Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a Schematic representation of domain architecture of VEGFR1 and VEGFR2. The transmembrane and juxtamembrane segments are labelled TM and JM, respectively. The C-terminal phosphotyrosine residues used for probing kinase activation are labelled. b Classical model of VEGFR activation in the presence of ligand (VEGF 165 ) . The schematics are made using Inkscape Ver 1.2 See Supplementary Fig. .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a – d Confocal images of VEGFR2 or VEGFR1 fused to mCherry in a low ( a , c ) and high ( b , d ) expressing CHO cell lines. The VEGFR expression level is shown in red (λ ex = 552 nm, λ em = 586-651 nm), and the phosphorylation status is shown in green (λ ex = 488, λ em = 505-531). Scale bar = 10 μm. e , f The expression level of VEGFR2 (panel e) or VEGFR1 (panel f) is plotted against the phosphorylation level of the corresponding tyrosine residues at the C-terminal tail. The low-expressing and high-expressing cells are indicated based on the mCherry intensity at the plasma membrane. Individual data points in the left panel represent the mean expression and phosphorylation level for the binned cells. The orange line represents the linear fitting of the individual data points in the ligand-dependent activation. The blue line in panel e represents the second-order polynomial fitting of the individual data points in the ligand-independent activation. In panel f, the blue line is the guiding line. The right panel represents the bar plot of the normalized phosphotyrosine levels. The phosphotyrosine level (FITC channel) is normalized with respect to the corresponding VEGFR expression level (mCherry channel) at the plasma membrane. In ( e ) (left), n = 85 (VEGFR2-VEGF 165 ), 89 (VEGFR2 + VEGF 165 ), and in ( f ) (left) n = 107 (VEGFR1-VEGF 165 ), 100 (VEGFR1 + VEGF 165 ) cells were examined over five independent experiments in ( e , f ) (right) Each bar represents the mean value of 30–40 cells in the bar plot. The error bar shows the standard deviation of data points. Data are presented as mean values ± SD from five independent experiments. g The immunoblot shows the representative phosphorylation level of VEGFR1 or VEGFR2 at the indicated time points after activating the transfected CHO cell line with 50 nM VEGF 165 . ( n = 3). h The plot of the phosphorylation level of respective C-terminal tyrosine residue as a function of time. The phosphorylation level is analyzed from the densitometric measurement of the Western blot shown in ( g ). The t 1/2 is determined by fitting the decay of the highest intensity observed to exponential decay. Data are presented as mean values ± SD from three independent experiments. All data were plotted using GraphPad Prism Ver 9.5.1. The confocal images were generated using Fiji Ver 1.54 f. The schematics were made using Inkscape Ver 1.2. Source data are provided as a Source Data file for panels e-h. See Supplementary Figs. and .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Expressing, Phospho-proteomics, Clinical Proteomics, Membrane, Activation Assay, Standard Deviation, Western Blot, Transfection, Residue, Generated

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a , b Schematic representation of VEGFR1 ( a ) and VEGFR2 ( b ) constructs used in this study. c The plot of Y1175 phosphorylation level against the expression level of the constitutively activated C482R mutant of VEGFR2 in the presence or absence of VEGF 165 . [ n = 73 (−VEGF 165 ) and 75 (+VEGF 165 ) cells examined over 5 independent experiments. Data are presented as mean values ± SD]. d The plot of Y1213 phosphorylation versus VEGFR1-C471R expression in the presence and absence of ligand. [ n = 70 (−VEGF 165 ) and 80 (+VEGF 165 ) cells examined over five independent experiments. Data are presented as mean values ± SD]. e The diffusion coefficient measured from FRAP studies of indicated constructs of VEGFR1 and VEGFR2 in the presence and absence of VEGF 165 are plotted. VEGFR1-GPA chimera and VEGFR1-GPA-G83I chimera represent dimer and monomer controls, respectively. Each data point in the box plot reflects the diffusion coefficient of the selected cell, and the black line indicates the mean value. n = 18 (VEGFR1-TM gPA-G83I ), 24 (VEGFR1-TM gPA ), 20 (VEGFR1-VEGF 165 ), 20 (VEGFR1 + VEGF 165 ) 24 (C471R-VEGF 165 ), 18 (C471R + VEGF 165 ), 25 (VEGFR2-VEGF 165 ), 18 (VEGFR2 + VEGF 165 ) 21 (C482R-VEGF 165 ), and 18 (C482R + VEGF 165 ) cells examined over 8 independent experiments. An unpaired two-tailed t -test was used to calculate significance. Boxplots represent quartiles. The data points outside the whisker range are set as outliers. The black line inside the box represents the median value. f , g The plot of the phosphorylation level of Y1175 in VEGFR2 ( f ) and Y1213 in VEGFR1 ( g ) against the indicated receptor expression level in the presence and absence of the ligand. In panel f, n = 72 (VEGFR1-VEGF 165 ), 70 (VEGFR1 + VEGF 165 ), and 74 (∆ECD-VEGFR1) cells were examined over four independent experiments. In ( g ), n = 70 (VEGFR2-VEGF165), 62 (VEGFR2 + VEGF165), and 91 (∆ECD-VEGFR2) cells were examined over four independent experiments. Data points are represented as mean values ± SD. All data were plotted using GraphPad Prism Ver 9.5.1. The boxplots were generated using Origin Pro 2020b. All the schematics and icons were designed using Inkscape Ver1.2. Source data are provided as a Source Data file for ( b – g ). See Supplementary Figs. , .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Construct, Phospho-proteomics, Expressing, Mutagenesis, Diffusion-based Assay, Two Tailed Test, Whisker Assay, Generated

