(Received for publication, June 7, 1995; and in revised form, July 7, 1995)
From the
Bone morphogenetic proteins (BMPs) comprise the largest
subfamily of TGF--related ligands and are known to bind to type I
and type II receptor serine/threonine kinases. Although several
mammalian BMP type I receptors have been identified, the mammalian BMP
type II receptors have remained elusive. We have isolated a cDNA
encoding a novel transmembrane serine/threonine kinase from human skin
fibroblasts which we demonstrate here to be a type II receptor that
binds BMP-4. This receptor (BRK-3) is distantly related to other known
type II receptors and is distinguished from them by an extremely long
carboxyl-terminal sequence following the intracellular kinase domain.
The BRK-3 gene is widely expressed in a variety of adult tissues. When
expressed alone in COS cells, BRK-3 specifically binds BMP-4, but
cross-linking of BMP-4 to BRK-3 is undetectable in the absence of
either the BRK-1 or BRK-2 BMP type I receptors. Cotransfection of BRK-2
with BRK-3 greatly enhanced affinity labeling of BMP-4 to the type I
receptor, in contrast to the affinity labeling pattern observed with
the BRK-1 + BRK-3 heteromeric complex. Furthermore, a
subpopulation of super-high affinity binding sites is formed in COS
cells upon cotransfection only of BRK-2 + BRK-3, suggesting that
the different heteromeric BMP receptor complexes have different
signaling potential.
Bone morphogenetic proteins (BMPs) ()are the largest
subfamily of growth factors in the TGF-
superfamily and have been
demonstrated to play important roles in endochondral bone formation and
embryogenesis(1, 2, 3, 4) . Like
other members of the TGF-
superfamily, BMPs appear to interact
with type I and type II receptors on the cell surface(5) .
Following the expression cloning of the type II receptors for activin
and TGF-
(6, 7) , an increasing number of
transmembrane serine/threonine (Ser/Thr) kinases have been identified
in mammals based on the conserved amino acid sequences in the
intracellular kinase domain(8, 9) . These include
three distinct mammalian type I receptors for BMPs that are
distinguished from the type I receptors for TGF-
(10, 11, 12) and activin (11, 13) by the capability of binding ligand on their
own when transfected into COS
cells(5, 14, 15) . BRK-1 (also known as
ALK-3, TFR11, and BMPR-IA) binds BMP-2 and BMP-4 more efficiently than
it binds BMP-7(5, 14, 15) . BRK-2 (also known
as ALK-6, BMPR-IB, and RPK-1(16) ) binds both BMP-4 and BMP-7
efficiently(15) . ActRI (also known as ALK-2 and SKR1) binds
both activin and BMP-7 but does not bind
BMP-4(15, 17) .
The only type II receptors that
have been identified for BMPs to date are from non-mammalian sources.
The product of the daf-4 gene from Caenorhabditis elegans binds both BMP-2 and BMP-4 (18) and forms a complex with
each of the BMP type I receptors in the presence of
ligand(5, 15) . The product of the punt gene
from Drosophila, originally identified as the Drosophila homologue of the activin type II receptor (AtrII (19) ),
has recently been demonstrated to bind BMP-2 and is required for
signaling by the Drosophila homologue of BMP-2 and BMP-4, the
product of the decapentaplegic gene(20, 21) .
The ability of the mammalian BMP type I receptors to form a complex
with the nematode Daf-4 type II receptor as well as with other proteins
having the expected size of the type II receptor in endogenous systems (5, 15) suggests the existence of mammalian BMP type
II receptors. Indeed, expression of a BMP type I receptor in COS cells
is insufficient to reproduce the high affinity binding observed in
endogenous systems, suggesting that the high affinity complex is
composed of both type I and type II receptor subunits(5) .
Since complex formation between the type I and type II receptors is a
prerequisite for signal transduction induced by TGF--related
ligands (13, 22) , identification of the mammalian BMP
type II receptor(s) is necessary in order to fully understand the
nature of the BMP receptor signaling complex.
