|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 36, 34181-34188, September 5, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface*
¶ ||
From the
Institute for Molecular Bioscience and
the Department of Biochemistry, The University
of Queensland, Brisbane, Queensland 4072, Australia
Received for publication, February 5, 2003 , and in revised form, June 10, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of co-expression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The TGF
1
superfamily of ligands have prominent roles in nearly all tissues during
development. There are over 30 members of this superfamily, including the
TGF isoforms, bone morphogenetic proteins (BMPs), and the closely
related growth and differentiation factors, activins, and nodals (reviewed in
Ref. 1). BMPs, like all members
of the TGF superfamily, are synthesized as preproteins, which are
subsequently cleaved by furin and related subtilisin-like convertases within
the Golgi apparatus at a conserved multibasic amino acid motif
(Arg-X-X-Arg) to yield a dimeric C-terminal mature domain
(2). Once secreted BMPs bind
and facilitate the formation of heteromeric complexes of type I and type II
serine/threonine kinase receptors
(1). These signal via
phosphorylation of Smad proteins, which can translocate into the nucleus and
effect gene regulation and expression (reviewed in Ref.
1). Although TGF-1, -2,
and -3 are secreted as a member of a large inactive complex containing the
latency-associated peptide and a latent TGF-binding protein, the BMPs
are currently thought to be processed and secreted as active mature molecules
and not complexed with a latent TGF-binding protein-like molecule.
However, like the TGFs, BMPs do not seem to act over a range of more
than one to two cells, suggesting that there is some mechanism of
"tethering" (3).
This limit in signaling range is in part influenced by the prodomain of BMPs
(3). The prodomain of the BMPs
also influences the efficiency of processing and the stability of the mature
BMP (2,
4). During development,
TGF superfamily members, and in particular BMPs, specify cell types by
forming precisely controlled concentration gradients. The amount of active
ligand is regulated via modulation of protein processing, receptor binding,
regulation of Smad function, and, as has been discovered recently,
extracellular antagonism of BMPs. One of the extracellular BMP antagonists is
Chordin (Chrd) and its homologue in Drosophila, short
gastrulation (sog)
(5–8).
The Chordin protein (CHRD) is a dorsalizing factor expressed by the organizer
in Xenopus, which functions by directly binding BMP4 to inhibit its
receptor binding (5,
8). Sog, the
functional homologue of Chrd in Drosophila, is expressed
ventrally and partly establishes the dorso-ventral body axis by inhibiting the
activity of dpp (most closely related to vertebrate BMP2/4)
(6). Overexpression of SOG
protein can inhibit BMP4 signaling in Xenopus, revealing functional
conservation of this interaction between vertebrates and invertebrates
(7). The BMP-antagonizing
activity of CHRD and SOG is itself regulated via secreted metalloproteases
related to BMP1 (9,
10). Xolloid in
Xenopus cleaves CHRD, effectively abolishing its ability to
antagonize BMP4 (9). Tolloid
functions in a similar way in Drosophila to cleave SOG
(10).
CHRD and SOG proteins are characterized by multiple cysteine-rich repeats,
similar to the von Willebrand Factor type C domain
(5). Such cysteine-rich repeats
(CRRs) have a conserved consensus sequence based on ten cysteines
(CXnWX4CX2CXCX6CX4CX4–6CX9–11CCPXC).
Specific individual CRRs in CHRD can bind directly to BMP4
(11). Recently, a number of
novel CRR-containing proteins have been identified, including Neuralin1 (also
known as Ventropin)
(12–14),
Drosophila Crossveinless2
(15), and Xenopus
Kielen (16). Although
possessing varying numbers of CRRs, all of these proteins bind to BMPs. CRR
motifs in other proteins mediate binding to other members of the TGF
superfamily. The CRR of procollagen type IIA can interact with TGF2 and
BMP2 (11,
17). It has therefore been
speculated that this domain has evolved to modulate interactions with
TGF superfamily members
(11). Connective tissue growth
factor contains one CRR and has been shown to antagonize BMP4 by inhibiting
receptor binding but acts synergistically with TGF1 to enhance receptor
binding (18). Therefore this
novel family of proteins appear critical in regulating the activity of
TGF/BMP signaling in development.
The Crim1 gene encodes a protein with six chordin-like CRRs, an
N-terminal insulin-like growth factor binding protein-like motif and a
putative transmembrane domain
(19). By analogy to other CRR
proteins, we have hypothesized that CRIM1 may also function as an antagonist
to one or more members of the TGF superfamily by direct binding via the
CRR repeats (19).
