0014-4827 .- 1090-2422. ; 339:2, s. 280-288
-
Journal article (peer-reviewed)abstract
- Malignant gliomas are among the most severe types of cancer, and the most common primary brain tumors. Treatment options are limited and the prognosis is poor. WNT-5A, a member of the WNT family of lipoglycoproteins, plays a role in oncogenesis and tumor progression in various cancers, whereas the role of WNT-5A in glioma remains obscure. Based on the role of WNT-5A as an oncogene, its potential to regulate microglia cells and the glioma-promoting capacities of microglia cells, we hypothesize that WNT-5A has a role in regulation of immune functions in glioma. We investigated WNT-5A expression by in silico analysis of the cancer genome atlas (TCGA) transcript profiling of human glioblastoma samples and immunohistochemistry experiments of human glioma tissue microarrays (TMA). Our results reveal higher WNT-5A protein levels and mRNA expression in a subgroup of gliomas (WNT-5A(high)) compared to non-malignant control brain tissue. Furthermore, we show a significant correlation between WNT-5A in the tumor and presence of major histocompatibility complex Class II-positive microglia/monocytes. Our data pinpoint a positive correlation between WNT-5A and a proinflammatory signature in glioma. We identify increased presence of microglia/monocytes as an important aspect in the inflammatory transformation suggesting a novel role for WNT-5A in human glioma.
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| 6. |
- Evangelou, Evangelos, et al.
(author)
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Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22
- 2011
-
In: Annals of the Rheumatic Diseases. - : BMJ. - 1468-2060 .- 0003-4967. ; 70:2, s. 349-355
-
Journal article (peer-reviewed)abstract
- Objectives Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. Methods A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. Results With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p = 9.2 x 10(-9)), thereby confirming its role as a susceptibility locus for OA. Conclusion The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
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| 7. |
- Fröjmark, Anne-Sophie, et al.
(author)
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Mutations in frizzled 6 cause isolated autosomal-recessive nail dysplasia
- 2011
-
In: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297 .- 1537-6605. ; 88:6, s. 852-860
-
Journal article (peer-reviewed)abstract
- Inherited and isolated nail malformations are rare and heterogeneous conditions. We identified two consanguineous pedigrees in which some family members were affected by isolated nail dysplasia that suggested an autosomal-recessive inheritance pattern and was characterized by claw-shaped nails, onychauxis, and onycholysis. Genome-wide SNP array analysis of affected individuals from both families showed an overlapping and homozygous region of 800 kb on the long arm of chromosome 8. The candidate region spans eight genes, and DNA sequence analysis revealed homozygous nonsense and missense mutations in FZD(6), the gene encoding Frizzled 6. FZD(6) belongs to a family of highly conserved membrane-bound WNT receptors involved in developmental processes and differentiation through several signaling pathways. We expressed the FZD(6) missense mutation and observed a quantitative shift in subcellular distribution from the plasma membrane to the lysosomes, where the receptor is inaccessible for signaling and presumably degraded. Analysis of human fibroblasts homozygous for the nonsense mutation showed an aberrant response to both WNT-3A and WNT-5A stimulation; this response was consistent with an effect on both canonical and noncanonical WNT-FZD signaling. A detailed analysis of the Fzd(6)(-/-) mice, previously shown to have an altered hair pattern, showed malformed claws predominantly of the hind limbs. Furthermore, a transient Fdz6 mRNA expression was observed in the epidermis of the digital tips at embryonic day 16.5 during early claw morphogenesis. Thus, our combined results show that FZD6 mutations can result in severe defects in nail and claw formation through reduced or abolished membranous FZD(6) levels and several nonfunctional WNT-FZD pathways.
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| 8. |
- Gratz, Lukas, et al.
(author)
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Pathway selectivity in Frizzleds is achieved by conserved micro-switches defining pathway-determining, active conformations
- 2023
-
In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
-
Journal article (peer-reviewed)abstract
- The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD(1-10)) paralogs and Smoothened, remains one of the most enigmatic GPCR families. This class mediates signaling predominantly through Disheveled (DVL) or heterotrimeric G proteins. However, the mechanisms underlying pathway selection are elusive. Here we employ a structure-driven mutagenesis approach in combination with an extensive panel of functional signaling readouts to investigate the importance of conserved state-stabilizing residues in FZD(5) for signal specification. Similar data were obtained for FZD(4) and FZD(10) suggesting that our findings can be extrapolated to other members of the FZD family. Comparative molecular dynamics simulations of wild type and selected FZD(5) mutants further support the concept that distinct conformational changes in FZDs specify the signal outcome. In conclusion, we find that FZD(5) and FZDs in general prefer coupling to DVL rather than heterotrimeric G proteins and that distinct active state micro-switches in the receptor are essential for pathway selection arguing for conformational changes in the receptor protein defining transducer selectivity. Signaling pathway selectivity downstream of GPCRs is not fully understood. Here, authors perform functional analysis of Frizzled mutants to uncover state-stabilizing residues or 'micro-switches' mediating selectivity towards Disheveled over G proteins.
