The present work describes 2 mutations in the BMP-like signaling molecule GDF5 that give rise to the human limb malformation syndromes BDA2 and SYM1, previously shown to be associated with mutations in BMPR1B
, respectively. Prediction of the GDF5 structure based on the known structure of the BMP2–BMPR1A complex implied that both mutations are located within the interaction interface of ligand and receptor. We therefore hypothesized that the GDF5 mutants display altered receptor-binding affinities and by this mechanism interfere with regular GDF5 signaling. We performed binding studies with the GDF5 mutants and showed that the L441P mutant has a dramatically reduced affinity to BMPR1B. In contrast, no difference in binding to NOG was observed, confirming that the gross tertiary structure of the mutant was not significantly altered. In accordance with these findings, we observed a severe reduction of chondrocyte differentiation and cartilage formation by the L441P mutant in our cell assays, suggesting lack of signaling through BMPR1B. However, the mutation is unlikely to result in complete inactivation because loss-of-function mutations in GDF5
cause BDC, a condition characterized by a distinct and more severe phenotype (33
Ligand cross-linking experiments showed that GDF5 binds efficiently to singly expressed type 2 and type 1b receptors, but positive signaling activity was only detected when both receptors were present (4
). More recent studies have shown that BMP receptors (types 1a, 1b, and 2) form homomeric and heteromeric complexes even in the absence of ligand and that both type 1 receptors have similar affinities to their ligands (34
). Since the L441P mutation affects only the type 1 receptor-binding site, the mutant should still be able to bind to the BMPR2 receptor and may consequently elicit a negative effect on the entire ligand-receptor complex. This effect is likely to be intensified by the fact that 75% of the GDF5 dimer molecules will contain at least 1 mutated molecule. The L441P mutation reported here to cause BDA2 was previously described in a cosanguineous Pakistani family with recessive DuPan syndrome (20
). This condition also belongs to the group of acromesomelic dysplasias but is less severe than the Grebe and Hunter-Thompson dysplasias. The DuPan phenotype resembles a homozygous loss-of-function mutation in BMPR1B
, as recently described (23
). Thus, the similarities between the L441P-associated phenotypes and those associated with BMPR1B
mutations are striking and strongly support our conclusion that the L441P mutation results in a selective loss of GDF5 signaling through the BMPR1B receptor. Differences between BDC and the L441P phenotype on one hand and DuPan and BMPR1B-associated phenotype on the other are likely to be due to the binding of GDF5 to other receptors and/or negative effects of the L441P mutant on the entire signaling cascade.
The R438L mutation described here causes a phenotype indistinguishable from that observed in individuals with NOG
). However, as shown in cell culture and by our in vitro binding assays, binding of the R438L mutant to NOG was unaltered in spite of the fact that R438 lies within the NOG interaction region, making it unlikely that the SYM1 phenotype described here is caused by an abnormal interaction of the mutant protein with NOG. Structural and mutational analysis of the BMP2 and BMPR1A binding sites revealed a specific type of protein-protein interface consisting of a large hydrophobic contact area (36
). The residues L51 and D53 (corresponding to L437 and S439 in GDF5, respectively) are particularly exposed and essential for binding to BMPR1A. Interestingly, R438 resides between these 2 residues within this receptor interaction site. R438 is conserved throughout species and within different GDFs but differs markedly in other BMPs, including the drosophila BMP analog DPP, in which the positively charged amino acid arginine is replaced by the nonpolar and hydrophobic alanine. In the mutant, R438 is replaced by the hydrophobic residue leucine, resulting in a GDF5 with BMP2-like properties. Mutagenesis of these sites in BMP2 has shown altered binding affinities for BMPR1A (5
). For example, converting L51 into proline resulted in a complete loss of binding to the type 1 receptor but normal binding to NOG and the type 2 receptor (36
). Our binding studies show that the R438L mutant is still able to bind to BMPR1B with high affinity. However, in contrast to WT GDF5, the mutant is also able to bind to BMPR1A, albeit with lower affinity than BMP2. Thus the R438L mutation converts GDF5 into a molecule with BMP2-like properties.
This hypothesis is supported by our cell assays. The R438L mutant is highly active in all tests and appears to be even more potent than WT GDF5. The major difference between the mutant and the WT protein, however, becomes apparent through testing the differentiation of C2C12 cells into osteoblasts versus myoblasts. C2C12 cells are mesenchymal progenitor cells that spontaneously differentiate into muscle cells when reaching confluence. Treatment with BMPs resulted in differentiation along the osteoblastic lineage, as shown by a drastic increase in ALP activity. GDF5 has no effect on this phenomenon, presumably because Bmpr1b is, in contrast to Bmpr1a, nearly absent in C2C12 cells (2
). In this assay, the R438L mutant behaves like BMP2, thus inducing osteoblastic differentiation and inhibiting muscle differentiation. We conclude that the R438L mutant GDF5 binds to both BMPR1A and BMPR1B, loses its preferential binding to BMPR1B, and thus takes on receptor affinities similar to those of BMP2.
The consequences of this double signaling activity of the R438L mutant can be 2-fold. First, as shown in our in vitro assays, the mutant is likely to be more active and will thus enhance the natural function of WT GDF5. Second, the activation of the BMP2/BMPR1A pathway may result in aberrant signaling interfering with the normal function of GDF5. In situ hybridization experiments on developing joints in the mouse indicated that signaling through the 1a receptor is possible within the Gdf5 expression domain. Bmp2-induced signaling was tightly regulated on the level of expression and the local release of inhibitors such as Chordin and Nog, indicating that the activation of aberrant Bmp2-like signaling is likely to result in a disturbance of joint formation. To study a gain of function in vivo, we overexpressed WT Gdf5 and mutant Gdf5 in chicken limb buds. We observed joint fusions and overall enlargements of the cartilaginous anlagen similar to that found in previous studies (10
) consistent with the proposed function of Gdf5 as a cartilage inducer. Similar results were obtained for WT Gdf5 and mutant Gdf5, indicating that the mutant proteins retained WT activity. Treatment of limb buds by implantation of BMP2-soaked beads resulted in the induction of apoptosis similar to that in vivo, in which BMPs are thought to be involved in the regulation of apoptosis in the interdigital mesenchyme (11
). In particular, Bmp2 is able to induce apoptosis through a Smad-independent, PKC-dependent signaling pathway (38
). We did not observe an increase in apoptosis when expressing WT Gdf5 and mutant Gdf5 in chick limbs, which shows that the increased activity of the R438L mutant had no major effect on this pathway. These results indicate that the R438L mutant elicits its pathology through a gain of function, probably by recruiting additional type 1a receptors expressed in the region at the critical time of joint formation. Thus, as shown schematically in Figure , the lack of joint formation observed in SYM1 was induced either by overactive GDF5 or by downregulated NOG. Both result in an imbalance of signal versus inhibitor, leading to the persistence of cartilage in the future joint interzone. In this model, proliferation and recruitment of mesenchymal cells caused by GDF5 is inhibited by NOG in the center of the joint interzone, resulting in dedifferentiation of interzone cells. The subsequent downregulation of NOG expression in these cells and the upregulation of BMP2 expression finally permit apoptosis and joint formation. In contrast, selective inhibition of GDF5 as in the L441P mutant primarily affects longitudinal growth and thus causes shortening or loss of individual skeletal elements.
Figure 7 Schematic drawing of proposed mechanism of metacarpophalangeal joint development and expression of Gdf5, Nog, and Bmp2 in mouse limbs corresponding to E12.5, E13.0, E13.5, and E14.5. Gdf5 induces longitudinal growth at the distal end of each element. (more ...)
The study of the mutations described here extends our knowledge of human GDF5-related phenotypes. Furthermore, the functional analysis showed that mutations in different genes of the same pathway can result in identical phenotypes, provided the mutations have similar effects on the affected signals. Inactivation of the ligand-receptor complex as in BMPR1B receptor mutants causes a BDA2 phenotype identical to the L441P mutation in GDF5 described here. Likewise, the phenotype caused by inactivation of the BMP-antagonist NOG can be mimicked by a gain-of-function mutation in GDF5 that results from a loss of signaling specificity. The present experiments localize some of the main determinants of GDF5-binding specificity, suggesting that GDF5 with altered binding affinities may function as an inhibitor of BMP signaling. The study of such mutated GDF5 peptides may improve our ability to make use of the pharmacological properties of GDF5 in the clinical applications of fracture healing and tendon and nerve regeneration.