TGF- signaling and regulation: Elucidating molecular mechanisms and pathogenic functions of the 'co-receptor' Cripto-1 and the receptor BMPRII

This project presents and applies a high-throughput, biosensor-based surface plasmon resonance (SPR) approach to interrogate bone morphogenetic protein (BMP) receptor interactions that underlie TGF-β superfamily signaling. The method is built around producing the extracellular, BMP-binding domains of receptors as human IgG1-Fc fusion proteins, which allows straightforward capture of receptors on the SPR sensor surface via anti-Fc antibodies (or alternatively protein A). The Fc-fusion strategy supports a highly reproducible and uniform surface regeneration protocol, preserves the activity of the receptor moiety, and enables repeated, comparable binding cycles. In a typical workflow, receptor-Fc fusion proteins are captured to approximately 150–300 response units (RUs), and BMP ligands are injected over the captured receptors at a single concentration in the range of roughly 60 - 100 nM (80 nM used as a representative concentration in examples). Using this single-concentration, high-throughput format, the assay permits rapid stratification of multiple BMPs into high-, medium-, and low-affinity binders and allows estimation of equilibrium dissociation constants (Kd) for high- and medium-affinity interactions with both good accuracy and high precision from single-injection binding curves.

The methodology includes a robust regeneration cycle that ensures reproducible reuse of sensor surfaces: bound BMP receptor complexes are eluted with magnesium chloride (MgCl2), followed by buffer re-equilibration, a water wash to avoid MgCl2 accumulation and microfluidic clogging, and final buffer equilibration to return the sensor baseline. This regeneration protocol consistently restores baseline response within ±5 RU across multiple cycles, enabling repeated capture, binding, and regeneration steps without compromising data quality. The assay can be configured on multi-channel chips to allow sequential capture of different receptor-Fc fusion proteins and parallel testing of many BMP family members; one example demonstrates capture of three distinct receptor-Fc proteins on a four-channel sensor chip and injection of 15 different TGF-β/BMP family members over BMPRII-Fc to survey binding profiles.

Applied specifically to BMPRII-Fc, the approach revealed distinct association and dissociation kinetics for different ligands and identified activin B and nodal as among the strongest BMPRII-Fc binders in the tested panel, producing response curves on the order of ~100 - 110 RU under the described conditions. The protocol's reproducibility and sensitivity make it a useful platform for systematically mapping ligand receptor specificities across TGF-β superfamily members, supporting mechanistic studies of receptor function and ligand selectivity. By enabling rapid, quantitative comparisons of binding behavior across receptors and ligands, this SPR-based high-throughput workflow provides a practical foundation for deeper investigations into BMP signaling, BMPRII function, and broader TGF-β pathway regulation.