BMP RECEPTOR BINDING PROTIENS

Bone morphogenetic proteins (BMPs) are members of the transforming

growth factor-b (TGF-) superfamily, which includes TGF- s, activins, inhibins,
and Mullerian inhibiting substance.31 BMPs play multiple roles in regulation of
growth, differentiation, and apoptosis of various cell types. They exhibit important
in vivo functions during embryonal development and tissue morphogenesis,
including bone and cartilage formation.
Members of the TGF- superfamily exert their biological effects via binding
to two types of serine/threonine kinase receptors (type I and type II), both of which
are required for signaling activity. The type II receptor transphosphorylates type I
receptor, leading to the activation of intracellular substrates, including Smad (Sma
and Mad) proteins. Since the type I receptor acts as a downstream component of
the type II receptor, specificities of the intracellular signals are determined by the
type I receptor. BMPs bind to three different type II receptors, i.e., BMP type II
receptor (BMPR-II) and activin type II and type IIB receptors (ActR-II and ActRIIB). Among type I receptors, BMP type IA (BMPR-IA, originally termed activin
receptor-like kinase 3 or ALK-3) and type IB receptors (BMPR-IB/ALK-6)
specifically bind BMPs.13 ALK-2 was originally identified as an activin type I
receptor (Act R-I), but recent findings have shown that it functions as a type I
receptor for certain types of BMPs, e.g.,BMP-7/OP-1 (osteogenic protein-1),
instead of activins.32 Smad proteins are signal transducers for the serine/threonine
kinase receptors. Below is discussed the signaling mechanism by Smad proteins,
focusing in particular on BMP signaling pathways.

Structures and Functions of Smad Proteins

Eight Smad proteins (Smads 1 through 8) have thus far been isolated in
mammals.33 Smads can be classified into three subtypes by structure and function,
i.e., receptor-regulated (or pathway-restricted) Smads (R-Smads), commonmediator Smads (co-Smads), and inhibitory Smads (anti-Smads). R-Smads are the
prototype of Smad proteins, which can be further classified into those activated by
BMP receptors and those activated by TGF-b and activin receptors. Smad 1, Smad
5, and Smad 8 (originally termed MADH 6) are R-Smads activated by BMP
receptors, whereas Smad 2 and Smad 3 are R-Smads activated by TGF-b and
activin receptors. Smad 4 is the only co-Smad identified in mammals. Smad 6 and
Smad 7 are anti-Smads.
In the NH2- and COOH-terminal regions, Smads have conserved regions
termed Mad homology-1 (MH1) and -2 (MH2) domains, respectively, which are
bridged by a linker region of variable length and amino acid sequence. Both MH1
and MH2 domains are observed in R- and co-Smads, but a typical MH1-like
structure is not found in anti-Smads. In addition, R-Smads, but not other types of
Smads, have a Ser-Ser-X-Ser (SSXS) motif in their most COOH-terminal regions.
R-Smads serve as direct substrates of type I serine/threonine kinase
receptors. They directly interact with and are phosphorylated at their COOHterminal SSXS motif by type I receptors.34 Activation of type I receptor by type II
receptor is required for direct interaction between type I receptor and R-Smad;
however, the kinase activity of type I receptor is not required for this interaction.35
Since R-Smads are rapidly released from type I receptor after phosphorylation,
their interaction can be detected in vitro only when type I receptor or R-Smads are
functionally inactive. After phosphorylation by serine/threonine kinase receptors,
R-Smads interact with coSmad to form hetero-oligomeric complexes, which then

translocate into the nucleus and regulate the transcription of various target genes.
Our recent finding, together with the finding that the MH2 domain of Smad 4
forms a trimer in solution, suggested that the hetero-oligomers may be
heterotrimers, composed of two and one molecules, or one and two molecules of
R-Smads and Smad 4, respectively.36 Although phosphorylated R-Smads can form
oligomers and translocate into the nucleus even in the absence of co-Smad, coSmad stabilizes the structures of the Smad oligomers and is thus required for
efficient transcriptional activity of the Smad complexes.
R-Smads activated by BMP receptors appear to be essential and probably
sufficient for the differentiation of osteoprogenitor cells into osteoblasts induced
by BMPs.24,34BMPs bind to three different type I receptors, i.e., ALK-3/BMPRIA, ALK-6/BMPR-IB, and ALK-2/ActR-I, which in turn activate Smads 1, 5, and
8. Smads 1 and 5 have been shown to induce the differentiation of C2C12 cells
even in the absence of stimulation by ligands or BMP receptors.

37

R-Smads are

observed throughout the cell in the absence of ligand stimulation and they cannot
efficiently induce differentiation of C2, C12 cells; however, R-Smads translocate
into the nucleus upon receptor activation, and induce cellular differentiation.
Nuclear translocation is thus one of the most critical events in the function of RSmads.

Activities of Smads in the Nucleus

In the nucleus, Smad proteins exert transcriptional activity through direct
binding to DNA as well as through association with other DNA-binding proteins.
The MH1 domain is responsible for the DNA-binding of Smad proteins.
Drosophila Mad, which is structurally similar to Smads 1, 5, and 8 was the first to
be shown to bind to DNA.38 Subsequently, Smad3 and Smad4have been shown to
bind to specific DNA sequences through the MH1 domains.39 Direct DNA-binding
has been demonstrated for Smad3 and Smad4, but not for Smad 2. Smads 2 and 3
are structurally and functionally highly similar to each other, but our recent data
revealed that a unique 30-amino acid region encoded by exon 3 of the Smad 2 gene
and located in the middle of the MH1 domain interferes with the binding of Smad
2 to DNA. This finding was further supported by analysis of the three-dimensional
structure of the MH1 domain of Smad 3. Smad 2 is functionally less potent than
Smad3 with regard to transactivation of p3TP-lux promoter. Smad 2 that lacks
exon 3 is able to bind to DNA, and thus has transcriptional activity as potent as that
of Smad 3. R-Smads activated by TGF- receptors specifically bind to the DNA
sequence AGAC or its complementary GTCT sequence. Drosophila Mad
binds to the GCCGNCGC sequence; however, the consensus sequence for the
binding of Smads 1, 5, and 8 remains to be determined.
Smads interact with specific DNA sequences via other DNA binding
proteins. Xenopus FAST-1 and its mammalian homologues associate with Smad 2,
and play important roles in the transcription of certain activin signals. Other
transcription factors, including c-Jun/c-Fos, 38have also been shown to interact
with Smad 3. Thus far, no proteins that specifically interact with R-Smads
activated by BMP receptors have been reported in mammals. Smads 2 and 3

interact with transcriptional coactivators, p300/CBP, upon ligand stimulation; this

interaction may be crucial for the efficient transcriptional activation of
targetgenes.40
The MH2 domain plays important roles in various functions of Smads,
including receptor interaction, oligomer formation, and transcriptional activation.
Interaction of R-Smads with FAST-1 as well as with p300/CBP is also mediated
through the MH2 domain of R-Smads. The MH2 domain appears to be important
for the interaction of Smads with most other proteins, although Smad 3 interacts
with c-Jun through the MH1 domain.41 R-Smads are activated by BMP receptors
in osteoblastic cells as well as in cells of many other types. Smads 1 and 5 can
efficiently induce the differentiation of C2C12 cells into osteoblast-like cells when
they translocate into the nucleus. However, most cells types expressing BMP
receptors and their downstream Smad proteins do not differentiate into osteoblastlike cells. It is possible that DNA-binding proteins interacting with Smads are at
least in part responsible for the determination of cellular fate after BMP
stimulation.