Bone morphogenetic proteins (BMP) are dimeric factors initiating several di
stinct signaling cascades by binding to two types of transmembrane serine/t
hreonine kinase receptors (BRI and BRII), and are thus regulating several s
teps in embryonal development and adult tissue homeostasis. BMP-2 contains
two symmetrical pairs of juxtaposed epitopes: the wrist epitope with high a
ffinity to BRI consists of residues from both BMP-2 monomers, while the knu
ckle epitope resembles the low affinity site for BRII and comprises residue
s from only one monomer. Here we generated heterodimeric BMP-2 muteins with
one monomer mutant in either epitope I for BRI (el-) or epitope II for BRI
I (eII-) and the second monomer wild type for receptor interactions (m-). T
hese muteins (B2el-/B2m- and B2eII-/B2m-) were analyzed by biosensor analys
is as well as by measuring their biological activity and compared to their
homodimeric forms (either wild type or mutant). Depletion of only one epito
pe II results in the loss of biological activity as measured by alkaline ph
osphatase (ALP) activity and Smad induced reportergene assays. However, dep
letion of only one epitope I shows a reduction of ALP activity to about 25%
, while the activation of the Smad pathway remained normal. Homomeric mutei
ns are non-functional for both Smad and ALP activation. This suggests that
two functional epitopes II have to be present on one BMP-2 molecule for rec
eptor activation. Futhermore, both pathways (Smad and ALP) are triggered di
fferently by distinct BMP-receptor complexes. Heteromeric BMP-2 mutants the
refore allow a distinguishable manipulation of either pathway and thus repr
esent important tools for the generation of specific BMP-2 antagonists or a
gonists.