Medical and Research Services, Veterans Affairs Medical Center, Seattle 98108; and Department of Medicine, University of Washington, Seattle, Washington 98493
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ABSTRACT |
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The finding that insulin-like growth factor
(IGF)-binding protein-5 (IGFBP-5) binding to mouse osteoblasts was
capable of being downregulated by IGFBP-5 suggested that the 420-kDa
membrane protein, which interacted with IGFBP-5, may be a signaling
receptor (Andress, D. L. J. Biol.
Chem. 270: 28289-28296, 1995). In the current
study, a carboxy-terminal IGFBP-5 peptide, IGFBP-5-(201218), which
was found to competitively inhibit
125I-IGFBP-5 binding and to
specifically bind to osteoblast monolayers, was used to affinity-purify
the 420-kDa membrane protein. Coincubation of the affinity-purified
membrane protein with
[32P]ATP resulted in
autophosphorylation at serine residues. Serine phosphorylation of the
420-kDa protein was enhanced by intact IGFBP-5, IGFBP-5-(1
169), and
IGFBP-5-(201
218). When the IGFBP-5 receptor was incubated with
dephosphorylated casein in the presence of
[32P]ATP, casein became
phosphorylated on serine residues. These data indicate that IGFBP-5
stimulates the phosphorylation of the IGFBP-5 receptor and suggest that
serine/threonine kinase activation may be important in mediating some
of the IGF-independent effects of IGFBP-5.
osteoblast
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS (IGFBPs) function to transport the IGFs and to modulate their activity within the circulation and at the cellular level (9). Many cell types produce IGFBPs, where they act in an autocrine/paracrine manner to inhibit or enhance localized IGF-stimulated effects. Although most studies have focused on the IGF-modulating role of this class of binding proteins, a few reports have described the independent effects of selected IGFBPs (3, 5, 15, 17). For example, the effects of IGFBP-3 in breast cancer (15) and prostate cancer (17) cell lines have been ascribed to the action of one or more cell surface-associated proteins that specify IGF-independent growth inhibition (16, 17). In addition, IGFBP-1 (10) and IGFBP-5 (4) have been shown to bind to cells in culture, although specific signaling receptors for these ligands were not described.
IGFBP-5 is secreted by osteoblast-like cells (3), and it has
IGF-independent stimulatory effects in mouse (4, 13), rat (18), and
human (13) osteoblast-like cells. Both IGFBP-5-(1169) (3) and intact
IGFBP-5 (13) stimulate osteoblast proliferation in addition to
enhancing IGF-I-stimulated proliferation. Moreover, intact
IGFBP-5 but not IGFBP-5-(1
169) stimulates growth hormone (GH) receptor activity in rat osteoblasts (18). Our recent findings that IGFBP-5 binds to a 420-kDa membrane protein on osteoblasts and
that its binding is rapidly downregulated by this ligand (2) suggested
that the membrane protein may function as a receptor. To further
investigate this possibility, we have examined specific regions of
IGFBP-5 for their ability to bind to the osteoblast surface and to
determine whether IGFBP-5 induces the phosphorylation of the 420-kDa
binding protein. We now show that at least one region of IGFBP-5
functions to mediate the binding of IGFBP-5 to osteoblasts
and that a peptide within this domain is capable of stimulating the
phosphorylation of the IGFBP-5 receptor, preferentially on serine
residues.
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EXPERIMENTAL PROCEDURES |
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Materials.
Recombinant forms of IGFBP-5, intact IGFBP-5 and IGFBP-5-(1169), were
expressed in baculovirus and purified as described previously (4).
IGFBP-5 peptides IGFBP-5-(81
95), IGFBP-5-(94
106), IGFBP-5-(109
121), IGFBP-5-(123
136), IGFBP-5-(138
152),
IGFBP-5-(167
185), IGFBP-5-(183
200), IGFBP-5-(201
218),
and IGFBP-5-(235
252) were synthesized and purified by reversed-phase
HPLC (Fred Hutchinson Cancer Center, Seattle, WA).
Na125I was purchased from
Amersham, and [32P]ATP
was obtained from ICN Radiochemicals. Intact
125I-IGFBP-5,
125I-IGFBP-5-(1
169), and
125I-IGFBP-5-(201
218) were
prepared using chloramine T as described (3); specific activities
ranged from 100 to 150 µCi/µg. Transforming growth factor-
1
(TGF-
1) was from R and D Systems (Minneapolis, MN), and IGFBP-6 was
from Chiron (Emeryville, CA). Collagenase was purchased from
Worthington. L-Lysine, casein,
and ninhydrin were purchased from Sigma Chemical, and Affigel was
obtained from Bio-Rad.