Crim1 shows developmentally regulated expression in the mouse embryo
localized to the developing kidney and testis
(20), the lens of the eye
(21), vibrissae, pinna, tooth
primordia, as well as specific regions within the central nervous system
(19). A study of the spatial
and temporal expression of Crim1, BMP2, and BMP7 during
mouse urogenital development, showed a pattern of overlapping expression for
these proteins implying a possible physiological interaction
(20). Here we show that CRIM1
interacts with members of the TGF family, including BMP4 and BMP7, when
co-expressed with these growth factors and that this interaction occurs via
the CRR repeat domains. Co-expression of CRIM1 with BMP4 or BMP7 results in a
reduction in the processing of pre-BMPs to their mature form and a consequent
reduction in the amount of BMP secretion. However, CRIM1 is trafficked with
its preprotein ligand to the plasma membrane, whereupon a proportion of the
complex remains tethered to the cell surface in an inactive preprotein form.
Hence CRIM1 can antagonize members of the TGF superfamily.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Construction of Mammalian Expression Vectors—A full-length human CRIM1 (GenBankTM accession number AF167706 [GenBank] ) expression construct was created from existing subclones, which covered the entire open reading frame of CRIM1. Intrinsic PmeI and DraII restriction enzyme sites were used for insertion into pCDNA3. An myc (EQKLI-SEEDL) or HA (YPYDVPDY) tag was introduced into CRIM1-Nmyc/HA constructs after residue 78 (Phe) by PCR. CRIM1-E was made by truncating the protein at residue 901, and inserting an myc or HA tag at the C terminus. BMP7preHA was derived from mouse BMP-7 (pMTAD1-BMP7myc, a gift of Dr. Elizabeth Robertson) by inserting the HA epitope after residue 87 (Tyr) as a linker in an intrinsic BsrGI site. Other constructs used in this study were human BMP4 (PMT21-BMP4myc, a gift of Dr. Tom Jessell), chicken Chordin (pMT21-CHRDHA, a gift of Dr. Kevin Lee), and human growth hormone receptor (pcDNA3-GHRHA, a gift of Prof. Michael Waters).
Generation of Anti-CRIM1 Antibodies—Polyclonal antibodies
were raised and purified as described previously
(26). 
-NC was raised
toward a synthetic peptide: KVCQPGYLNILVSKASGKPGEC (Chiron Technologies).
-CC was raised toward a glutathione S-transferase-conjugated
C-terminal epitope of mouse CRIM1 (residues 998–1036; GenBankTM
accession number AF168680
[GenBank]
), which was cloned into pGEX as described previously
(26).
Cell Culture and Transfections—COS7 cells were transfected when at 50–70% confluence with 1 µg of DNA (or as indicated), in 3 µl of FuGENE6 (Roche Applied Science). Transfected cells were incubated in Dulbecco's modified Eagle's medium plus 10% fetal calf serum or OPTI-MEM (Invitrogen) for serum-free conditions for 2–3 days. Conditioned media was harvested and concentrated 10x using Amicon Ultra concentraters (Millipore).
Cell Fractionation and Western Blotting—CRIM1-Nmyc-transfected COS7 cells were fractionated into light membrane, heavy membrane, and soluble fractions as previously described (22). Western blotting, of fractionated samples, cellular extracts, or media as described below, was performed as described previously (26).
Immunofluorescence of Cells—Immunofluorescence on cell lines using the antibodies indicated was performed as described previously for permeabilized cells (26). For cell surface binding, antibody incubations were performed on live cells incubated on ice to inhibit internalization. Cells were finally fixed in 4% paraformaldehyde for 30 min. Antibody labeling was captured on an Olympus AX70 compound fluorescence microscope.
Protein Expression and Glycosylation Assay—For production of
CRIM1, CHRD, BMP4, or BMP7 for in vitro binding studies, transiently
transfected COS7 cells were incubated in serum-free OPTI-MEM (Invitrogen) for
2 days. Conditioned media was concentrated 1:10 and snap-frozen. Conditioned
media from transfections using pcDNA3 without insert were used as a control.
CRIM1-E protein was expressed by using a stable Chinese hamster ovary cell
line. The protein was purified from conditioned media on an -myc
affinity column. The -myc produced by the hybridoma 9E10 was
cross-linked to protein A-agarose (Sigma) using the method supplied with the
cross-linker (Pierce). Protein concentrations were estimated against samples
of known concentration by Western blotting. N-Glycosidase S was used
to determine N-linked glycosylation according to the manufacturer's
instructions (Roche Applied Science).
