Alk1-ED and Alk3-ED variants were generated by site directed mutagenesis of the corresponding expression plasmids using PCR (Quikchange, Stratagene, LaJolla, CA). All variants were expressed and purified as described for the corresponding wild type protein. Biacore 3000 biosensor instrument was used for quantitative measurement of the receptor-ligand interactions. BMP-9 and BMP-2 were diluted in 0.1 M acetic acid to a final concentration 2 – 6 μg/mL and coupled to the carboxymethylated dextran chip surface (CM5, GE Healthcare, Piscataway, NJ) using amine-coupling methods. The chip surface was first activated by injecting 35 μL of N-hydroxysuccinimide (NHS) and 1-ethyl-3′-(3-diethylaminopropyl) carbodiimide hydrochloride (EDC) followed by injection of 5 – 15 μL of the ligand to achieve the desired surface density (600–800 RU for BMP-9 and 2500 RU for BMP-2). Remaining activated groups were blocked by injecting 35 μL ethanolamine. One of the flow cells was activated and blocked without injecting any ligand as a reference surface. All injected samples were dialyzed against HBS-EP buffer (10 mM Hepes, 3 mM EDTA 500 mM NaCl, .002% surfactant P20, pH 7.4), centrifuged for 10 min at 50,000 x g, passed through a 0.2 mm filter prior to injection into the SPR instrument. Protein concentrations were determined based on the calculated extinction coefficient at 280 nm and the measured absorbance at this wavelength immediately prior to injection. Six or more concentrations of two-fold serial dilutions of the wild type and the variants of Alk1 and Alk3 were injected over the chip surface at a flow rate of 5 – 10 μL/min. All the injections were performed at room temperature were preceded by a brief injection of 2.5 M guanidine hydrochloride (20 μL/min for 12 seconds) to regenerate the surface. Instrument noise was removed by referencing the data against three or more buffer blank injections, while background signal was eliminated by referencing the data against a blank flow cell. Kds were determined by fitting the equilibrium binding response, Req, as a function of the injected receptor concentration, [R], to Req = (Rmax × [R])/(Kd + [R]) using the program Kaleidagraph (Synergy Software, Reading, PA).