Cell culture. Neonatal mouse osteoblasts were released from calvaria with collagenase, as previously described (3), and grown for 1 wk in DMEM and 10% FCS. Confluent cells were then released by trypsin and plated onto 48-well plates (Costar) containing 10% FCS.
IGFBP-5 binding to osteoblasts.
Confluent monolayers of osteoblasts in 48-well plates were incubated in
serum-free medium for 24 h. The cells were washed with PBS and then
incubated in assay buffer (20 mM HEPES, 0.1 mg/ml BSA, pH 7.0) for 2 h
at 4°C with intact
125I-IGFBP-5,
125I-IGFBP-5-(1169), or
125I-IGFBP-5-(201
218) in the
absence or presence of varying concentrations of unlabeled intact
IGFBP-5, IGFBP-5-(1
169), and IGFBP-5-(201
218). At the end of the
incubation period, the buffer was removed, and the cells were rinsed
with PBS and solubilized with 1 N NaOH. Radioactivity of the cell
lysates was quantified and specific binding was determined.
Preparation of extracellular matrix. Confluent osteoblasts were removed with trypsin-EDTA, plated onto 48-well plates, and grown to confluence over 72 h. Cells were then rinsed with ice-cold PBS, and the cell membranes were extracted in 1% Triton X-100-PBS for 10 min on ice, followed by removal of nuclei and cytoplasm with 25 mM ammonium acetate, pH 9.0, for 10 min (2). The remaining extracellular matrix (ECM) was rinsed with PBS and immediately used for binding studies.
IGFBP-5-(201218) affinity purification of the 420-kDa membrane
protein.
Membranes from mouse osteoblasts were prepared as previously described
(2). Briefly, confluent primary cultures of osteoblasts were detached
with 1 mM EDTA and pelleted; the cell pellet was resuspended in 10 mM
sodium phosphate, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 0.15 M NaCl, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 2 mM iodoacetic acid. After
sonication, the cell lysates were centrifuged at 12,000 g for 30 min, and the supernatant was
centrifuged at 40,000 g for 1 h. The
pellet was then suspended in 50 mM HEPES, pH 7.4, 0.15 M NaCl, 1 mM
PMSF, and 2 mM magnesium sulfate and was centrifuged at 40,000 g for 1 h. Membrane proteins were
extracted in 50 mM HEPES, pH 7.4, 1% Triton X-100, 0.15 mM NaCl, 1 mM
PMSF, and 2 mM magnesium sulfate overnight at 4°C and were
recovered in the supernatant after centrifugation at 12,000 g. The membrane preparation was
applied to an IGFBP-5-(201
218) affinity column [10 mg of
IGFBP-5-(201
218) bound to Affigel-10] overnight and equilibrated in 50 mM HEPES, pH 7.4, 1% Triton X-100. The column was
washed with 50 ml of equilibration buffer, followed by 50 ml of 50 mM
HEPES, pH 7.4, 0.05% Triton, 0.25 M NaCl, and 1 mM PMSF. Bound protein
was eluted with 8 ml of 50 mM HEPES, 0.05% Triton X-100, 1.5 M NaCl,
and 1 mM PMSF and concentrated with a Centricon-30 filtration device.
The eluted protein was separated on a 5% SDS-polyacrylamide gel under
reducing conditions and stained with silver. A similar membrane
affinity purification was also attempted using an IGFBP-5-(183
200)
affinity column.
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Phosphorylation of the 420-kDa IGFBP-5 receptor.
The affinity-purified IGFBP-5 receptor was preincubated without and
with intact IGFBP-5, IGFBP-5-(1169), IGFBP-5-(183
200), and
IGFBP-5-(201
218) at 0°C for 30 min in 20 mM HEPES, pH 7.4, 3.3%
glycerol, and 0.08% Triton X-100 (14). The phosphorylation reaction
was started by adding 5 µCi/5 µM [32P]ATP, 0.1%
mercaptoethanol, and 2.5 mM MnCl2.
After 20 min at 0°C, the phosphorylated IGFBP-5 receptor was
analyzed by 5% SDS-PAGE, and phosphoimages (Bio-Rad) of the dried gels
were obtained.
Phosphorylation of casein. IGFBP-5 receptor phosphorylation of casein was determined by incubating dephosphorylated casein without and with the affinity-purified IGFBP-5 receptor in 20 mM HEPES, pH 7.4, 3.3% glycerol, and 0.08% Triton X-100 at 0°C for 30 min. Phosphorylation was then started by adding 5 µCi [32P]ATP, 0.1% mercaptoethanol, and 2.5 mM MnCl2 for 20 min on ice. Phosphorylation products were separated through a 4-15% gradient SDS-polyacrylamide gel and identified by phosphoimaging of the dried gels.