Immunoprecipitation—For in vitro binding
experiments, conditioned concentrated media (see above) were incubated in 200
µl of 0.5% bovine serum albumin, 5 mM Tris-HCl, pH 8.0, for3hon
ice. Cross-linking was performed where indicated using disuccinimidyl suberate
(Pierce) according to the manufacturer's instructions. For immunoprecipitation
of co-expressed samples, media was used without concentration.
Immunoprecipitations were performed by the addition of 5 µl of -myc
antibody and a 2-h incubation at room temperature, followed by the addition of
40 µl of protein A-Sepharose (50% slurry, Sigma) and a further 1-h
incubation. The beads were pelleted and washed four times with PBS 0.1% Triton
X-100 and once with PBS. Protein was eluted by addition of SDS-PAGE loading
buffer (0.625 M Tris-HCl, pH 6.8, 4% SDS, 10%
-mercaptoethanol, 40% glycerol, 0.05% bromphenol blue) and boiled for 5
min. For cellular extracts, transfected cells were suspended in 600 µl of
lysis buffer (0.05 M Tris, 0.15 M NaCl, pH 8.5, Complete
protease inhibitors (Roche Applied Science), 0.5% Triton X-100).
Immunoprecipitation was carried out as above, except that beads were pelleted
and washed in lysis buffer three times, and twice in PBS.
Biotinylation—Cell surface proteins were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin according to the manufacturer's instructions (Pierce).
Explant Culture—Embryonic mouse kidneys were dissected at 11.5 days postcoitum (dpc) and grown in culture medium containing Dulbecco's modified Eagle's medium/F-12, 20 mM glutamine, 5 µg/ml holo-Transferrin as described in a previous study (27). Conditioned medium (5 µl) was added where indicated. For immunofluorescence analyses, explants were fixed in 100% ice-cold methanol, 10 min, washed in PBS, and permeabilized with 1% Triton X-100 in PBS. Antibody incubations were performed for 1 h at 37 °C in 1% bovine serum albumin in PBS. Primary antibodies used were WT1 (DAKO, 1/100 dilution) and calbindin 28K (Sigma, 1/200 dilution). Secondary antibodies were Cy3-conjugated anti-rabbit IgG and Alexa-conjugated anti-mouse IgG. Ureteric tip and nephron number were counted manually, and statistics were performed using InStat3. Due to variability in the age of explants between litters, one-way analysis of variance could only be performed within a given experiment.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CRIM1 Is a Type I Transmembrane Protein—Sequence analysis of the CRIM1 open reading frame reveals a C-terminal transmembrane domain, suggesting the large N-terminal CRR-containing domain localizes in the extracellular space (19). To test this, we designed mammalian expression constructs of human full-length CRIM1, differentially tagged with myc or HA epitope tags (CRIM1-Nmyc/HA). We also produced myc- and HA-tagged constructs with a deletion of the potential transmembrane domain and C-terminal sequence (CRIM1-Emyc/HA) (Fig. 1A). Polyclonal antibodies were raised in rabbits to epitopes in the N and C termini of CRIM1 (
-NC and -CC,
respectively) (Fig.
1A). Western blotting with -myc, -NC, and
-CC of cellular extracts of COS7 cells transfected with the CRIM1
constructs identified bands of 
140 kDa for CRIM1-Nmyc and 120
kDa for CRIM1-Emyc (Fig. 1,
B and C). CRIM1-E was detected with the
-NC antibody (Fig.
1C), but the -CC epitope is deleted in this
construct. A sequence of LVXLPX-DESK (where X is
presumably cysteine) was obtained after N-terminal amino acid sequencing of
purified CRIM1-E, which confirms a functional signal peptide sequence with a
cleavage site between amino acids Ala-34 and Leu-35.
|
To analyze subcellular localization we fractionated CRIM1-transfected cells
to determine if it was localized to membrane fractions. CRIM1 was detected
predominantly in the light membrane fraction, which corresponds to the plasma
membrane compartment. A smaller concentration was evident in the heavy
membrane fraction, which corresponds to intracellular trafficking compartments
(Fig. 1D)
(22). To examine potential
plasma membrane localization and orientation, we analyzed
CRIM1-Nmyc transfected cells by immunofluorescence. In transfected
cells that had been permeabilized by treatment with Triton X-100, there was a
strong signal from antibodies to the N-terminal domain (-myc) and the
C-terminal domain (-CC) (Fig. 1,
E and F). Without permeabilization, only the
N-terminal domain appeared to be accessible to antibody binding, suggesting
the C-terminal was located within the cell
(Fig. 1, G and
H). In cells transfected with the deletion construct
CRIM1-E, no cell surface localization of CRIM1-E was evident
(Fig. 1J), yet
immunofluorescence of permeabilized cells clearly showed the protein was
expressed (Fig. 1I),
suggesting that without the transmembrane domain CRIM1 is secreted. Therefore
the CRIM1 protein adopts a type I transmembrane conformation with the
cysteine-rich N-terminal end localized to the extracellular space
(Fig. 1K).
