A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

Abstract

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.

Characterization of Fibrodysplasia Ossificans Progessiva relevant Acvr1/Acvr2 Activin receptors in medaka (Oryzias latipes)

“…To investigate whether type II receptor domains that are implicated in human FOP pathology are conserved in medaka, we analyzed ligand binding domains (LBDs) by sequence comparison and performed a ligand surface binding assay (LSBA) [ 24 ] using fluorescently labelled hsActivin A-Cy5 ( Fig 5 , S6 Fig ). Sequence comparison of the hsACVR2A LBD with that of olaAcvr2ab highlighted subtle differences in two hydrophobic amino acids (Phe61 hsACVR2A / Tyr61 olaAcvr2ab, Ile83 hsACVR2A / Met83 olaAcvr2ab).…”

“…Together, this suggests that Activin A binding to medaka olaAcvr2ab, olaAcvr2ba and olaAcvr2bb is likely to be similar as in the human counterparts. To test this hypothesis, we performed LSBA [ 24 ] using COS7-cells that transiently expressed Halo-tagged hsACVR2A, hsACVR2B, and olaAcvr2ab, olaAcvr2ba and olaAcvr2bb. As expected from previous studies, strong hsActivin A-Cy5 binding was observed for cells expressing hsACVR2A-Halo and hsACVR2B-Halo but not the non-binding receptor hsTGFBR2-Halo ( Fig 5B , S6B Fig ).…”

“…By this, the gain of function mutation allows Activin A induced SMAD1/5/8 signaling [ 14 ]. Since hsACVR1 alone is a weak Activin A binder [ 24 ], hsACVR2B is crucial in mediating Activin A binding towards hsACVR1. Here, hsACVR2B binds hsActivin A with high affinity and subsequently interacts with T203 within the GS-box of hsACVR1 [ 52 ].…”

“…To investigate whether type II receptor domains that are implicated in human FOP pathology are conserved in medaka, we analyzed ligand binding domains (LBDs) by sequence comparison and performed a ligand surface binding assay (LSBA) [24]…”

Uncovering Tumor‐Promoting Roles of Activin A in Pancreatic Ductal Adenocarcinoma

“…[ 6 ] Activin A acts through its binding to activin type 2 receptors (ACVR2A or ACVR2B), which recruits type 1 receptors to form a receptor complex. [ 7 ] Then, intracellular signaling transductions are mediated by two pathways: the canonical pathway, where SMAD3 is phosphorylated and recruits SMAD4 to acquire transcriptional activity, and the noncanonical pathway, where subfamilies of the mitogen‐activated protein kinases including p38 activate downstream molecules. [ 8 ] SMAD3‐dependent signaling is indispensable for the proliferation of ovarian granulosa cells by activin A, and the downstream targets, Inha and Fst , feedback inhibits activin A‐mediated proliferation.…”