Phosphoamino acid analysis. Phosphoamino acid analysis was performed using two-dimension thin-layer chromatography as described by Duclos et al. (6). After SDS-polyacrylamide gel analysis of the phosphorylated products, the bands of interest were cut out of the dried gel and incubated overnight in 10% isopropanol-10% acetic acid. The gel pieces were then incubated twice in 50% methanol for 90 min each and dried by speed-vac for 1 h. Gel pieces were incubated in 500 µl 25 mM ammonium bicarbonate containing 50 ng/ml trypsin overnight at 37°C, at which time 500 µl of 25 mM ammonium bicarbonate were added for 2 h at 37°C (8). The supernatant was removed and dried by speed-vac, and the sample was resuspended in 6 N hydrochloric acid for 2 h at 110°C. After drying, the sample was resuspended in 10 µl of water containing 10 µg each of phosphoserine, phosphothreonine, and phosphotyrosine. Phenol red was added to the sample before its application to a sheet of cellulose acetate (Kodak), which was run in the first dimension for 5 h using isobutyric acid-0.5 M ammonium dihydroxide (5:3). The cellulose acetate was then dried overnight, and the second dimension was run with isopropanol-hydrochloric acid-water (7:1.5:1.5) as the solvent. The cellulose sheets were air dried, sprayed with ninhydrin to identify the phosphoamino acid standards, and then dried at 110°C for 30 min before identification of the 32P-labeled amino acids with the phosphoimager.
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RESULTS |
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Competition binding studies using different IGFBP-5 peptides were
performed to identify potential sites within IGFBP-5 that may be
responsible for mediating its attachment to the osteoblast surface. In
comparing unlabeled IGFBP-5-(1169) and intact IGFBP-5 for their
ability to compete for intact
125I-IGFBP-5 binding (Fig.
1A),
we found that IGFBP-5-(1
169) was ineffective as a competitor.
However, when
125I-IGFBP-5-(1
169) binding was
examined, both unlabeled intact IGFBP-5 and IGFBP-5-(1
169) competed
for binding, although IGFBP-5-(1
169) was a more effective competitor
(Fig. 1B). lt was unclear from these
studies whether conformational differences due to truncation of IGFBP-5
can fully explain the differences in competitive binding. Thus, to
further investigate whether amino acids within other portions of
IGFBP-5 may be important in mediating its binding to osteoblasts,
synthetic peptides from the variable region and from the carboxy
terminus were used to compete for intact
125I-IGFBP-5 binding. As shown in
Table 1, of the peptides tested, IGFBP-5-(201
218) was
the major inhibitor of intact
125I-IGFBP-5 binding. Whereas most
of these peptides are relatively neutral, the exceptions are
IGFBP-5-(235
252), which is acidic, and IGFBP-5-(138
152) and
IGFBP-5-(201
218), which are basic. Thus, because IGFBP-5-(138
152)
did not inhibit intact
125I-IGFBP-5 binding
substantially, the effect of IGFBP-5-(201
218) may not be solely
attributable to its basic charge.
To more thoroughly evaluate the role of IGFBP-5-(201218), competition
binding studies were performed to evaluate the relative effectiveness
of this peptide to function as a competitor for intact
125I-IGFBP-5 and
125I-IGFBP-5-(1
169) binding to
osteoblast monolayers. On a molar basis, lGFBP-5-(201
2l8) was
~100-fold less effective than unlabeled intact IGFBP-5 in competing
with intact 125I-IGFBP-5 for
binding sites (Fig.
2A), and
it was ~1,000-fold less effective than unlabeled IGFBP-5-(1
169) in
competing with 125I-IGFBP-5-(1
169) binding.
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Because these data suggested that IGFBP-5-(201218) may bind directly
to the osteoblast surface, we evaluated this possibility by incubating
mouse osteoblasts and osteoblast-derived ECM with 125I-IGFBP-5-(201
218) to
determine its specificity for binding. As shown in Fig.
3
(inset), almost all of the
125I-IGFBP-5-(201
218)
specifically bound to the monolayers compared with its binding to
osteoblast-derived ECM. Competition binding studies with unlabeled
IGFBP-5-(201
218) confirmed its specificity for cell binding.
