The BMP-7 pd interacts with the growth factor at sites close to the type II receptor binding sites. The extracellular domains of the receptors BMPRII, ActRIIA, BMPRIA, and BMPRIB (R&D Systems) were used as soluble ligands in ELISA binding assays with coated BMP-7 substrates. Binding of these ligands to the separated gfd (open circles) was compared with binding to the BMP-7 complex (filled squares). Binding of the receptor ligands was detected with monoclonal antibodies specific for each receptor (R&D Systems). Each data point represents the average value of a triplicate. Error bars are shown.

Binding of BMPRII to the BMP-7 complex results in displacement of the BMP-7 pd. (a) Reference runs of BMP-7 complex in TBS (bands at 70 kDa represent unprocessed BMP-7 full-length monomer), BMP-7 pd in TBS (immunoblotted with anti-BMP-7 pd antibody), and BMPRII (immunoblotted with anti-BMPRII antibody; R&D Systems). (b) Left panel: blots with anti-BMP-7 gfd antibody; right panel: blots with anti-BMP-7 pd antibody. At a BMP-7 complex/BMPRII molar ratio of 1:0.25, the BMP-7 gfd signal is shifted farther down in the gradient, resulting in three individual peaks (fractions 8, 10, and 12). Fractions 8 and 9 showed no pd signal (right side), indicating the presence of a freed BMP-7 gfd bound to the receptor domain. Immunoblotting for BMPRII showed the presence of the receptor in the same fractions as the freed BMP-7 gfd. At a molar ratio of 1:0.5 (BMP-7 complex/BMPRII), the equilibrium is shifted more toward the state of freed gfd-receptor complex and almost no free BMP-7 complex is detectable.

ActRIIA also displaces the BMP-7 pd. (a) Sucrose gradient of ActRIIA together with the BMP-7 gfd. Immunoblotting with anti-BMP-7 gfd showed a main peak in fractions 8 and 9, indicating the formation of a receptor-gfd complex, (b) Interaction of BMP-7 complex with ActRIIA. Immunoblotting of sucrose gradient fractions revealed the appearance of four peaks: free complex (fractions 11–14); complex bound to the receptor molecule (fractions 10–12); free gfd bound to the receptor (fractions 7–9); and high-molecular-weight complexes, caused by dimerization of the Fc domains of the receptor molecules, possibly consisting of two BMPRII-Fc dimers and multiple BMP-7 gfds (fractions 3–5).

Type I receptors, such as BMPRIA, do not displace the BMP-7 pd. (a) BMP-7 complex with BMPRIA at a molar ratio of 1:0.25. Samples were analyzed by immunoblotting for the BMP-7 gfd (left panel) or for the BMP-7 pd (right panel). Two individual overlapping peaks were recognizable: complex bound to receptor (fractions 9–11) and free complex (fractions 11–14). (b) Cartoon representation of the different species forming the two distinctive peaks in (a). At a BMP-7 complex/BMPRIA ratio of 1:0.25, BMP-7 complex species bound to the receptor were found next to free BMP-7 complex. A model of the BMP-7 complex with its type I receptor domains is shown. The pd does not obstruct the interaction site for the type I receptor ectodomains. Binding of one receptor molecule induces little to no displacement of the BMP-7 pd.

BIAcore studies of BMP-7 growth factor and BMP-7 complex show similar binding affinity to immobilized BMPRII or ActRIIA. BMP-7 pd binds with slightly lower affinity to the BMP-7 growth factor. (a) Interaction of BMP-7 gfd or complex in concentrations of 0–80 nM diluted in HBS-EP with 500 RU of BMPRII (upper panel) or ActRIIA (lower panel) immobilized on a CM5 chip. Nonspecific binding to the control surface has been subtracted at a molar ratio of 1:1. (b) Interaction of BMP-7 gfd in concentrations of 0–80 nM diluted in HBS-EP with BMP-7 pd (500 RU) immobilized on a CM5 chip.

BMP-7 complex and free BMP-growth factor are equally active. (a) BMP-2, BMP-7 gfd, and BMP-7 complex were added for 1 day to C3H/10T1/2 cells that were transiently transfected with an Msx2 reporter construct. Both BMP-7 species showed the same activation of Msx2 promoter-TK-luciferase reporter gene activity over basal levels for all concentrations (3.85–30.8 nM). BMP-2 served as a positive control, whereas BSA served as a negative control. (b) BMP-7 complex (7cplx) and BMP-7 (7gfd) were added in equal molar amounts increasing from 0.32 nM (corresponding to 10 ng/ml of BMP-7 gfd and 30 ng/ml of BMP-7 complex) to 3.2 nM to ATDC5 chondroprogenitor cells and incubated for 1 h. For positive and negative controls, cells were treated with equal amounts of BMP-2 gfd (2gfd; R&D Systems) and similar or higher nanograms of BSA. Smad phosphorylation, detected by Western blotting, was induced to the same extent by both BMP-7 species. Ponceau S stain shows the total transferred proteins, (c) ATDC5 cells were treated with BMP-7 at a constant concentration of 3.2 nM (100 ng/ml of BMP-7 gfd and 300 ng/ml of BMP-7 complex). Cells were examined after 20 min or up to 6 h by Western blotting for pSmad1/5/8. BMP-7 gfd and BMP-7 complex showed equivalent kinetics and persistence of pSmads. (d) C2C12 cells were also treated with BMPs in a similar time course experiment as in (c). Total RNA was harvested, and quantitative real-time RT-PCR assays were performed in triplicate for Id3, a BMP-responsive gene. The data are representative of two independent experiments. RNA levels of the ARBP PO gene, which are constant during treatment, were used to standardize samples. Fold expression values were calculated relative to the BSA-treated sample for each time point. BMP-7 gfd and BMP-7 complex resulted in equal induction of Id3.

