TGF-beta superfamily ligands are homo- or heterodimeric and recruit two type I and two type II Ser/Thr kinase receptors to initiate a transmembrane signaling cascade. Even with the known structure of the homodimer ligands in complex with extracellular domains of both receptor types, the sequential assembly of the signaling complex with its cognate receptors in the cell membrane remains elusive. We generated a bone morphogenetic protein-2/-6 heterodimer carrying two asymmetric interfaces for each receptor type. We demonstrate that the heterodimer possesses high affinity to both receptor types and increased Smad1-dependent signaling activity by both cell-based and chondrogenesis assays. Furthermore, we find that the minimal signaling complex consists of two type II receptors and one type I receptor per dimer. Our study reveals how the engineered heterodimers may use their independent binding interfaces to differentially recruit the different receptors for each receptor type to create new biological properties.
"The cells were incubated at 37°C under a humidified condition of 5% CO2 and routinely subcultured using trypsine-EDTA when the cell density reached around 80% confluence. Smad1-dependent luciferase report assays were performed as previously described [18,24,35]. In short, cultured C2C12 cells were trypsinized, washed once with PBS, resuspended in OptiMEM (Invitrogen, USA) plus 0.1% FBS and seeded in 96-well plates at 15,000 cells per 80 μL per well. "
[Show abstract][Hide abstract] ABSTRACT: The development of chemical refolding of transforming growth factor-beta (TGF-beta) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-beta superfamily ligands could not be refolded readily by the same methods.
Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9.
MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-beta superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-beta chimeras for activity screen is also discussed.
"Several studies have explored the importance of the mode of binding of BMPs to receptor subunits. The cellular response to BMPs has been shown to depend on the mode of receptor oligomerization [17,18,34]. BMP binding to preformed receptor complexes drives Smad-dependent, transcriptional pathways, whereas BMP-induced receptor subunit assembly leads to non-transcriptional responses, such as cytoskeletal rearrangements [17,18]. "
[Show abstract][Hide abstract] ABSTRACT: In dorsal spinal neurons and monocytes, bone morphogenetic protein (BMP)7 activates distinct transduction pathways, one leading to inductive specification and the other to axon orientation and chemotaxis. BMP7-evoked induction, also stimulated by the closely related BMP6, acts through a Smad cascade, leading to nuclear signaling, and is not BMPR subunit selective. Orientation is evoked by BMP7, but not by BMP6, through PI3K-dependent cytoskeletal activation mediated by the type II BMPRs, ActRIIA and BMPRII and is independent of the Smad cascade. The responses can be stimulated concurrently and suggest that BMP7, but not BMP6, can selectively activate BMPR subunits that engage the divergent paths. Although structural and biochemical analyses of selected BMP/BMPR interfaces have identified key regions of interaction, how these translate into function by related BMPs is poorly understood. To determine the mechanisms underlying the distinct activities of BMP7 and the disparate properties of BMP7 and BMP6 in spinal cord development, we have performed a family-wide structure/function analysis of BMPs and used the information to predict and test sites within BMPs that may control agonist properties, in particular the ability of a BMP to orient axons, through interactions with BMPRs.
We demonstrate that whereas all BMPs can induce dorsal neurons, there is selectivity in the ability also to orient axons or evoke growth cone collapse. The degree to which a BMP orients is not predictable by overall protein similarity with other BMPs but comparison of sequences of potent and weakly orienting BMPs with that of the non-orienting BMP6 revealed three candidate positions within the BMPs at which the amino acid residues may confer or obstruct orienting ability. Residue swapping analysis has identified one residue, Gln48 in BMP6, that blocks axon orienting ability. Replacing Gln48 with any of the amino acids present at the equivalent residue position in the orienting subset of BMPs confers orienting activity on BMP6. Conversely, swapping Gln48 into BMP7 reduces orienting ability. The inductive capacity of the BMPs was unchanged by these residue swaps.
The results suggest that the presence of the Gln48 residue in BMP6 is structurally inhibitory for BMP/BMPR interactions that result in the activation of intracellular signaling, leading to axon orientation. Moreover, since residue 48 in BMP7 and the corresponding residue in BMP2 are important for type II BMPR binding, our results provide a basis for a mechanistic understanding of the diverse activities of BMPs in spinal cord development.
Neural Development 05/2012; 7(1):16. DOI:10.1186/1749-8104-7-16 · 3.45 Impact Factor
"In addition, the minimal receptor complex required for BMP versus TGF-β signaling appears to differ. While a heterotrimeric (type I:type II:type II) BMP receptor complex is minimally required to transduce BMP signals (Isaacs et al., 2010), autonomously functioning TβRI:TβRII (type I:type II) heterodimers have been shown to be sufficient for the transduction of TGF-β signals (Huang et al., 2011). "
[Show abstract][Hide abstract] ABSTRACT: Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal dominant disorder characterized by episodic deposition of heterotopic bone in place of soft connective tissue. All FOP-associated mutations map to the BMP type I receptor, ALK2, with the ALK2(R206H) mutant form found in the vast majority of patients. The mechanism(s) regulating the expressivity of hyperactive ALK2(R206H) signaling throughout a patient's life is not well understood.
In Drosophila, human ALK2(R206H) receptor induces hyperactive BMP signaling. As in vertebrates, elevated signaling associated with ALK2(R206H) in Drosophila is ligand-independent. We found that a key determinant for ALK2(R206H) hyperactivity is a functional type II receptor. Furthermore, our results indicate that like its Drosophila ortholog, Saxophone (Sax), wild-type ALK2 can antagonize, as well as promote, BMP signaling.
The dual function of ALK2 is of particular interest given the heterozygous nature of FOP, as the normal interplay between such disparate behaviors could be shifted by the presence of ALK2(R206H) receptors. Our studies provide a compelling example for Drosophila as a model organism to study the molecular underpinnings of complex human syndromes such as FOP.
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