High-throughput, biosensor-based approach to examine bone morphogenetic protein (BMP)–receptor interactions

Abstract

Binding of a BMP to its cognate cell surface receptors is the initiating step in the BMP signaling cascade. Thus, knowing which BMP–receptor complexes form is vital for understanding the physiological activities of a particular BMP. Here, we describe a surface plasmon resonance (SPR)-based, high-throughput approach that allows fast identification and evaluation of BMP–receptor complexes. Briefly, the extracellular, BMP-binding domains of receptors are produced as human IgG1-Fc-fusion proteins. The Fc moiety enables simple capture of the Fc-receptor-fusion protein on the sensor chip, supports a highly reproducible, uniform approach of surface regeneration, and ensures full activity of the receptor moiety. BMPs are injected over the captured receptors at one concentration (approximately 60–100 nM), permitting stratification of high-affinity, medium-affinity, and low-affinity binders. Using this concentration range, equilibrium dissociation constants for high-affinity and medium-affinity binders can be estimated with good accuracy and with great precision from the single injection binding curves.