Characterization and Cloning of a Receptor for BMP-2 and BMP-4 from NIH 3T3 Cells

Abstract

The bone morphogenetic proteins (BMPs) are a group of transforming growth factor ,B (TGF-Pi)-related factors whose only receptor identified to date is the product of the daf-4 gene from Caenorhabditis elegans. Mouse embryonic NIH 3T3 fibroblasts display high-affinity 125I-BMP-4 binding sites. Binding assays are not possible with the isoform 125I-BMP-2 unless the positively charged N-terminal sequence is removed to create a modified BMP-2, 125I-DR-BMP-2. Cross-competition experiments reveal that BMP-2 and BMP-4 interact with the same binding sites. Affinity cross-linking assays show that both BMPs interact with cell surface proteins corresponding in size to the type I (57-to 62-kDa) and type II (75-to 82-kDa) receptor components for TGF-, and activin. Using a PCR approach, we have cloned a cDNA from NIH 3T3 cells which encodes a novel member of the transmembrane serine/threonine kinase family most closely resembling the cloned type I receptors for TGF-,B and activin. Transient expression of this receptor in COS-7 cells leads to an increase in specific 125I-BMP4 binding and the appearance of a major affinity-labeled product of -64 kDa that can be labeled by either tracer. This receptor has been named BRK-1 in recognition of its ability to bind BMP-2 and BMP-4 and its receptor kinase structure. Although BRK-1 does not require cotransfection of a type II receptor in order to bind ligand in COS cells, complex formation between BRK-1 and the BMP type II receptor DAF-4 can be demonstrated when the two receptors are coexpressed, affinity labeled, and immunoprecipitated with antibodies to either receptor subunit. We conclude that BRK-1 is a putative BMP type I receptor capable of interacting with a known type II receptor for BMPs.The transforming growth factor ,B (TGF-,B) superfamily contains a large number of growth, differentiation, and morphogenetic cytokines that are active as homo-or heterodimers.This superfamily includes the TGF-1 family, the activin family, the Mullerian inhibiting substance (MIS), and the bone morphogenetic protein (BMP)/Vg family, which composes the largest group (reviewed in reference 39). Human BMP-2 and BMP-4 and their Drosophila homolog dpp are highly related (74 to 76% sequence identity [72]), as are BMP-5, BMP-6, and BMP-7 and their Drosophila homolog 60A (69 to 73% sequence identity [21]). Given the high degree of structural similarity among these family members, it is expected that their receptors will also form a family of related molecules. activin (2, 40) have been cloned. It has recently been determined that the product of the daf-4 gene from Caenorhabditis elegans, which is involved in both inhibition of dauer larva formation and exit from the dauer stage, is a type II receptor for BMP-2 and BMP-4 (24). Each of these receptors is predicted to be a transmembrane serine/threonine kinase. Ligand binding to each of these type II receptors in a variety of cell types does not appear to require coexpression of additional receptor components, indicating that type II receptors are capable of...

A Kinase Domain-truncated Type I Receptor Blocks Bone Morphogenetic Protein-2-induced Signal Transduction in C2C12 Myoblasts

“…2 This suggests that the BMP-2 signals are transduced by the complex of BMPR-IA and BMPR-II in these cells. It was reported that both BMPR-IA and BMPR-IB formed complexes with BMPR-II or DAF-4, a type II receptor for a homologue of BMP-2/BMP-4 in C. elegans, in a ligand-dependent manner when overexpressed together on the surface of COS cells (21,22,(25)(26)(27). In contrast to the truncated BMPR-IA, the kinase domain-truncated BMPR-IB did not inhibit the BMP-2 signaling in C2C12 cells even if its mRNA was abundantly expressed.…”

“…The structure of the type I receptor is conserved in all type I receptors of the TGF-␤ superfamily, suggesting that the BMP signals are transduced by the BMP type I receptor like those of TGF-␤. Two type I receptors (BMPR-IA and BMPR-IB) and one type II receptor (BMPR-II) have been identified for BMP-2 and BMP-4 (21)(22)(23)(24)(25)(26)(27). BMP-7 can interact with activin type I and type II receptors in addition to BMP-2/BMP-4 receptors (26,28).…”

The BMP signaling and in vivo bone formation

“…BMP signals are mediated by type II and type I serine/ threonine kinase receptors. Three type I receptors have been shown to bind BMP ligands, type IA and IB BMP receptors (BMPR-IA or ALK-3 and BMPR-IB or ALK-6) and type IA activin receptor (ActR-IA or ALK-2) (Koenig et al, 1994;ten Dijke et al, 1994;Macias-Silva et al, 1998). Three type II receptors for BMPs have also been identified and they are type II BMP receptor (BMPR-II) and type II and IIB activin receptors (ActR-II and ActR-IIB) (Yamashita et al, 1995;Nohno et al, 1995;Rosenzweig et al, 1995;Kawabata et al, 1995).…”

Human Bone Morphogenetic Protein 2 Contains a Heparin‐Binding Site which Modifies Its Biological Activity

“…Physical binding studies employing IT-BMP-2 revealed dissociation constants, Kd, of 2-5 nM with the type I1 daf-4 receptor from Cuenorhubditis elegans (Estevez et al, 1993). Type I receptors expressed in COS cells, showed K,, values of 0.2-1 nM (Graff et al, 1994;Koenig et al, 1994;Penton et al, 1994;Yamaji et al, 1994). Binding affinities were found to be increased after cotransfection with daf-4 (Brummel et al, 1994).…”