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a Schematic representation of VEGFR1 and VEGFR2 constructs used in this study. b Immunoblot showing the phosphorylation of Y1213 in the indicated constructs of VEGFR1. The expression level of the VEGFR1 is determined using an antiHA antibody. The bar plot in the lower panel represents the relative Y1213 phosphorylation level determined from densitometric analysis. Data are presented as mean values ± SD from three independent experiments. An unpaired two-tailed t-test is used to calculate the significance. c The plot of the Y1213 phosphorylation level against the expression level of VEGFR1 ΔECD and wt from the single-cell assay. n = 48 (VEGFR1-VEGF 165 ), 69 (VEGFR1 + VEGF 165 ), 67 (∆ECD-VEGFR1), 75(∆ECD-VEGFR1 TM ) 91 (∆ECD-VEGFR1 JM ), and 95 (∆ECD-VEGFR1 TMJM ) cells were examined over four independent experiments. Data points are presented as mean values ± SD. d Sequence alignment of TM segment of VEGFR1 and VEGFR2. The amino acid residues at the VEGFR2 ligand-independent and dependent dimer interface are colored red and blue, respectively. Below, is the cartoon of the ligand-independent and dependent VEGFR2 TM dimer . e The plot of Y1213 phosphorylation versus the expression level of indicated VEGFR1 constructs. n = 63(VEGFR1-VEGF 165 ) and 73(VEGFR1TM VEGFR2 -VEGF 165 ), 80(VEGFR1TM VEGFR2 + VEGF 165 ) cells examined over 5 independent experiments. Data are presented as mean values ± SD. f The dimerization propensity of indicated VEGFR1 constructs is probed from the diffusion coefficient measured by the FRAP experiment. n = 20(VEGFR1-VEGF 165 ), n = 20(VEGFR1 + VEGF 165 ), 33(VEGFR1TM VEGFR2 -VEGF 165 ), 17(VEGFR1TM VEGFR2 + VEGF 165 ) cells examined over 10 independent experiments. An unpaired two-tailed t -test was used to calculate significance. Boxplots represent quartiles, and whiskers correspond to range. The data points outside the whisker range are set as outliers. The black line in the box represents the median value. All data were plotted using GraphPad Prism Ver 9.5.1. The boxplots were generated using Origin Pro 2020b. All the schematics and icons were designed using Inkscape Ver1.2. Source data are provided as a Source Data file for the panels ( b , c , e , f ). See Supplementary Fig. .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Construct, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test, Sequencing, Diffusion-based Assay, Whisker Assay, Generated

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a Sequence alignment of VEGFR1 and VEGFR2 JM segments. The domain boundaries are defined based on FMS-like tyrosine kinase 3 structure (PDB ID: 1RJB ) . b – e The upper panel shows the schematic diagram of the JM-KD construct used for MD simulation. The bottom panel is the space-filled model of VEGFR1 and VEGFR2 constructs. The electrostatic surface potentials are colored blue and red for the positive and negatively charged sidechains, respectively. The polar uncharged residues are colored grey. The electrostatic interactions (electrostatic latch) between JM and the C-lobe of the kinase domain are shown in the inset. The space-filled model was generated by using PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC. See Supplementary Fig. .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Sequencing, Construct, Generated