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| 9. |
- Grätz, Lukas, et al.
(author)
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NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
- 2023
-
In: British Journal of Pharmacology. - : John Wiley & Sons. - 0007-1188 .- 1476-5381.
-
Journal article (peer-reviewed)abstract
- Background and PurposeWnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer.Experimental ApproachSW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization.Key ResultsWith this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells.Conclusions and ImplicationsBinding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion.
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| 10. |
- Kilander, Michaela B. C., et al.
(author)
-
Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6
- 2014
-
In: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 28:5, s. 2293-2305
-
Journal article (peer-reviewed)abstract
- Frizzleds (FZDs) are classified as G-protein-coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD(6) regulates convergent extension movements, and its C-terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD(6), Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD(6) precouples to G(i1) and G(q) but not to G(oA),G(s), and G(12) proteins. G-protein coupling is measured as a 10-20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross-linking. The FZD(6) Arg511Cys mutation is incapable of G-protein precoupling, even though it still binds DVL. Using both FRAP and Forster resonance energy transfer (FRET) technology, we showed that the FZD(6)-G(i1) and FZD-G(q) complexes dissociate on WNT-5A stimulation. Most important, G-protein precoupling of FZD(6) and WNT-5A-induced signaling to extracellular signal-regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD(6)-G-protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G-protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G-protein, and DVL function.Kilander, M. B. C., Petersen, J., Andressen, K. W., Ganji, R. S. Levy, F. O., Schuster, J., Dahl N., Bryja, V., Schulte, G. Disheveled regulates precoupling of heterotrimeric G proteins to Frizzled 6.
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| 11. |
- Kinsolving, Julia, et al.
(author)
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Structural and functional insight into the interaction of Clostridioides difficile toxin B and FZD7
- 2024
-
In: Cell Reports. - 2211-1247. ; 43:2
-
Journal article (peer-reviewed)abstract
- The G protein -coupled receptors of the Frizzled (FZD) family, in particular FZD1,2,7, are receptors that are exploited by Clostridioides difficile toxin B (TcdB), the major virulence factor responsible for pathogenesis associated with Clostridioides difficile infection. We employ a live -cell assay examining the affinity between full-length FZDs and TcdB. Moreover, we present cryoelectron microscopy structures of TcdB alone and in complex with full-length FZD7, which reveal that large structural rearrangements of the combined repetitive polypeptide domain are required for interaction with FZDs and other TcdB receptors, constituting a first step for receptor recognition. Furthermore, we show that bezlotoxumab, an FDA -approved monoclonal antibody to treat Clostridioides difficile infection, favors the apo-TcdB structure and thus disrupts binding with FZD7. The dynamic transition between the two conformations of TcdB also governs the stability of the pore -forming region. Thus, our work provides structural and functional insight into how conformational dynamics of TcdB determine receptor binding.
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| 12. |
- Pennells, Lisa, et al.
(author)
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Equalization of four cardiovascular risk algorithms after systematic recalibration : individual-participant meta-analysis of 86 prospective studies
- 2019
-
In: European Heart Journal. - : Oxford University Press (OUP). - 0195-668X .- 1522-9645. ; 40:7, s. 621-
-
Journal article (peer-reviewed)abstract
- Aims: There is debate about the optimum algorithm for cardiovascular disease (CVD) risk estimation. We conducted head-to-head comparisons of four algorithms recommended by primary prevention guidelines, before and after ‘recalibration’, a method that adapts risk algorithms to take account of differences in the risk characteristics of the populations being studied.Methods and results: Using individual-participant data on 360 737 participants without CVD at baseline in 86 prospective studies from 22 countries, we compared the Framingham risk score (FRS), Systematic COronary Risk Evaluation (SCORE), pooled cohort equations (PCE), and Reynolds risk score (RRS). We calculated measures of risk discrimination and calibration, and modelled clinical implications of initiating statin therapy in people judged to be at ‘high’ 10 year CVD risk. Original risk algorithms were recalibrated using the risk factor profile and CVD incidence of target populations. The four algorithms had similar risk discrimination. Before recalibration, FRS, SCORE, and PCE over-predicted CVD risk on average by 10%, 52%, and 41%, respectively, whereas RRS under-predicted by 10%. Original versions of algorithms classified 29–39% of individuals aged ≥40 years as high risk. By contrast, recalibration reduced this proportion to 22–24% for every algorithm. We estimated that to prevent one CVD event, it would be necessary to initiate statin therapy in 44–51 such individuals using original algorithms, in contrast to 37–39 individuals with recalibrated algorithms.Conclusion: Before recalibration, the clinical performance of four widely used CVD risk algorithms varied substantially. By contrast, simple recalibration nearly equalized their performance and improved modelled targeting of preventive action to clinical need.