In this paper, we
describe the cloning and characterization of a human BMP type II
receptor, which we term BRK-3 (BMP Receptor Kinase-3) that is identical
in sequence to a novel receptor Ser/Thr kinase isolated on the basis of
its ability to interact with type I receptor kinase domains in the
yeast two hybrid system(23) . As is observed for the activin
and TGF- type II receptors(6, 7) , BRK-3 binds
ligands on its own when expressed in COS cells. However, in contrast to
what is established for other type II receptors, cross-linking of
I-labeled BMP-4 to this human BMP type II receptor is not
observed in the absence of a BMP type I receptor. We demonstrate that,
while BRK-3 is capable of forming a complex with either the BRK-1 or
BRK-2 BMP type I receptors, a high affinity complex is formed only when
BRK-2 is coexpressed with BRK-3 in COS cells.
cDNA libraries of human skin fibroblasts were constructed
in the gt10 vector using SuperScript Choice System (Life
Technologies, Inc.), yielding 8
10
independent
recombinants. Hybridization screening with one of the PCR clones was
carried out as described(24) . Nucleotide sequence was
determined on both strands using an ABI DNA sequencer 373 after
subcloning several restriction fragments into suitable vectors such as
M13mp18/mp19.
To isolate a novel human receptor protein kinase related to
type II receptors for TGF- and activin, we first employed PCR to
amplify cDNA from human skin fibroblasts. In addition to the human
activin type II receptor cDNA(25) , we obtained a
289-nucleotide PCR fragment encoding a portion of a novel Ser/Thr
kinase. We isolated several overlapping cDNAs from human fibroblast
libraries by hybridization screening. Analysis of the entire coding
sequence reveals the BRK-3 protein to be a 1038-amino acid member of
the transmembrane Ser/Thr kinase family, identical to the T-ALK
receptor recently identified from a HeLa cell cDNA
library(23) , although we have isolated additional 5`- and
3`-noncoding regions of the cDNA. The sequence of BRK-3 has been
deposited to the GSDB/DDBJ/EMBL/NCBI data bases under the accession
number D50516.
Several structural features suggest that the BRK-3
protein is a type II receptor for a member of the TGF-
superfamily. First, both an upstream Cys box in the extracellular
domain and an intracellular GS domain that are conserved only in type I
receptor sequences(5, 9, 22) are absent in
the BRK-3 sequence. Second, the sequence of the BRK-3 kinase domain is
more closely related to other type II receptors, particularly the
MISRII(26, 27) , activin type II (6) and IIB (28) receptors, than it is to the type I receptors (data not
shown and (23) ). Third, BRK-3 has a unique 533-amino acid
carboxyl-terminal tail rich in Ser, Thr, and Pro residues that is
absent in the type I receptors.
Fig. 1shows the tissue specific expression of the BRK-3 gene as determined by Northern blot analysis in different human tissues. Four distinct transcripts of >10, 8, 6.5, and 5 kilobase pairs were detected in almost all tissues examined, except liver, leukocyte, testis, and thymus, where the longest transcript of the BRK-3 gene was not detected. The BRK-3 gene was expressed abundantly in lung, placenta, and testis. The relative abundance of the three major transcripts (>10, 8, and 5 kilobase pairs) varied depending on the tissue and may reflect tissue-specific processing of the transcripts.
Figure 1: Northern blot analysis of BRK-3 mRNA. Each lane contains 2 µg of poly(A) RNA from the indicated human tissues. Size markers are indicated on the left. As a control, human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was used in the repeated hybridization of the same blots.
To determine whether
the BRK-3 protein binds BMP-4, COS-7 cells were transfected with tBRK-3
alone or in combination with either the BRK-1 or BRK-2 type I receptor,
and whole cell binding to a single concentration of I-labeled BMP-4 was measured (Fig. 2). tBRK-3
binds
I-labeled BMP-4 by itself when expressed alone in
COS cells. Binding of
I-labeled BMP-4 is greatly enhanced
by coexpression of tBRK-3 with BRK-2 and to a lesser extent by
coexpression of tBRK-3 with BRK-1. These increases in binding could be
due either to an increase in binding affinity or to an additive effect
of binding to the different receptor proteins. In contrast to the whole
cell binding experiments, affinity labeling experiments reveal that
tBRK-3 is not efficiently cross-linked by
I-labeled BMP-4
when expressed alone in COS-1 cells (Fig. 3A). However,
coexpression of tBRK-3 with either BRK-1 or BRK-2 reveals a new band at
94 kDa, in addition to the cross-linked type I receptor bands at
75-78 kDa. (All approximate molecular weights are assumed to
include the molecular weight of the BMP-4 monomer.) Allowing for the
presence of three potential N-glycosylation sites in the BRK-3
extracellular domain, the new
94-kDa band is consistent with the
predicted molecular weight of tBRK-3, cross-linked to the BMP-4
monomer. Interestingly, coexpression of BRK-2 with BRK-3 results in a
more intense labeling of the type I receptor band, in contrast to what
is observed when the BRK-1 type I receptor is coexpressed with BRK-3.