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: a Schematic representation of chimeric constructs and mutants of VEGFR1 used in this study. b , c Concentration-dependent activation of VEGFR1 constructs is determined using a single-cell assay in the presence and absence of ligands. In panel ( b ), n = 51 (VEGFR1-VEGF 165 ), 107 (VEGFR1-JM VEGFR2 -VEGF 165 ) and 86 (VEGFR1-JM VEGFR2 + VEGF 165 ) cells examined over six independent experiments. In panel c, n = 55 (VEGFR1-VEGF 165 ), 95 (VEGFR1-TMJM VEGFR2 -VEGF 165 ), and 84 (VEGFR1-TMJM VEGFR2 + VEGF 165 ) cells examined over six independent experiments. Data are presented as mean values ± SD. d The plot of Y1213 phosphorylation versus the expression level of indicated VEGFR1 constructs. n = 56 (VEGFR1-VEGF 165 ), 84 (VEGFR1 3 M -VEGF 165 ), and 70 (VEGFR1 ∆SSS - VEGF 165 ), 83 (VEGFR1 3 M ∆SSS - VEGF 165 ), 107 (VEGFR1-JM VEGFR2 - VEGF 165 ) and 65 (VEGFR1 3 M ∆SSS + VEGF 165 ) cells examined over six independent experiments. Data are presented as mean values ± SD. e The dimerization propensity of the indicated VEGFR1 construct is probed from the diffusion coefficient measured using the FRAP experiment. n = 18 (VEGFR1-TM gPA-G83I ), 24 (VEGFR1-TM gPA ), 23 (VEGFR1-JM VEGFR2 -VEGF 165 ), 18 (VEGFR1-JM VEGFR2 + VEGF 165 ) 20 (VEGFR1-TMJM VEGFR2 - VEGF 165 ) and 17 (VEGFR1-TMJM VEGFR2 + VEGF 165 ) cells examined over eight independent experiments. An unpaired two-tailed t-test was used to calculate significance. Boxplots represent quartiles, and whiskers correspond to range. The lower whisker shows the 5th percentile, and the upper whisker shows the 95th percentile. The black line in the box represents the mean value. f The relative fraction of phosphorylated Y1213 for the indicated VEGFR1 construct upon VEGF 165 stimulation is shown as a bar diagram. The fraction phosphorylated was obtained from the slope of the ligand-dependent activation of the respective VEGFR1 construct, as described in panels ( b , c , d ), and normalized against the wt data. Data are presented as mean values ± SD from three independent experiments. An unpaired two-tailed t -test was used to calculate significance. (VEGFR1-JM VEGFR2 * p = 0.0108, VEGFR1 3 M ns p = 0.1827, VEGFR1 ∆SSS * p = 0.0101, VEGFR1 3 M ∆SSS * p = 0.0172, VEGFR1-TMJM VEGFR2 * p = 0.0286). g The densitometric analysis of the Y1213 phosphorylation level at the indicated time points for the wt (blue) and chimeric construct (magenta) of VEGFR1 (Fig. and Supplementary Fig. ). The phosphorylation level at each time point is normalized against the highest intensity observed for the respective data set. The error bar represents the standard deviation of three independent experiments. Data are presented as mean values ± SD. h The rate of phosphorylation (left panel) and phosphorylation t 1/2 (right panel) of the indicated VEGFR constructs are determined from the densitometric analysis of ligand-dependent activation, as described in Fig. and Fig. 6g and Supplementary Fig. . The error bar represents the standard deviation of three independent experiments. Data are presented as mean values ± SD from three independent experiments. An unpaired two-tailed t-test was used to calculate the significance. All the data were plotted using GraphPad Prism Ver 9.5.1. The boxplots were generated using Origin Pro 2020b. All the schematics and icons were designed using Inkscape Ver1.2. Source data are provided as a Source Data file for panels b-h. See Supplementary Figs. , .

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Construct, Concentration Assay, Activation Assay, Phospho-proteomics, Expressing, Diffusion-based Assay, Two Tailed Test, Whisker Assay, Standard Deviation, Generated

Journal: Nature Communications

Article Title: Molecular basis of VEGFR1 autoinhibition at the plasma membrane

doi: 10.1038/s41467-024-45499-2

Figure Lengend Snippet: Right: The ligand binding to the ECD induces receptor dimerization and rearranges the TM-JM segment. Slow release of JM inhibition in VEGFR1 leads to transient tyrosine phosphorylation at the C-terminal tail. Faster release of JM inhibition in VEGFR1 chimera and the mutants remodels the tyrosine phosphorylation to be sustained. Left: Ligand-independent activation of VEGFR1 is suppressed due to a delicate balance between the slow release of JM inhibition and protein tyrosine phosphatase (PTP) activity . The schematics and icons were designed using Inkscape Ver1.2.

Article Snippet: The cDNA encoding wild-type human VEGFR1 (Residue number 1–1338; pDONR223-FLT1 was a gift from William Hahn & David Root; Addgene plasmid # 23912) was subcloned into pcDNA-mCherry vector between the HindIII and Kpn1 restriction sites.

Techniques: Ligand Binding Assay, Inhibition, Phospho-proteomics, Activation Assay, Activity Assay