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| 13. |
- Petersen, Julian, et al.
(author)
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Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling
- 2017
-
In: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8:1
-
Journal article (peer-reviewed)abstract
- G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They can exist and act as dimers, but the requirement of dimers for agonist-induced signal initiation and structural dynamics remains largely unknown. Frizzled 6 (FZD6) is a member of Class F GPCRs, which bind WNT proteins to initiate signaling. Here, we show that FZD6 dimerizes and that the dimer interface of FZD6 is formed by the transmembrane a-helices four and five. Most importantly, we present the agonist-induced dissociation/re-association of a GPCR dimer through the use of live cell imaging techniques. Further analysis of a dimerization-impaired FZD6 mutant indicates that dimer dissociation is an integral part of FZD6 signaling to extracellular signal-regulated kinases1/2. The discovery of agonistdependent dynamics of dimers as an intrinsic process of receptor activation extends our understanding of Class F and other dimerizing GPCRs, offering novel targets for dimerinterfering small molecules.
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| 14. |
- Schulte, Barbara, et al.
(author)
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Wither the Social Sciences? On Excellence, Rankings, and other Trifles
- 2015
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In: Samhällsvetenskapliga fakulteten i Lund – en vital 50-åring. En jubileumsskrift. - 9789176233597 ; , s. 528-539
-
Book chapter (pop. science, debate, etc.)abstract
- I argue in this chapter that it is essential for the social sciences to provide explanations and possibly also critical reflections as to how, why and to what end rankings and assessments are imbued with social (and other) values in different contexts. For too long we have delib- erately avoided scrutinizing the very mechanisms that have been framing and often also constraining our ways of ‘doing social science’ (including teaching and research). Therefore it is important to include in our research agenda the things that determine the present and future of our academic disciplines.
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| 15. |
- Schulte, Gunnar
(author)
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Adenosine receptor signaling and the activation of mitogen-activated protein kinases
- 2002
-
Doctoral thesis (other academic/artistic)abstract
- The nucleoside adenosine is present in all cells and body fluids of all living organisms; its production, both intracellularly and extracellularly, is tightly coupled to energy consumption resulting in increasing extracellular adenosine levels with increased energy consumption. Adenosine is metabolized by adenosine deaminase and adenosine kinase. Deamination leads to the production of inosine, which appears in concentrations similar to those of adenosine. Adenosine exerts modulatory effects on many body functions and is known to regulate, for example, the cardiovascular and central nervous system, pain modulation, mast cells and immune function. These physiological effects are mediated by four pharmacologically and biochemically distinct adenosine receptors, which belong to the family of G protein coupled receptors activating heterotrimeric G proteins. The adenosine A, and A3 receptors generally couple to Gj proteins, whereas the adenosine A2A and A2B receptors activate Gs proteins. In order to describe adenosine receptor pharmacology and signaling the recombinant human adenosine receptors were stably transfected into Chinese hamster ovary (CHO) cells, which normally do not express adenosine receptors. This is of advantage for the description of single adenosine receptor subtypes since sufficiently specific pharmacological tools are not available and since most cells express more than one adenosine receptor subtype. Furthermore, this recombinant cellular system allows the comparison of the human adenosine receptors in front of a similar cellular background. Human adenosine receptors are, indeed, activated by adenosine, whereas its metabolite inosine is only active at the adenosine A, and A3 receptor, and even there inosine has low potency and efficacy. Thus, adenosine is the main ligand for adenosine receptors. Moreover, adenosine A, and A2A receptors may be constitutively activated at basal adenosine levels (30 - 300 nM), whereas the A2B and A3 receptor are only activated when adenosine levels are increased. Inosine - at high concentrations - may have effects at the adenosine A3 receptor, at least where it is highly expressed. Adenosine affects cell growth positively and negatively. An important role in mitogenesis is played by the family of m itogen -activated protein kinases (MAPK) and we show that all adenosine receptors expressed in CHO cells mediate signaling to the MAPK extracellular signal-regulated kinase 1/2 (ERK1/2). ERK1/2 phosphorylation is time- and dose-dependently increased upon stimulation with the unspecific adenosine receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) and with the endogenous ligand adenosine. Adenosine receptors showed different efficacy in mediating ERK1/2 phosphorylation: A3>A1>A2A>A2B. The EC50 value for the adenosine A, A2A, and A3 receptor mirrored that for effects on e.g. adenylyl cyclase, but NECA-induced ERK1/2 phosphorylation in CHO A2B cells was half-maximal already at 20 nM, compared to a half-maximal cAMP production at 1.4 muM. Due to the high efficacy and the high potency of agonists to induce ERK1/2 activation via the adenosine A3 and A2B receptor, respectively, we specifically examined these intracellular signaling pathways, using both pharmacological and molecular biological tools. Pharmacological tools such as kinase and adenylyl cyclase inhibitors, however, caused unexpected problems. Due to structural similarity, inhibitors binding to the ATP-binding site of such enzymes may also bind to and block the ligand-binding site of adenosine receptors, which could be shown by competitive radioligand binding experiments. The adenosine A3 receptor signals to ERK1/2 by the release of beta-gamma subunits from pertussis toxin sensitive G proteins, activation of phosphatidyl i nositol-3- kinase (P13K), the small GTP-binding protein Ras and the MAPKK MEK. The Gs-coupled adenosine A2B receptor, on the other hand, accomplishes ERK1/2 activation in a cAMP-dependent but cAMP-dependent protein kinase (PKA)-independent manner. Furthermore, this pathway uses P13K and transactivation of the epidermal growth factor receptor, whereas activation of the small GTP-binding protein Rap1 is not necessary for ERK1/2 activation. Signaling to the cAMP response element binding protein (CREB) and the stress-activated protein kinase p38 involves cAMP and PKA - independent of P13K. The molecular aspects of adenosine receptor signaling in vivo are still obscure, but the knowledge of intracellular signaling pathways activated by single adenosine receptor subtypes in recombinant cell systems may present some support for mechanisms in the in vivo situation.
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| 16. |
- Strakova, Katerina, et al.
(author)
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Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization
- 2018
-
In: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 293:48, s. 18477-18493
-
Journal article (peer-reviewed)abstract
- Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1(WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD(6). Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD(6) C terminus, is efficiently phosphorylated by casein kinase 1 is an element of (CK1 is an element of) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1-mediated FZD(6) phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1 is an element of-targeted phosphomo-tif reduced FZD(6) surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD(6). Phos-pho-Ser-648 FZD(6) immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD(6) protein expression, suggesting that FZD(6) phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD(6) in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD(6) rather than asymmetric protein distribution accounts for polarized receptor signaling.
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| 17. |
- Strakova, Katerina, et al.
(author)
-
The tyrosine Y250(2.39) in Frizzled 4 defines a conserved motif important for structural integrity of the receptor and recruitment of Disheveled
- 2017
-
In: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 38, s. 85-96
-
Journal article (peer-reviewed)abstract
- Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The Interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y250(2.39)) in the intracellular loop 1 (ILl) of human FZD(4). We have found Y250(2.39) to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y250(2.39)F, impaired in DVL2 binding, was defective in both beta-catenin-dependent and beta-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gan and G alpha(13) and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y250(2.39) is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y250(2.39) and H348(4.46) plays a role in specifying downstream signaling pathways induced by the receptor.
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| 18. |
- Wright, Shane C., et al.
(author)
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FZD(5) is a G alpha(q)-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs
- 2018
-
In: Science Signaling. - : AMER ASSOC ADVANCEMENT SCIENCE. - 1945-0877 .- 1937-9145. ; 11:559
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Journal article (peer-reviewed)abstract
- Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD(5)) is one of the most studied class F GPCRs that promote the functional inactivation of the beta-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD(5) has not been shown to activate heterotrimeric G proteins. Here, we show that FZD(5) exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD(5) activated G alpha(q) and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD(5) is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.
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