The
150-kDa band that is most apparent in the BRK-2 + BRK-3
cotransfected cells may represent cross-linking with endogenous BRK-3
(see below). The nature of the minor
110-kDa band that is observed
when BRK-3 is coexpressed with either type I receptor is unknown and
could be due either to differential glycosylation or cross-linking of
the ligand dimer to tBRK-3.
Figure 2:
Whole cell binding of I-labeled BMP-4 in COS cells expressing tBRK-3, BRK-1,
and BRK-2. Bars represent specific binding to COS-7
cells(5) , normalized to cell number. Error bars represent the standard error of the mean of six to nine
determinations. Binding was obtained using a tracer concentration of
130-210 pM. BRK-3* represents the truncated
form of BRK-3 (tBRK-3).
Figure 3:
Affinity labeling of the BMP receptors in
COS cells expressing BRK-3 in combination with different BMP type I
receptors. A, tBRK-3 does not cross-link to I-labeled BMP-4 on its own when expressed in COS cells.
COS-1 cells were affinity labeled with 270 pM tracer, as
described under ``Experimental Procedures.'' The experiment
was repeated once with similar results. B, tBRK-3 forms a
complex with either BRK-1 or BRK-2 in the presence of
I-labeled BMP-4. COS-1 cells were affinity-labeled with
350 pM tracer and immunoprecipitations were performed using
antibodies for the BMP type I receptor kinase domain as described under
``Experimental Procedures.'' Antiserum 1379 was used in lanes 1, 2, 3, and 7, whereas antiserum 1380 was used
in lanes 4-6 and 8. The experiment was repeated
three additional times with similar
results.
Immunoprecipitation of cells
cotransfected with tBRK-3 and either BRK-1 or BRK-2 resulted in the
appearance of the same additional bands apparent in the cross-linking
studies, indicating that tBRK-3 formed a complex with either type I
receptor in the presence of I-labeled BMP-4 (Fig. 3B). Furthermore, an additional band of
150-180 kDa is apparent in the BRK-2 receptor
immunoprecipitates (Fig. 3B), suggesting that an
additional protein exists in the complex of BRK-2 with tBRK-3 in the
presence of
I-labeled BMP-4. Interestingly, this
additional protein corresponds in size to the fulllength BRK-3 receptor
cross-linked to the BMP-4 monomer, (
)and may represent the
endogenous COS cell BRK-3 that exists in the complex with the truncated
type II receptor in the presence of ligand and the type I receptor.
When affinity labeling to the BRK-2 + BRK-3 receptor complex was
performed with
I-labeled BMP-4 in the absence and
presence of 10 nM BMP-2, DR-BMP-2(5) , or BMP-4,
binding was completely abolished to all bands in the BRK-2 + BRK-3
complex, whereas 50 nM TGF-
was completely
ineffective, indicating that this receptor complex specifically binds
either BMP-2 or BMP-4, but is not accessible to TGF-
(data not shown). Finally, the differential change in affinity
labeling of the BRK-1 versus BRK-2 band that is observed in
the type I-BRK-3 receptor complex is in contrast to what is observed
when DAF-4 is cotransfected with either type I receptor, where an
increase in affinity labeling of either type I receptor is observed (Fig. 3B). These results suggest that, whereas similar
increases in either binding affinity or affinity labeling occur when
DAF-4 is the type II receptor, tBRK-3 forms different complexes with
the two different BMP type I receptors.