CRIM1 Can Be Cleaved to Secrete a Soluble Ectodomain—
Because CHRD and other CRR-containing proteins encode secreted molecules, we
determined whether a secreted form of CRIM1 exists. Media harvested from COS7
cells transfected with full-length CRIM1 (CRIM1-Nmyc) showed a
small amount of CRIM1 in the media (Fig.
1B). The molecular mass of this form was found to be
10–15 kDa smaller than from cellular lysates. Although
CRIM1-Nmyc from cell lysates was able to be immunoprecipitated by
both -NC and -CC CRIM1 antibodies, only the -NC antibody
was able to immunoprecipitate the secreted form of CRIM1-Nmyc from
the conditioned media (Fig.
1L). These results suggest that CRIM1 is cleaved to
release the N-terminal cysteine-rich ectodomain.
CRIM1 Is N-Glycosylated—Careful analysis of cell lysates and
media from COS7 cells transfected with CRIM1-Emyc suggested a minor
molecular weight disparity. To determine if this was due to N-linked
glycosylation, we treated CRIM1-Emyc from media with
N-glycosidase. This resulted in a molecular mass change of 10
kDa suggesting that CRIM1 is N-glycosylated
(Fig. 1M). Analysis of
the amino acid sequence of CRIM1 suggests that there are five potential
N-glycosylation sites (NX(S/T), where X is not P)
in the extracellular domain (Fig.
1N).
CRIM1 and Members of the TGF Superfamily Can
Interact—Strong evidence has suggested that CHRD-like CRR proteins
bind to members of the TGF superfamily, particularly the BMPs
(11). Due to the similarity of
CRIM1 CRRs to CHRD, we have focused our analysis on BMP4
(8,
11,
12,
14). We have also investigated
the possibility of interaction with BMP7, which shows overlapping sites of
expression with CRIM1 in the developing kidney
(19,
20). We first tested whether
CRIM1 ectodomain protein harvested in media from cells transfected with
CRIM1-EHA or CRIM1-NHA could bind mature BMPs similarly
secreted into the media from BMP-transfected cells. After incubation of CRIM1
(5–10 nM) and BMP4 or BMP7 (2–10 nM) at room
temperature for 1 h, there was no evidence of binding
(Fig. 2A). Under
similar binding conditions, CHRD was co-immunoprecipitated together with BMP4,
and weakly with BMP7 (Fig.
2A), as previously described
(8). To exclude the possibility
that the absence of observable binding was due to a low affinity interaction
between CRIM1 and BMP4, chemical cross-linking of the proteins in solution was
also performed. Although the presence of cross-linked dimeric BMP4, BMP7, and
BMP4·CHRD complexes analyzed under reducing conditions showed that the
cross-linking was effective, no binding of CRIM1 to BMP4 was apparent
(Fig. 2B). This
suggests that the recombinant CRIM1 ectodomain does not bind BMP4 and BMP7
directly in solution. We next determined if CRIM1 could interact with BMPs
intracellularly when transiently co-transfected in COS7 cells.
CRIM1-NHA was co-immunoprecipitated with BMP4myc and
BMP7myc (Fig.
2C). In addition, the unprocessed form of BMP4
(Fig. 2D) was
co-immunoprecipitated with CRIM1-NHA. This suggests that
full-length CRIM1 can bind to the precursor form of BMP4.
|
Because CRIM1 can be secreted into the media as an ectodomain, we determined if this form was still bound to BMPs. Both CRIM1-EHA and CRIM1-NHA secreted into media were able to be co-immunoprecipitated with an anti-myc antibody from conditioned media if co-expressed with BMP4myc or BMP7myc (Fig. 2E). CRIM1-Nmyc was only weakly co-immunoprecipitated, but this may be due to the low amount of secreted CRIM1 (Fig. 1A). This suggests that CRIM1 can be secreted into the media as a complex with BMPs.
