Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2022.04.11.487861

Quantitation of FGFR3 signaling via GRB2 recruitment on micropatterned surfaces

Hartl, I.·Brumovska, V.·Striedner, Y.·Yasari, A.·Schuetz, G. J.·Sevcsik, E.·Tiemann-Boege, I.
View original source ↗Export metadata ↓

Abstract

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some of these mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. Receptor activation was then quantified via the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 patterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) in the wildtype (WT), as well as in different FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, K650E). Our data showed that the addition of ligands increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This may be explained by the different receptor populations probed, since the micropatterning method specifically reports on signaling events at the plasma membrane. Graphical AbstractSpecifications: The maximum size of the image should be 200 x 500 pixels with a minimum resolution of 300 dpi, using Arial font with a size of 10-16 points; Preferred file types: TIFF, EPS, PDF or MS Office files O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/487861v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4a279eorg.highwire.dtl.DTLVardef@6f35d1org.highwire.dtl.DTLVardef@13661e7org.highwire.dtl.DTLVardef@155b480_HPS_FORMAT_FIGEXP M_FIG C_FIG Research HighlightsO_LIQuantification of FGFR3 signaling in live cells on micropatterned surfaces C_LIO_LIAnalysis of GRB2 recruitment to the mature receptor at the plasma membrane C_LIO_LILigand-independent kinase activation of FGFR3 mutants C_LIO_LIActivation of FGFR3 at the cell surface can be different than in bulk cell extracts C_LI

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hartl, I., Brumovska, V., Striedner, Y., Yasari, A., Schuetz, G. J., Sevcsik, E., Tiemann-Boege, I.. 2022-04-11. Quantitation of FGFR3 signaling via GRB2 recruitment on micropatterned surfaces. https://doi.org/10.1101/2022.04.11.487861

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Engineered Miniature ParTasR Enables Efficient PAMless Genome Editing

TIGR-Tas system holds great promise for genome-engineering applications owing to its compact size and PAMless targeting capability. However, its low editing efficiency in mammalian cells severely limits its broader utility. Here, we engineered a highly active variant of ParTasR, termed eTasR, through structure-guided rational design of ParTasR combined with fusion to the exonuclease TREX2. eTasR increased mammalian editing efficiency from the original 4.29% to 48.94% with an average 26.52-fold increase, significantly surpassing the efficiency of SpRY, a reported version of PAM-free nuclease. More importantly, single-AAV delivery enabled efficient Hpd disruption in vivo, resulting in robust phenotypic rescue. Thus, eTasR provides a compact, PAMless and highly efficient platform for genome editing and therapeutic applications.

molecular biology↗
bioRxiv2026

Enhancing pancreatic islet cell survival via NMDAR antagonist-driven ROR1 activation as revealed by phosphoproteomics

NMDA receptors (NMDARs) control function and survival not only of neurons, but also of insulin-producing pancreatic beta cells. Since insufficient insulin secretion and beta cell death are key events during diabetes development, targeting NMDARs offers a therapeutic approach. Specifically, the NMDAR antagonist dextrorphan (DXO) increases glucose-stimulated insulin secretion in clinical trials. Furthermore, treatment with DXO or optimized DXO derivatives prevents pancreatic islet cell death upon diabetogenic insults. Nonetheless, the signaling pathways induced by NMDAR inhibition are poorly understood. Here, we use complementary approaches, phosphoproteomics and tyrosine kinase profiling, to demonstrate that DXO and its derivatives rapidly regulate multiple intracellular signaling clusters, including mTOR and ERK signaling, in pancreatic islets. Notably, mTOR inhibition, but not ERK inhibition, is sufficient to negate DXO's protective effects. Further, we show that activation of Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) is a universal event upon NMDAR inhibition in islets. Simultaneous knockdown of Ror1/ROR1 and its paralogue Ror2/ROR2 compromises the viability of both mouse and human islets, highlighting the ROR1/2 signaling axis as a promising avenue for enhancing beta cell survival.

molecular biology↗
bioRxiv2026

An ssRNA Phage Single-gene Lysis Protein Targeting MraY Reveals Convergent Evolution Across the RNA-DNA Phage Boundary

Small lytic phages of the ssRNA Leviviricetes and ssDNA Microviricetes achieve host lysis by expression of a single protein, designated as an Sgl protein. Four ssRNA Sgls have been shown to inhibit steps in peptidoglycan biosynthesis: three target MurJ, the Lipid II flippase, and one targets MurA, the committed step of the pathway. A fifth Sgl, the E protein of the canonical ssDNA microvirus {varphi}X174, inhibits MraY, which catalyzes the synthesis of the first lipid-linked intermediate. This inhibition requires SlyD, a cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase. The Sgl of the Caulobacter ssRNA phage {varphi}Cb5 (Cebevirus halophobicum) has not been characterized. Here, we present physiological evidence that Sgl{varphi}Cb5 targets MraY. Moreover, we show that, despite the absence of detectable sequence similarity, Sgl{varphi}Cb5 shares several features with the {varphi}X174 E protein, including the distribution of hydrophobic and charged residues, a proposed N-out, C-in membrane topology, and retention of lytic activity in an Sgl{varphi}Cb5 -LacZ fusion. Furthermore, expression of the Caulobacter cytoplasmic FKBP-type peptidyl-prolyl cis-trans isomerase CC2799 enhances Sgl{varphi}Cb5 -mediated lysis in E. coli. These parallels between Sgl function in a DNA phage and an RNA phage either represent remarkably consonant convergent evolution or some evolutionary cross-over event for which there is currently no precedent.

molecular biology↗