In order to address the
possibility that the differential affinity labeling of the BRK-1 or
BRK-2 receptor bands that is observed in the presence of BRK-3 is
reflective of a differential nature of the receptor complex that is
dictated by the type I receptor, the binding affinity of BMP-4 to the
two different receptor complexes was examined. Fig. 4A compares the affinity of BMP-4 in cells transfected with either
BRK-1 alone to that in cells transfected with BRK-1 + BRK-3. No
change in binding affinity for BMP-4 is observed (IC = 1.24
10
M for BRK-1
alone versus 1.25
10
M in
cells cotransfected with BRK-1 + BRK-3). Although the binding
affinity of BMP-4 is similar in cells transfected with either BRK-1 or
BRK-2 alone, cotransfection of BRK-3 with BRK-2 results in an increase
in the binding affinity of BMP-4 (IC
= 4.58
10
M for BRK-2 alone versus IC
values of 3.35
10
M and 1.34
10
M in
cells cotransfected with BRK-2 + BRK-3 (Fig. 4B)).
Furthermore, the formation of a super-high affinity binding site
population (where the data are best described by a two binding site
model for the competitor, p < 0.0001) is observed only in
cells cotransfected with the BRK-2 type I receptor and the BRK-3 type
II receptor.
Figure 4:
BMP-4 binds to the different receptor
complexes with differential affinity. A, comparison of BMP-4
binding affinity in COS-7 cells transfected with BRK-1 alone or with
BRK-1 + tBRK-3. Whole cell binding was performed with 107 pMI-labeled BMP-4. The points represent the average of
triplicate determinations. Error bars represent the standard
error of the mean. The curves represent the optimal fit of the data to
a one binding site model. The IC
values are reported in
the text. B, comparison of BMP-4 binding affinity in COS-7
cells transfected with BRK-2 alone or with BRK-2 + tBRK-3. Whole
cell binding was performed with 150 pM
I-labeled
BMP-4. The points represent the average of triplicate determinations. Error bars represent the standard error of the mean. The
curves represent the optimal fits of the data, to a one binding site
model (BRK-2) or to a two binding site model (BRK-2 + tBRK-3). The two binding site model assumes that the tracer binds
to both sites with similar affinity, but the unlabeled ligand
distinguishes between the two affinity
sites.
We have isolated a new member of the receptor Ser/Thr protein
kinase superfamily that is structurally related to the type II
receptors for members of the TGF- superfamily. When expressed in
COS cells, this human type II receptor is capable of forming
differential heteromeric complexes with either the murine BRK-1 or the
chicken BRK-2 type II receptors in the presence of BMP-4. It has been
demonstrated previously that murine and human BMP type I receptors form
a complex with the nematode DAF-4 type II
receptor(5, 15) , that a dominant negative construct
of the murine BMP type I receptor BMPR-IA/TFR11/BRK-1 alters
dorsal-ventral patterning when expressed in ventral blastomeres of Xenopus embryos(14) , that the chicken BMP-related
ligand dorsalin-1 induces alkaline phosphatase activity in murine bone
marrow stromal cells(29) , and that the Drosophila BMP-2 and BMP-7 homologues, dpp and 60A, induce ectopic bone
formation in rats(30) . Given the high degree of sequence
conservation among the ligands and receptors of this family, it is
unlikely that the observed differential binding properties for the type
I-type II receptor complexes described herein are due to the species
differences of the type I receptors. Rather, the differential binding
properties described below most likely reflect the different signaling
potential of the receptor complexes, which has been demonstrated for
the TGF-
and activin receptor systems to be dependent on the type
I receptor in the complex(13) .
The mammalian BRK-3 receptor
binds BMP ligands on its own when transfected into COS cells, as is
observed for mammalian type II receptors for
TGF-(7) , activin(6, 28) ,
MIS(27) , and with the nematode BMP type II receptor
DAF-4(18) . The finding that BRK-3 binds ligands on its own in
COS cells, coupled with the finding that both truncated BRK-3 and a
protein that corresponds in size to the full length BRK-3
can be brought down in a complex with the type I receptor when
BRK-2 and truncated BRK-3 are cotransfected into COS cells (Fig. 3B) is consistent with the model that these
receptors are heterotetramers, composed of pre-existing homodimers of
the type II
receptor(31, 32, 33, 34, 35, 36) .