CRIM1 Associates with BMP7 at the Cell Surface—Because CRIM1
associates with BMPs in the cell, we used immunofluorescence analysis on
non-permeabilized cells to determine if BMP7myc is tethered to the
cell surface when co-expressed with full-length CRIM1 (CRIM1-NHA).
When BMP7 is expressed alone, or co-expressed with human growth hormone
receptor (GHRHA), there is no BMP7-specific cell surface staining
(Fig. 2, F and
G). However, when BMP7 was co-expressed with full-length
CRIM1, BMP7 was detected on the cell surface
(Fig. 2, H and
I). To determine whether CRIM1 was interacting with the
mature or precursor form of BMP7 at the cell surface, an HA tag was introduced
into the N-terminal precursor domain of the BMP7 construct.
Co-immunofluorescence was performed on non-permeabilized COS7 cells
co-transfected with this construct and CRIM1 using a CRIM1 antibody
(-CN) and -HA. The results in
Fig. 2 (J and
K) show that the BMP7 precursor was present on the cell
surface of CRIM1-expressing cells.
Biotinylation assays were used to investigate the levels of cell-surface pre-BMP7 in the presence and absence of CRIM1. COS7 cells were co-transfected with CRIM1 and BMP7 or empty vector and BMP7. Cell surface proteins were biotinylated and precipitated with streptavidin-agarose. Precipitated proteins were analyzed by Western blotting and densitometry. Fig. 3 (A and B) show that the CRIM1-producing cells retained a higher percentage of pre-BMP7 on the cell surface than non-expressing cells. To determine whether the tethered pre-BMP7 can be removed from the cell surface, COS7 cells co-transfected with CRIM1 and BMP7 or empty vector and BMP7 were subjected to a 5-min PBS wash followed by a 5-min DTT (20 mM) wash. The washes were concentrated and analyzed for the presence of pre-BMP7 by Western blotting. Fig. 3C shows that a greater amount of pre-BMP7 was washed from the surface of CRIM1-producing cells, with further pre-BMP7 released from the CRIM1-producing cells after DTT treatment. These results are consistent with those of the previous experiment showing that CRIM1-producing cells retain more BMP7 on the cell surface and that the retention involves a conformation reliant on oxidized cysteine residues.
|
The CRR Repeats in CRIM1 Interact with BMP7—To determine whether the CRR repeats are responsible for the interaction of CRIM1 with BMPs, as in CHRD (11), a series of CRIM1 deletion constructs were made (shown schematically in Fig. 3D). These constructs were co-transfected with BMP7 in COS7 cells and the interaction investigated by immunoprecipitation (Fig. 3E). The constructs in which one of the two CRR domains were deleted (D2 and D4) interact poorly with BMP7 compared with the full-length CRIM1 and the other deletion constructs. These results suggest that the two blocks of CRR repeats, CRR1–2 and CRR3–6, bind cooperatively to ligand.
CRIM1 Inhibits Expression of BMP4 and BMP7—One unexpected
observation of CRIM1-BMP co-transfections compared with BMP transfections
alone was that the level of expression of the BMP4 or BMP7 was reduced when
co-expressed with CRIM1. To confirm that this was a specific effect of CRIM1
expression and not an artifact due to co-expression of two constructs, COS7
cells were co-transfected with BMP7myc or BMP4 myc and
equimolar amounts of either CRIM1myc, GHRHA, or CRIM1-D2
myc. Cell lysates were analyzed by Western blotting using an
-myc antibody and then stripped and reprobed with the HA antibody to
ensure co-expression of GHRHA. The level of BMP7 and BMP4
expression in the lysate was reduced markedly when co-expressed with CRIM1 but
not with GHR or with CRIM1 D2 (Fig.
4A). COS7 cells were co-transfected with BMP7 and
increasing concentrations (0.5–3 µg) of CRIM1 construct. Analysis of
the conditioned media showed that secretion of BMP7 was reduced in a
dose-dependent manner with increasing concentration of CRIM1
(Fig. 4B).
|
CRIM1 Co-localizes with BMP7 Intracellularly—Because CRIM1 appeared to interact with the BMPs intracellularly during processing, it would follow that the two proteins should be trafficked through the same intracellular compartments. Immunofluorescence of permeabilized COS7 cells that had been transfected with either CRIM1HA or BMP7 myc showed co-localization of these proteins with GM130, a Golgi-specific antibody (Fig. 4, C–F). COS7 cells co-transfected with CRIM1HA and BMP7myc showed marked co-localization (Fig. 4, G and H).
