scholarly journals The biophysical basis of receptor tyrosine kinase ligand functional selectivity: Trk-B case study

2020 ◽  
Vol 477 (23) ◽  
pp. 4515-4526 ◽  
Author(s):  
Fozia Ahmed ◽  
Michael D. Paul ◽  
Kalina Hristova
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Biological Effects ◽  
Membrane Receptors ◽  
Live Cells ◽  
Published Data ◽  
Receptor Kinase ◽  
Neurotrophin 3 ◽  
Trk B

Tropomyosin receptor kinase B (Trk-B) belongs to the second largest family of membrane receptors, Receptor Tyrosine Kinases (RTKs). Trk-B is known to interact with three different neurotrophins: Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-4 (NT-4), and Neurotrophin-3 (NT-3). All three neurotrophins are involved in survival and proliferation of neuronal cells, but each induces distinct signaling through Trk-B. We hypothesize that the different biological effects correlate with differences in the interactions between the Trk-B receptors, when bound to different ligands, in the plasma membrane. To test this hypothesis, we use quantitative FRET to characterize Trk-B dimerization in response to NT-3 and NT-4 in live cells, and compare it to the previously published data for Trk-B in the absence and presence of BDNF. Our study reveals that the distinct Trk-B signaling outcomes are underpinned by both different configurations and different stabilities of the three ligand-bound Trk-B dimers in the plasma membrane.

Role of Grb2 in EGF-stimulated EGFR internalization

2002 ◽  
Vol 115 (9) ◽  
pp. 1791-1802 ◽  
Author(s):  
Tetsuo Yamazaki ◽  
Kristien Zaal ◽  
Dale Hailey ◽  
John Presley ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Growth Factor Receptor ◽  
Sh3 Domain ◽  
Membrane Receptors ◽  
Live Cells ◽  
Dependent Manner ◽  
C Terminus ◽  
Downstream Effectors ◽  
Transferrin Uptake

Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area. Within 1 minute of EGF stimulation, Grb2 redistributed to the plasma membrane where it bound EGFR-CFP in an SH2 dependent manner. The plasma membrane then began to dynamically ruffle, and Grb2-YFP and EGFR-CFP were found to internalize together in large macropinocytic structures. These structures were morphologically distinct from conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited transferrin uptake, had no effect on EGF-induced internalization of EGFR. YFP-tagged Grb2 containing an inhibitory mutation in either N- or C-SH3 domain redistributed to the plasma membrane upon EGF stimulation, but the macropinocytic structures containing Grb2-YFP and EGFR-CFP did not translocate inward and appeared to remain tethered to the plasma membrane. This suggested that the Grb2 SH3 domain was responsible for coupling the membranes containing EGFR with downstream effectors involved in internalization of these membranes. Transferrin uptake was unaffected in the presence of all of the SH3 domain mutants, consistent with the EGF-stimulated EGFR internalization pathway being clathrin-independent. These results demonstrate a role for Grb2 in events associated with a macropinocytic internalization pathway for EGFR in activated cells.

scholarly journals Lysine residues form an integral component of a novel NH2-terminal membrane targeting motif for myristylated pp60v-src.

1992 ◽  
Vol 119 (2) ◽  
pp. 415-425 ◽  
Author(s):  
L Silverman ◽  
M D Resh
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Lysine Residues ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Contact Mechanism

Association of pp60v-src with the plasma membrane is fundamental to generation of the transformed phenotype. Although myristylation of pp60v-src is required for interaction with a membrane-bound receptor, the importance of NH2-terminal amino acids in receptor binding has not yet been uncoupled from their role in signaling myristylation. Using chimeric src proteins, peptides identical or related to the NH2 terminus of src, and site-directed mutagenesis, we demonstrate that NH2-terminal lysines in conjunction with myristate are essential for membrane localization. Subsequent to NH2-terminal interaction with the "src receptor," internal regions of the src protein also participate in membrane binding. This novel NH2-terminal motif and internal contact mechanism may direct other members of the src family of tyrosine kinases to their membrane receptors.

