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.
Keywords: Activin; Bone morphogenetic protein; Protein–protein interaction s; Surface plasmon resonance; TGF-β.