Taken together our results suggest that CRIM1 interacts with the BMP7 precursor intracellularly, and this interaction results in decreased production of BMPs. The interaction with pre-BMPs appears to be maintained during trafficking of CRIM1 to the cell surface, where pre-BMP7 remains tethered to the cell surface.
CRIM1 Antagonizes BMP4 —To examine the functional effect of CRIM1 tethering pre-BMP to the cell surface we used a kidney explant assay. BMP4 is known to retard the development of embryonic mouse kidneys grown in culture in a dose-dependent manner (23). Kidneys were dissected from embryonic mice at 11.5 or 12 dpc and were grown in culture for 3 days. Conditioned media from COS7 cells transfected with empty vector alone, BMP4 plus empty vector, BMP4 plus CRIM1, or BMP4 plus D2 were added to the cultures (Fig. 5, A–D). Kidneys grown in BMP4-conditioned media displayed reduced ureteric tree branching and reduced numbers of comma and S-shaped bodies compared with kidneys grown in control or CRIM1-conditioned media. BMP4 plus CRIM1-conditioned media showed a reduced BMP4 effect (Fig. 5, E and F), whereas BMP4 plus CRIM1 D2-conditioned media showed an intermediate phenotype (Fig. 5F). Hence CRIM1 can ameliorate the effect of BMP4. Removal of CRR1 and -2 decreases the ability of CRIM1 to act in this way. Analysis of the conditioned media by Western blotting and densitometric analysis (Fig. 5, G and H) showed that the media from the CRIM1-expressing cells contained less mature BMP4. These results support our other analyses and suggest that in vivo the effect of CRIM1 on BMP expression and secretion may result in a reduced concentration of active mature BMPs available to the tissues.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Proteins containing chordin-like CRRs are an important and growing family of extracellular regulators of the TGF
superfamily. Most identified to
date are secreted diffusible proteins, such as CHRD and SOG, which inhibit BMP
function. Unlike CHRD and SOG, we have shown that CRIM1 is an
N-glycosylated transmembrane protein predominantly localized to the
cell membrane, but that a proportion of CRIM1 can be cleaved and released from
the cell membrane as a secreted ectodomain. We also demonstrate that CRIM1 can
interact intracellularly with BMP4 or BMP7, supporting the hypothesis that it
will act as a mediator of TGF superfamily ligand activity, although not
defining the specific physiological ligand. Binding of Crim1 appears to affect
BMP production in three ways. There is a decrease in the production and
processing of preprotein to mature BMP, the secretion of mature BMP is
reduced, and a proportion of the BMP released from the cell remains associated
with CRIM1, either tethered to the cell surface or as a complex in the media.
These represent novel modes of BMP antagonism. We have verified that it is the CRR repeats of CRIM1 that mediate the binding of BMPs, because CRIM1 D2 (CRRs 1 and 2 deleted) and D4 (CRRs 3–6 deleted) interact only weakly with BMP7. CHRD and other CRR-containing proteins bind to the mature, secreted form of BMP via one or more CRR. Unlike these proteins CRIM1 binds to pre-BMPs. However, because the CRR repeats of CRIM1 are necessary for binding, it is likely that interaction is still between the CRRs and the mature domain of the dimeric BMP precursor.
The intracellular interaction of CRIM1 and BMPs appears to be specific and not due to an artifact of overexpression. A nonspecific interaction between CRIM1 and BMPs within the cell would suggest the proteins were misfolded. However, the editing machinery within the cell would target the misfolded proteins for degradation within the ER (24). CRIM1 and BMP7 are clearly trafficked from the ER through the Golgi and can be secreted. Furthermore, the two CRIM1 deletion constructs D2 and D4 interact with BMP7 only weakly, whereas GHR does not interact with BMP7 at all. That BMP expression is reduced in the presence of CRIM1 also appears to be specific for CRIM1, because it does not occur with CRIM1 D2 or GHR.
The apparent role of CRIM1 in the retardation of ligand secretion is not unique. The fibroblast growth factor receptor (FGFR, also called E selectin) is localized both to the Golgi and the cell membrane. Within the cell, FGFR inhibits the secretion of its ligand, fibroblast growth factor (FGF), and maintains an intracellular pool of FGF (25). Co-expression of CRIM1 with BMPs resulted in the reduced production of BMPs. CRIM1 may inhibit processing of pre-BMPs to BMPs and thereby target the pre-BMPs for degradation or, like the FGFR, CRIM1 may sequester pre-BMPs into an intracellular pool that is limited in capacity. Once the pool is full, further expression may be inhibited. Whatever the mechanism may be, the overall effect of CRIM1 co-expression with BMPs is to reduce the amount of active mature BMPs secreted into the media, effectively resulting in antagonism.
