scholarly journals CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action

2021 ◽  
Author(s):  
Carl W White ◽  
Laura E Kilpatrick ◽  
Natasha Dale ◽  
Rekhati S Abhayawardana ◽  
Sebastian Dekkers ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Mechanism Of Action ◽  
Viral Infections ◽  
Resonance Energy Transfer ◽  
Antimicrobial Properties ◽  
Resonance Energy ◽  
Endogenous Inhibitor ◽  
Functional Responses ◽  
Intact Cells ◽  
Mucosal Tissues

CXCL17 is the most recently described chemokine. It is principally expressed by mucosal tissues, where it facilitates chemotaxis of monocytes, dendritic cells, and macrophages and has antimicrobial properties. CXCL17 is also implicated in the pathology of inflammatory disorders and progression of several cancers, as well as being highly upregulated during viral infections of the lung. However, the exact role of CXCL17 in health and disease is largely unknown, mainly due to a lack of known molecular targets mediating CXCL17 functional responses. Using a range of bioluminescence resonance energy transfer (BRET) based assays, here we demonstrate that CXCL17 inhibits CXCR4-mediated signalling and ligand binding. Moreover, CXCL17 interacts with neuropillin-1, a VEGFR2 co-receptor. Additionally, we find CXCL17 only inhibits CXCR4 ligand binding in intact cells and demonstrate that this effect is mimicked by known glycosaminoglycan binders, surfen and protamine sulfate. This indicates that CXCL17 inhibits CXCR4 by a unique mechanism of action that potentially requires the presence of a glycosaminoglycan containing accessory protein. Altogether, our results reveal that CXCL17 is an endogenous inhibitor of CXCR4 and represents an important discovery in our understanding of the (patho) physiological functions of CXCL17 and regulation of CXCR4 signalling.

scholarly journals Association with Coregulators Is the Major Determinant Governing Peroxisome Proliferator-activated Receptor Mobility in Living Cells

2006 ◽  
Vol 282 (7) ◽  
pp. 4417-4426 ◽  
Author(s):  
Cicerone Tudor ◽  
Jérôme N. Feige ◽  
Harikishore Pingali ◽  
Vidya Bhushan Lohray ◽  
Walter Wahli ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Molecular Mechanisms ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Peroxisome Proliferator Activated Receptors

The nucleus is an extremely dynamic compartment, and protein mobility represents a key factor in transcriptional regulation. We showed in a previous study that the diffusion of peroxisome proliferator-activated receptors (PPARs), a family of nuclear receptors regulating major cellular and metabolic functions, is modulated by ligand binding. In this study, we combine fluorescence correlation spectroscopy, dual color fluorescence cross-correlation microscopy, and fluorescence resonance energy transfer to dissect the molecular mechanisms controlling PPAR mobility and transcriptional activity in living cells. First, we bring new evidence that in vivo a high percentage of PPARs and retinoid X receptors is associated even in the absence of ligand. Second, we demonstrate that coregulator recruitment (and not DNA binding) plays a crucial role in receptor mobility, suggesting that transcriptional complexes are formed prior to promoter binding. In addition, association with coactivators in the absence of a ligand in living cells, both through the N-terminal AB domain and the AF-2 function of the ligand binding domain, provides a molecular basis to explain PPAR constitutive activity.

scholarly journals Visualizing conformational dynamics of proteins in solution and at the cell membrane

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Sharona E Gordon ◽  
Mika Munari ◽  
William N Zagotta
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Computational Method ◽  
Intact Cells ◽  
Distance Distributions ◽  
Amber Codon Suppression ◽  
Noncanonical Amino Acid

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

scholarly journals Imaging kinase–AKAP79–phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

2002 ◽  
Vol 160 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Seth F. Oliveria ◽  
Lisa L. Gomez ◽  
Mark L. Dell'Acqua
Keyword(s):  
Signal Transduction ◽  
Glutamate Receptors ◽  
Direct Evidence ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adaptor Proteins ◽  
Living Cells ◽  
Intact Cells ◽  
Anchoring Protein

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A–kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

scholarly journals The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2

2004 ◽  
Vol 167 (6) ◽  
pp. 1241-1253 ◽  
Author(s):  
Minsoo Kim ◽  
Christopher V. Carman ◽  
Wei Yang ◽  
Azucena Salas ◽  
Timothy A. Springer
Keyword(s):  
Cell Adhesion ◽  
Ligand Binding ◽  
Immune Cell ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Dynamic Regulation ◽  
Affinity Modulation ◽  
Leukocyte Function

Dynamic regulation of integrin adhesiveness is required for immune cell–cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function–associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

scholarly journals Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Aβ Secretion

2006 ◽  
Vol 281 (43) ◽  
pp. 32240-32253 ◽  
Author(s):  
Mikko Hiltunen ◽  
Alice Lu ◽  
Anne V. Thomas ◽  
Donna M. Romano ◽  
Minji Kim ◽  
...  
Keyword(s):  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Protein Trafficking ◽  
Resonance Energy Transfer ◽  
Neuronal Cell ◽  
Resonance Energy ◽  
Precursor Protein ◽  
Intact Cells ◽  
App Trafficking ◽  
Intracellular Compartments

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with α-, β-, or γ-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (α- and β-forms). Moreover, UBQLN1 knockdown increased levels of secreted Aβ40 and Aβ42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic “gatekeeper” that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Aβ.

scholarly journals Bioluminescence resonance energy transfer identify scaffold protein CNK1 interactions in intact cells

FEBS Letters ◽  
2004 ◽  
Vol 579 (3) ◽  
pp. 648-654 ◽  
Author(s):  
Marco A. Lopez-Ilasaca ◽  
Julio C. Bernabe-Ortiz ◽  
Soon-Young Na ◽  
Victor J. Dzau ◽  
Ramnik J. Xavier
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Scaffold Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Intact Cells
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