We cannot say at the present time whether the BRK-3 type II receptor
homodimer is formed in the absence of ligand.
In contrast to other
type II receptors, cross-linking to BRK-3 is not evident in
the absence of the type I receptor (Fig. 3A). A
sequential binding model for TGF- and activin receptors has been
proposed wherein the ligand first binds to a preexisting type II
receptor homodimer, with subsequent association of the type I receptor
into the
complex(22, 31, 32, 35, 36) .
It has also been proposed that recruitment of the type I receptor into
the complex causes a conformational change in either the ligand or the
receptor such that the ability of the lysines on the ligand and the
receptor to cross-link is altered, resulting in an increased
cross-linking efficiency of the type I versus the type II
receptors(37, 38) . In contrast to the TGF-
and
activin receptor systems, it appears that the conformational change in
the BMP ligand-receptor complex that occurs when both the type I and
type II receptors contact the ligand results in an increased tendency
for the ligand to be cross-linked to the BMP type II receptor.
Additionally, in contrast to the TGF-
and activin receptor
systems, both BMP type I (5) and type II receptors are capable
of binding BMP ligand on their own when transfected into COS cells.
Hence, it cannot be said at the present time whether the same
sequential binding model applies to the BMP receptor complex.
Nevertheless, the distinction between the ability to bind and
to cross-link to the receptor appears to be an important one
for the BRK-3 mammalian BMP type II receptor, and it appears that a
conformational change in either the ligand or the type II receptor is
taking place as the type I receptor is recruited into the complex.
While this does not seem to be the case for the nematode BMP type II
receptor DAF-4, which does cross-link to BMPs in the absence of a
cotransfected type I receptor(18) , it is interesting that a
similar result is obtained for the Drosophila Punt receptor,
in which cross-linking to
I-labeled activin is apparent
in the absence of a cotransfected activin type I receptor (19) , whereas cross-linking to
I-labeled BMP-2
is not apparent in the absence of a cotransfected Drosophila BMP type I receptor(21) , implying a differential
distribution of lysines at the ligand-type II receptor interface for
activin versus BMPs.
We also observe a dramatic difference
in the binding affinity of BMP-4 to the different type I-BRK-3 type II
receptor complexes (Fig. 4). While both the BRK-1 and BRK-2 BMP
type I receptors are capable of forming a complex with BRK-3, as
evidenced by immunoprecipitation studies (Fig. 3B), an
increase in binding affinity of BMP-4 is only observed when the
BRK-2-BRK-3 complex is formed in COS cells. After submission of this
manuscript, Liu et al.(39) reported the cloning of an
alternatively spliced human BMP type II receptor that is 10 amino acids
shorter than the artificially truncated form of human BRK-3 studied
here. Affinity labeling studies with I-labeled BMP-2 and
I-labeled BMP-7 demonstrate complex formation of BRK-3
with ALK-3 (the human homologue of BRK-1), ALK-6 (the murine homologue
of BRK-2), and ActRI/SKR1 in the presence of both ligands but the
largest signal was observed when BMP-7 was the ligand and ActRI was the
type I receptor(39) . Interestingly, BMP-2 did not signal at
all when BRK-1 or BRK-2 was the type I receptor. These authors did not
report the binding affinity of these ligands to the different receptor
complexes. The increase in binding affinity that we observe with BMP-4
in the presence of the appropriate receptor components is analogous to
what is observed for other multicomponent cytokine receptor systems, in
which it is the high affinity state of the receptor that is responsible
for signaling(40) . We predict that the BRK-2-BRK-3 receptor
complex will represent a functional signaling complex for low
concentrations of BMP-4, whereas the BRK-1-BRK-3 complex will be
insufficient to transduce the BMP-4 signal at low concentrations of
ligand. It therefore follows that a requirement for formation of a
functional receptor signaling complex for BMPs will not only be that
the individual receptor subunits bind ligand and form a complex, but
that they form a high affinity complex in the presence of the
appropriate ligand. In addition, the signaling receptor subunits will
need to be colocalized in the same cell or tissue in vivo in
order to represent a physiologically relevant receptor complex.
Investigations of the signaling potential of the different BMP receptor
complexes, as well as the colocalization of the individual BMP receptor
subunits in vivo are currently in progress.