The function of tethering of BMPs to the cell surface by Crim1 is unclear,
as is the role of cleavage of the ectodomain. Tethering by CRIM1 may act to
increase the effective half-life of BMPs by facilitating their slow and
prolonged release from the surface, may regulate the distance over which BMPs
can act, or may deliver BMPs to receptors on the cell surface. Curiously,
however, our biotinylation studies suggest that it is the pre-BMP form of
BMPs, which are tethered to the cell. CRIM1 cleavage does appear to occur and
complexes between BMP and cleaved CRIM1 do exist in the media of
co-transfected cells. This might again support the concept of delivery to
receptors on another cell, restriction of the distance over which BMPs can
act, or regulation of half-life in solution. Repression of secretion,
tethering, and binding to the ectodomain may represent different actions
tailored for different ligands. Another CRR-containing protein, connective
tissue growth factor, has already been shown to bind both TGF1 and BMP4,
delivering the former to its receptor and therefore acting as a facilitator
but antagonizing the binding of the latter, thereby acting as an antagonist
(18). It is possible that
there are multiple roles, both positive and negative, being played by Crim1
depending upon the particular ligand bound. Further studies will be needed to
clarify the versatility of Crim1 with respect to TGF superfamily ligand
binding. However, these studies suggest that Crim1, unlike the CRR-containing
proteins previously described, may function by reducing the production and
secretion of active BMPs from the cells in which it is expressed. This would
provide a novel level of regulation of BMP action during development and
beyond.
|
FOOTNOTES |
|---|
In memory of our colleague Dr. Toshiya Yamada, 1960–2001.
* This work was supported in part by the National Health and Medical Research
Council (NMHRC) of Australia (Grant 102578) and the Sylvia and Charles Viertel
Charitable Trust. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
¶ Holds an Australian Postgraduate Award.
|| An NHMRC Senior Research Fellow and a Sylvia and Charles Viertel Senior Research Fellow. To whom correspondence should be addressed. Tel.: 61-7-3365-4494 (ext. 1819); Fax: 61-7-3365-4388; E-mail: M.Little{at}imb.uq.edu.au.
1 The abbreviations used are: TGF, transforming growth factor ;
BMP, bone morphogenetic protein; CHRD, chordin; CRR, cysteine-rich repeat;
GHR, growth hormone receptor; FGFR, fibroblast growth receptor; DTT,
dithiothreitol; PBS, phosphate-buffered saline; HA, hemagglutinin.
|
ACKNOWLEDGMENTS |
|---|
We thank Michael Waters, Tom Jessell, Kevin Lee, and Elizabeth Robertson for expression constructs.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
- Miyazono, K., Kusanagi, K., and Inoue, H. (2001) J. Cell. Physiol. 187, 265–276[CrossRef][Medline] [Order article via Infotrieve]
- Constam, D. B., and Robertson, E. J. (1999)
J. Cell Biol. 144,
139–149
[Abstract/Free Full Text] - Jones, C. M., Armes, N., and Smith, J. C. (1996) Curr. Biol. 6, 1468–1475[CrossRef][Medline] [Order article via Infotrieve]
- Dick, A., Hild, M., Bauer, H., Imai, Y., Maifeld, H., Schier, A. F., Talbot, W. S., Bouwmeester, T., and Hammerschmidt, M. (2000) Development 127, 343–354[Abstract]
- Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. K., and Derobertis, E. M. (1994) Cell 79, 779–790[CrossRef][Medline] [Order article via Infotrieve]
- Francois, V., Solloway, M., O'Neill, J. W., Emery, J., and Bier, E.
(1994) Genes Dev.
