Engineering synthetic antibody binders for allosteric inhibition of prolactin receptor signaling

Background: Many receptors function by binding to multiple ligands, each eliciting a distinct biological output. The extracellular domain of the human prolactin receptor (hPRL-R) uses an identical epitope to bind to both prolactin (hPRL) and growth hormone (hGH), yet little is known about how each hormone binding event triggers the appropriate response. Findings: Here, we utilized a phage display library to generate synthetic antibodies (sABs) that preferentially modulate hPRL-R function in a hormone-dependent fashion. We determined the crystal structure of a sAB-hPRL-R complex, which revealed a novel allosteric mechanism of antagonism, whereby the sAB traps the receptor in a conformation more suitable for hGH binding than hPRL. This was validated by examining the effect of the sABs on hormone internalization via the hPRL-R and its downstream signaling pathway. Conclusions: The findings suggest that subtle structural changes in the extracellular domain of hPRL-R induced by each hormone determine the biological output triggered by hormone binding. We conclude that sABs generated by phage display selection can detect these subtle structural differences, and therefore can be used to dissect the structural basis of receptor-ligand specificity. Keywords: Prolactin signaling, Synthetic antibody, Phage display, Alloster

Quantitative localization microscopy with a single fluorophore per labeled site.

<p>(a) Three state model with rates. (b) Cutout of total image of sparsely distributed DNA oligomers on glass labeled with single Alexa Fluor 647 dyes showing well-isolated clusters of localizations. (c) Cumulative number of localizations and single-exponential fit. (d) Correlation parameter <i>Q</i> determined from the spatial image correlations and fit with switching model shows agreement with the ground truth value determined from the cluster analysis. The estimated value for the average number of localizations ⟨<i>M</i>(<i>t</i>)⟩ shows agreement with the ground truth value determined from the cluster statistics. (e-g) Histograms of the number of localizations accumulated per cluster and model prediction at three time points during the image acquisition.</p

Quantitative localization microscopy with heterogeneous labeling density.

<p>(a) Overview image (pixel size 10 nm, clipped for visibility) and (b) zoomed inset (pixel size 4 nm) of the dashed white box in (a) of secondary antibody-Alexa Fluor 647 labeled Nup153 protein of the NPC in the nuclear membrane with non-specifically bound (secondary) antibodies outside the nuclear membrane region. (c) The correlation parameter Q for the region inside the nuclear membrane (red box) is higher than outside (blue box) due to the tight clustering of the secondary antibodies labeling the Nup153 proteins. The relative number of accumulated localizations at each time point is similar, indicating that the bleaching behavior is similar and the sources of the localizations are identical in both regions.</p

Quantitative localization microscopy of NB-labeled Seh1 in the NPC.

<p>(a) FCS-analysis of NB stoichiometry indicating there is a single fluorophore per NB. (b) Cutout of quantitative localization microscopy image of NB-labeled Seh1 in the NPC (<i>k</i>_<i>bl</i> = 4.8 × 10⁻³/s and <i>M</i>_∞ = 5.0). The numbers at the green boxes indicate the estimated number of NBs within the box. (c) Histogram of the estimated number of NBs per NPC.</p

Quantitative localization microscopy with multiple emitters per labeled site.

<p>(a) Number of localizations per neutravidin tetramer as a function of DOL as estimated from the image correlations and the ground truth values from cluster analysis, showing good agreement. (b) Fitted bleach rate <i>k</i>_<i>bl</i> and switching rate <i>k</i>_<i>sw</i> = <i>M</i>_∞<i>k</i>_<i>bl</i> as a function of DOL values for the same data, indicating independent activation and bleaching per label. Error bars indicate the standard deviation among samples at the same DOL. (c) Image of IgE receptors on the membrane of RBL cells labeled with primary antibodies with a DOL of 1.5 (<i>k</i>_<i>bl</i> = 9.1 × 10⁻³/s and <i>M</i>_∞ = 2.3).</p

Parameters used for filtering localization events.

<p>Localizations were filtered for the minimum number of photons per event before grouping, minimum number of photons per event after grouping, the maximum duration of the event after grouping, and the maximum width (FWHM) of the Gaussian fitted to the spot.</p

Tyrosine-protein kinase Yes controls endothelial junctional plasticity and barrier integrity by regulating VE-cadherin phosphorylation and endocytosis

Abstract Vascular endothelial (VE)-cadherin in endothelial adherens junctions is an essential component of the vascular barrier, critical for tissue homeostasis and implicated in diseases such as cancer and retinopathies. Inhibitors of Src cytoplasmic tyrosine kinase have been applied to suppress VE-cadherin tyrosine phosphorylation and prevent excessive leakage, edema and high interstitial pressure. Here we show that the Src-related Yes tyrosine kinase, rather than Src, is localized at endothelial cell (EC) junctions where it becomes activated in a flow-dependent manner. EC-specific Yes1 deletion suppresses VE-cadherin phosphorylation and arrests VE-cadherin at EC junctions. This is accompanied by loss of EC collective migration and exaggerated agonist-induced macromolecular leakage. Overexpression of Yes1 causes ectopic VE-cadherin phosphorylation, while vascular leakage is unaffected. In contrast, in EC-specific Src deficiency, VE-cadherin internalization is maintained and leakage is suppressed. In conclusion, Yes-mediated phosphorylation regulates constitutive VE-cadherin turnover, thereby maintaining endothelial junction plasticity and vascular integrity