A Photoactivable Formaldehyde Donor with Fluorescence Monitoring Reveals Threshold to Arrest Cell Migration

2019 ◽  
Author(s):  
Lukas P Smaga ◽  
Nicholas W Pino ◽  
Gabriela E Ibarra ◽  
Vishnu Krishnamurthy ◽  
Jefferson Chan
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Fluorescence Enhancement ◽  
Biological Effects ◽  
Cellular Systems ◽  
Live Cells ◽  
First Report ◽  
Cell Lysate ◽  
Intracellular Release ◽  
Biological Analytes

Controlled light-mediated delivery of biological analytes enables the investigation of highly reactivity molecules within cellular systems. As many biological effects are concentration dependent, it is critical to determine the location, time, and quantity of analyte donation. In this work, we have developed the first photoactivatable donor for formaldehyde (FA). Our optimized photoactivatable donor, photoFAD-3, is equipped with a fluorescence readout that enables monitoring of FA release with a concomitant 139-fold fluorescence enhancement. Tuning of photostability and cellular retention enabled quantification of intracellular FA release through cell lysate calibration. Application of photoFAD-3 uncovered the concentration range necessary for arresting wound healing in live cells. This marks the first report where a photoactivatable donor for any analyte has been used to quantify intracellular release.

scholarly journals Plasma membrane glycosphingolipid signaling: a turning point

2021 ◽  
Author(s):  
Elena Chiricozzi
Keyword(s):  
Plasma Membrane ◽  
Chemical Synthesis ◽  
Bioinformatic Analysis ◽  
Membrane Receptors ◽  
Outer Side ◽  
Neuronal Homeostasis ◽  
Extracellular Stimuli ◽  
Conceptual Shift ◽  
Plasma Membrane Receptors ◽  
Key Events

AbstractPlasma membrane interaction is highly recognized as an essential step to start the intracellular events in response to extracellular stimuli. The ways in which these interactions take place are less clear and detailed. Over the last decade my research has focused on developing the understanding of the glycosphingolipids-protein interaction that occurs at cell surface. By using chemical synthesis and biochemical approaches we have characterized some fundamental interactions that are key events both in the immune response and in the maintenance of neuronal homeostasis. In particular, for the first time it has been demonstrated that a glycolipid, present on the outer side of the membrane, the long-chain lactosylceramide, is able to directly modulate a cytosolic protein. But the real conceptual change was the demonstration that the GM1 oligosaccharide chain is able, alone, to replicate numerous functions of GM1 ganglioside and to directly interact with plasma membrane receptors by activating specific cellular signaling. In this conceptual shift, the development and application of multidisciplinary techniques in the field of biochemistry, from chemical synthesis to bioinformatic analysis, as well as discussions with several national and international colleagues have played a key role.

scholarly journals The Expression of the Cancer-Associated lncRNA Snhg15 Is Modulated by EphrinA5-Induced Signaling

2021 ◽  
Vol 22 (3) ◽  
pp. 1332
Author(s):  
Daniel Pensold ◽  
Julia Gehrmann ◽  
Georg Pitschelatow ◽  
Asa Walberg ◽  
Kai Braunsteffer ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Membrane Receptors ◽  
In Vitro Assays ◽  
Eph Receptor ◽  
Medulloblastoma Cell Line ◽  
And Migration

The Eph receptor tyrosine kinases and their respective ephrin-ligands are an important family of membrane receptors, being involved in developmental processes such as proliferation, migration, and in the formation of brain cancer such as glioma. Intracellular signaling pathways, which are activated by Eph receptor signaling, are well characterized. In contrast, it is unknown so far whether ephrins modulate the expression of lncRNAs, which would enable the transduction of environmental stimuli into our genome through a great gene regulatory spectrum. Applying a combination of functional in vitro assays, RNA sequencing, and qPCR analysis, we found that the proliferation and migration promoting stimulation of mouse cerebellar granule cells (CB) with ephrinA5 diminishes the expression of the cancer-related lncRNA Snhg15. In a human medulloblastoma cell line (DAOY) ephrinA5 stimulation similarly reduced SNHG15 expression. Computational analysis identified triple-helix-mediated DNA-binding sites of Snhg15 in promoters of genes found up-regulated upon ephrinA5 stimulation and known to be involved in tumorigenic processes. Our findings propose a crucial role of Snhg15 downstream of ephrinA5-induced signaling in regulating gene transcription in the nucleus. These findings could be potentially relevant for the regulation of tumorigenic processes in the context of glioma.

Testing biological effects of hand-washing grey water for reuse in irrigation on an urban farm: a case study

2013 ◽  
Vol 34 (4) ◽  
pp. 545-551 ◽  
Author(s):  
Mohammad Zain Khan ◽  
Yei Lin Sim ◽  
Yang Jian Lin ◽  
Ka Man Lai
Keyword(s):  
Biological Effects ◽  
Hand Washing ◽  
Grey Water ◽  
Urban Farm
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