8,
2602–2616
[Abstract/Free Full Text] - Schmidt, J., Francois, V., Bier, E., and Kimelman, D. (1995) Development 121, 4319–4328[Abstract]
- Piccolo, S., Sasai, Y., Lu, B., and De Robertis, E. M. (1996) Cell 86, 589–598[CrossRef][Medline] [Order article via Infotrieve]
- Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L., and De, R. E. (1997) Cell 91, 407–416[CrossRef][Medline] [Order article via Infotrieve]
- Marques, G., Musacchio, M., Shimell, M. J., Wunnenbergstapleton, K., Cho, K., and Oconnor, M. B. (1997) Cell 91, 417–426[CrossRef][Medline] [Order article via Infotrieve]
- Larrain, J., Bachiller, D., Lu, B., Agius, E., Piccolo, S., and De Robertis, E. M. (2000) Development 127, 821–830[Abstract]
- Nakayama, N., Han, C. E., Scully, S., Nishinakamura, R., He, C., Zeni, L., Yamane, H., Chang, D., Yu, D., Yokota, T., and Wen, D. (2001) Dev. Biol. 232, 372–387[CrossRef][Medline] [Order article via Infotrieve]
- Coffinier, C., Tran, U., Larrain, J., and De Robertis, E. M. (2001) Mech. Dev. 100, 119–122[CrossRef][Medline] [Order article via Infotrieve]
- Sakuta, H., Suzuki, R., Takahashi, H., Kato, A., Shintani, T.,
Iemura, S., Yamamoto, T. S., Ueno, N., and Noda, M. (2001)
Science 293,
111–115
[Abstract/Free Full Text] - Conley, C. A., Silburn, R., Singer, M. A., Ralston, A., Rohwer-Nutter, D., Olson, D. J., Gelbart, W., and Blair, S. S. (2000) Development 127, 3947–3959[Abstract]
- Matsui, M., Mizuseki, K., Nakatani, J., Nakanishi, S., and Sasai,
Y. (2000) Proc. Natl. Acad. Sci. U. S. A.
97,
5291–5296
[Abstract/Free Full Text] - Zhu, Y., Oganesian, A., Keene, D. R., and Sandell, L. J.
(1999) J. Cell Biol.
144,
1069–1080
[Abstract/Free Full Text] - Abreu, J. G., Ketpura, N. I., Reversade, B., and De Robertis, E. M. (2002) Nat. Cell Biol. 4, 599–604[Medline] [Order article via Infotrieve]
- Kolle, G., Georgas, K., Holmes, G. P., Little, M. H., and Yamada, T. (2000) Mech. Dev. 90, 181–193[CrossRef][Medline] [Order article via Infotrieve]
- Georgas, K., Bowles, J., Yamada, T., Koopman, P., and Little, M. H. (2000) Dev. Dyn. 219, 582–587[CrossRef][Medline] [Order article via Infotrieve]
- Lovicu, F. J., Kolle, G., Yamada, T., Little, M. H., and McAvoy, J. W. (2000) Mech. Dev. 94, 261–265[CrossRef][Medline] [Order article via Infotrieve]
- Piper, R. C., Hess, L. J., and James, D. E. (1991) Am. J. Physiol. 260, C570–C580
- Martinez, G., Mishina, Y., and Bertram, J. F. (2002) Int. J. Dev. Biol. 46, 525–533[Medline] [Order article via Infotrieve]
- Ruddon, R. W., Sherman, S. A., and Bedows, E. (1996) Protein Sci. 5, 1443–1452[Medline] [Order article via Infotrieve]
- Kohl, R., Antoine, M., Olwin, B. B., Dickson, C., and Kiefer, P.
(2000) J. Biol. Chem.
275,
15741–15748
[Abstract/Free Full Text] - Little, M. H., Wilkinson, L., Brown, D. L., Piper, M., Yamada, T., and Stow, J. L. (2001) Am. J. Physiol. 281, C486–C495
- Piper, M., Georgas, K., Yamada, T., and Little, M. (2000) Mech. Dev. 94, 213–217[CrossRef][Medline] [Order article via Infotrieve]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Nystrom, K. Hultenby, S. Ek, J. Sjolund, H. Axelson, K. Jirstrom, M. A. Saleem, K. Nilsson, and M. E. Johansson CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney Nephrol. Dial. Transplant., July 1, 2009; 24(7): 2038 - 2044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. S. Butler, R. A. Dean, E. M. Tam, and C. M. Overall Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding Mol. Cell. Biol., August 1, 2008; 28(15): 4896 - 4914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wilkinson, T. Gilbert, G. Kinna, L.-A. Ruta, D. Pennisi, M. Kett, and M. H. Little Crim1KST264/KST264 Mice Implicate Crim1 in the Regulation of Vascular Endothelial Growth Factor-A Activity during Glomerular Vascular Development J. Am. Soc. Nephrol., June 1, 2007; 18(6): 1697 - 1708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Sun, F.-F. Zhuang, J. E. Mullersman, H. Chen, E. J. Robertson, D. Warburton, Y.-H. Liu, and W. Shi BMP4 Activation and Secretion Are Negatively Regulated by an Intracellular Gremlin-BMP4 Interaction J. Biol. Chem., September 29, 2006; 281(39): 29349 - 29356. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |