Receptor oligomerization and beyond: a case study in bone morphogenetic proteins

Abstract

Background: Transforming growth factor (TGF)beta superfamily members transduce signals by oligomerizing two classes of serine/threonine kinase receptors, termed type I and type II. In contrast to the large number of ligands only seven type I and five type II receptors have been identified in mammals, implicating a prominent promiscuity in ligand-receptor interaction. Since a given ligand can usually interact with more than one receptor of either subtype, differences in binding affinities and specificities are likely important for the generation of distinct ligand-receptor complexes with different signaling properties.

Results: In vitro interaction analyses showed two different prototypes of binding kinetics, 'slow on/slow off' and 'fast on/fast off'. Surprisingly, the binding specificity of ligands to the receptors of one subtype is only moderate. As suggested from the dimeric nature of the ligands, binding to immobilized receptors shows avidity due to cooperative binding caused by bivalent ligand-receptor interactions. To compare these in vitro observations to the situation in vivo, binding studies on whole cells employing homodimeric as well as heterodimeric bone morphogenetic protein 2 (BMP2) mutants were performed. Interestingly, low and high affinity binding sites were identified, as defined by the presence of either one or two BMP receptor (BMPR)-IA receptor chains, respectively. Both sites contribute to different cellular responses in that the high affinity sites allow a rapid transient response at low ligand concentrations whereas the low affinity sites facilitate sustained signaling but higher ligand concentrations are required.

Conclusion: Binding of a ligand to a single high affinity receptor chain functioning as anchoring molecule and providing sufficient complex stability allows the subsequent formation of signaling competent complexes. Another receptor of the same subtype, and up to two receptors of the other subtype, can then be recruited. Thus, the resulting receptor arrangement can principally consist of four different receptors, which is consistent with our interaction analysis showing low ligand-receptor specificity within one subtype class. For BMP2, further complexity is added by the fact that heterooligomeric signaling complexes containing only one type I receptor chain can also be found. This indicates that despite prominent ligand receptor promiscuity a manifold of diverse signals might be generated in this receptor limited system.

Figure 1

Experimental layout. Model of biosensor experiments with ligands as analyte passed over immobilized receptor ectodomains (ECDs) (a), receptor ECDs passed over immobilized ligands (b) and ternary complexes formed by perfusing an immobilized type I receptor with the ligand plus the ECD of a type II receptor (c).

Figure 2

Influence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for ligand-receptor interaction. Binding affinities of the ligands bone morphogenic protein (BMP)2, BMP7 and growth and differentiation factor (GDF)5 to the immobilized type I receptors BMP receptor (BMPR)-IA and BMPR-IB (a) and those of the same ligands plus activin A to the type II receptors activin receptor (ActR)-II, ActR-IIB, and BMPR-II (b) are depicted as bar diagrams. The data represent mean values of two individual experiments using six different ligand concentrations. Standard deviations are indicated by error bars.

Figure 3

Binding of radiolabeled proteins on cell surfaces. (a) Dose-dependent binding of iodinated core bone morphogenic protein (wild type) (coreBMP2wt) to C2C12 cells (total binding, black squares). Unspecific binding as determined by addition of a 1,000-fold excess of cold ligand (blue diamonds) was subtracted resulting in specific binding of the ligand (red stars). (b) Comparison of specific binding of coreBMP2wt to COS-7 cells transfected with either BMP receptor (BMPR)-IA or activin receptor (ActR)-IIB or cotransfected with both receptors chains. (c) Specific binding of the iodinated heterodimeric coreBMP2/L51P mutant to BMPR-IA transfected COS-7 cells using ligand concentration up to 4 nM. (d) Specific binding of a radiolabeled anti-BMPR-IA Fab fragment to either untransfected (black squares) or BMPR-IA transfected (red asterisks) COS-7 cells. At all cases specific binding was fitted to a one-site binding model resulting in the indicated values for K_D and B_max.

Figure 4

Binding of radiolabeled ligands using higher ligand concentrations. (a) Specific binding of core bone morphogenic protein (wild type) (coreBMP2wt) to BMP receptor (BMPR)-IA transfected results in a biphasic binding curve with a pronounced break (marked by arrow). Fitting the curve separately (0 to 500 pM and 1,000 to 4,000 pM) to a one-site binding model the indicated values for K_D and B_max were achieved. (b) Specific binding of coreBMP2wt to COS-7 cells transfected with activin receptor (ActR)-IIB and (c) to untransfected C2C12 cells.

Figure 5

Biological activity of bone morphogenic protein (BMP)2 variants. The dose-dependent induction of alkaline phosphatase (ALP) activity in serum starved C2C12 cells is shown for the indicated ligands BMP2 wild type (BMP2wt) (black squares), coreBMP2wt (red triangles) and the heterodimeric coreBMP2wt/L51P variant (blue asterisks). The background absorption at 405 nm of 0.09 ± 0.0075 was not subtracted to indicate the signal to noise ratio.

Figure 6

SMAD phosphorylation. (a) A concentration-dependent phosphorylation of SMAD-1 mediated by bone morphogenic protein (BMP)2 is shown at the indicated timepoints in C2C12 cells by western blotting. Analysis of SMAD-1 and actin levels acts as loading control. (b) Time-dependent phosphorylation of SMAD-1 by BMP2 at a concentration of 0.3 nM. (c) Diagram of the time-dependent phosphorylation levels of SMAD-1 without ligand or induced by BMP2 at the indicated concentrations. The data were obtained by scans of western blot exposures. pSMAD signals were quantified and normalized to total SMAD-1 levels using the software ImageJ. (d) Concentration-dependent phosphorylation of SMAD-1 mediated by heterodimeric core BMP wild type (coreBMP2wt)/L51P.

Figure 7

Inhibition of the SMAD and p38 mitogen-activated protein (MAP) kinase pathway. Alkaline phosphatase (ALP) assays were carried out using C2C12 cells in the absence or presence of 250 nM of bone morphogenic protein (wild type) (BMP2wt). (a) Dorsomorphin (DM) or (b) SB203580 was added at the indicated timepoints. The background absorption at 405 nm of 0.09 ± 0.0075 was not subtracted to indicate the signal to noise ratio.