BMPRII binds the BMP-7 complex in solution. BMP-7 gfd and BMP-7 complex were each dialyzed with BMPRII against TBS and were then applied to a 5%–20% sucrose gradient. Results were compared with reference runs with BMP-7 gfd alone or BMP-7 complex alone. Fractions were collected starting with fraction 1 (20%) to fraction 28 (5%) and were analyzed by immunoblotting with monoclonal anti-BMP-7 gfd antibody (R&D Systems). (a) Left panel: BMP-7 gfd (0.79 µM) was dialyzed together with BMPRII at a gfd/receptor molar ratio of 1:2.5, showing a signal in fractions 6–9 (peak fraction, 7); right panel: BMP-7 gfd in TBS sediments in fractions 18–22 (peak fractions, 20 and 21). (b) Left panel: BMP-7 complex (0.53 µM; molecular mass = ∼94.6 kDa) dialyzed at a molar ratio of 1:2.5 with BMPRII in TBS shows two signals in fractions 2 and 3 and fractions 6–11 (double peak), indicating three possible peaks; right panel: BMP-7 gfd (molecular mass=31.6 kDa) signals of BMP-7 complex in TBS were detected in fractions 11–14 (peak fractions, 12 and 13). Signals ∼60–70 kDa represent small amounts of unprocessed BMP-7 full-length monomer detectable only by blotting.

BMPRII displaces the BMP-7 pd as a dimer from the complex. (a) BMP-7 complex (0.53 µM) incubated with 1.32 µM BMPRII (1:2.5) and analyzed by immunoblotting for the BMP-7 gfd and the BMP-7 pd (left panel) and for the BMP-7 pd only (right panel) after velocity sedimentation. Three individual but overlapping peaks were recognizable, indicating the formation of three species: BMP-7 gfd bound to the BMPRII (fractions 5–8); BMP-7 complex bound to the receptor (fractions 9–13); and freed pd dimer (fractions 14–19). (b) Cartoon representation of a dynamic equilibrium of the different tentative interaction intermediates forming the three distinctive peaks in (a): BMP-7 gfd species with no attached pd bound to receptor; BMP-7 complex bound to the receptor; and freed BMP-7 pd, which is displaced from the complex by the BMPRII. Each BMP gfd has two independent interaction sites for its type I (solid circle) and type II (dashed circle) receptors.

The reconstituted GDF-8 complex does not bind to its type II receptor, ActRIIB. GDF-8 complex was successfully reconstituted by dialyzing the gfd together with its pd (both components were purchased from R&D Systems). The reconstituted GDF-8 complex (0.1 µM) sample was dialyzed together with ActRIIB (0.05 µM) in TBS. The signals for the GDF-8 gfd (upper left panel) and pd (upper right panel) as well as for the receptor (lower left panel) remain unaffected in the presence of each other, compared with the reference runs (ActRIIB alone, lower right panel; reconstituted GDF-8 complex alone, fractions 10–15; GDF-8 pd alone, fractions 19–222), indicating that no higher-molecular-weight complex was formed and no interaction could be detected.

BMPRII and BMP-7 pd compete for the BMP-7 growth factor. (a) Competition ELISA between BMP-7 pd and BMPRII. BMP-7 pd (separated from the BMP-7 complex) was immobilized on a microtiter plate using mAb2 (anti-BMP-7 pd) as a capture antibody. BMP-7 gfd was incubated either in increasing amounts (0–0.5 µM) without BMPRII (filled black squares connected by solid line, left) or at a constant concentration of 0.5 µM in the presence of increasing amounts of BMPRII (0–2.0 µM BMPRII; empty cycles connected by dashed line, right). The signals represent amounts of BMP-7 gfd detected by using a biotinylated polyclonal anti-BMP-7 gfd antibody. (b) Displacement of the gfd from a preformed BMPRII-BMP-7 complex by the pd. BMPRII was coated at a concentration of 0.1 µM, and BMP-7 gfd was subsequently incubated at a constant concentration of 0.125 µM. Increasing amounts of BMP-7 pd were added in a second incubation step (0–2.0 µM BMP-7 pd; black squares connected by solid line) or, as a control, no BMP-7 pd (empty circles connected by dashed line). BMP-7 gfd was detected using the same antibody as in (a).