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Because these studies indicated that the 201-218 region within the
carboxy terminus of IGFBP-5 is one of the sites that interacts with the
osteoblast surface, we prepared an IGFBP-5-(201218) affinity column
to determine whether it would capture the 420-kDa osteoblast membrane
protein previously recognized as a binding site for intact IGFBP-5 and
for IGFBP-5-(1
169) (2). Osteoblast membrane preparations were applied
to the affinity column, and after a wash with buffer containing 0.25 M
NaCl, protein was eluted with 1.5 M NaCl, concentrated, and separated
on a 4-15% SDS-PAGE. Shown in Fig. 4
is the silver stain of the 420-kDa protein that was eluted from the
affinity column. Its size is identical to the osteoblast membrane
protein we previously recovered using an intact IGFBP-5
affinity column (2). We did not detect the 420-kDa protein after
applying similar amounts of membrane protein to the IGFBP-5-(183
200)
affinity column (data not shown).
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Our previous results with this membrane protein suggested that it not
only functions to bind IGFBP-5 but may also act as a receptor for this
ligand, because 125I-IGFBP-5
binding was rapidly downregulated by prior exposure to unlabeled
IGFBP-5 and internalization of both intact
125I-IGFBP-5 and
125I-IGFBP-5-(1169) could be
demonstrated (2). To evaluate further the possibility that the 420-kDa
membrane protein behaved as a receptor, we asked whether it was capable
of becoming phosphorylated in response to IGFBP-5. Affinity-purified
preparations of the 420-kDa membrane protein were incubated without and
with intact IGFBP-5, IGFBP-5-(1
169), and
IGFBP-5-(201
218). As shown in Fig. 5,
intact IGFBP-5 (lane 1) and
IGFBP-5-(1
169) (lane 2) caused an
increase in [32P]ATP labeling of the
420-kDa membrane protein. IGFBP-5-(201
218) also stimulated
phosphorylation of the membrane protein (Fig. 5B) at concentrations of 2 µg/ml
(lane 2) and 20 µg/ml
(lane 3). The stimulatory effect was
specific, because phosphorylation was not stimulated with 20 µg/ml
IGFBP-5-(183
200) or with 10 nM IGF-I, IGFBP-6, or TGF-
(data not
shown). Constitutive autophosphorylation was present in
lane 3 (Fig.
5A) and in lane
1 (Fig. 5B).
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To determine the type of phosphorylation induced by IGFBP-5,
phosphoamino acid analysis of the IGFBP-5 receptor was performed after
stimulation with IGFBP-5-(201218). As shown in Fig.
6, two-dimensional thin-layer
chromatography of tryptic digests of the
32P-labeled IGFBP-5 receptor
revealed that the 32P label
comigrated with serine residues in the control (unstimulated) receptor
preparation and that serine phosphorylation was enhanced on exposure to
20 µg/ml IGFBP-5-(201
218). Because some serine/threonine kinases
can be stimulated by lysine (20), we used lysine to determine whether
it could also stimulate phosphorylation and found that it stimulated
the 32P labeling of serine and
threonine residues of the IGFBP-5 receptor.
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To demonstrate that the IGFBP-5 receptor could function as a protein kinase, casein was tested as a possible substrate for phosphorylation. Dephosphorylated casein became phosphorylated only in the presence of the IGFBP-5 receptor (Fig. 7A), and tryptic digestion of 32P-labeled casein revealed that serine residues were predominantly phosphorylated (Fig. 7B).
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DISCUSSION |
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Our previous results demonstrating the binding and internalization of
intact IGFBP-5 and IGFBP-5-(1169) in neonatal mouse osteoblasts
suggested that these ligands were interacting with a putative receptor
(2). The 420-kDa osteoblast membrane protein, which was partially
purified using an intact IGFBP-5 affinity column in those studies, is
now also shown to be retrievable from an IGFBP-5-(201
218) affinity
column. Because intact IGFBP-5, IGFBP-5-(1
169), and
IGFBP-5-(201
218) all stimulate the phosphorylation of this membrane
protein, and it in turn is capable of phosphorylating casein, these
data indicate that intact as well as truncated versions of IGFBP-5 bind
to a cell surface receptor that then stimulates serine/threonine kinase
activity. This is the first indication that an IGFBP can directly
stimulate protein phosphorylation and adds to the data showing that
selected members of this family of proteins have effects on cells that
are not mediated by the IGFs.
The finding that different regions of IGFBP-5 are capable of
stimulating IGFBP-5 receptor activity suggests that there may be more
than one site on IGFBP-5 that interacts with the receptor. Because not
all of the regions within IGFBP-5 were tested for binding activity, we
can only speculate that amino acids within the 1-81 and
153-169 regions may mediate the binding of IGFBP-5-(1169). Similarly, we cannot exclude the possibility that amino acids within
positions 170-200 and 219-234 may also be involved with binding the carboxy terminus of IGFBP-5 to its receptor. The finding that IGFBP-5-(201
218) was a weak competitor of IGFBP-5-(1
169) binding suggests that basic residues within IGFBP-5-(1
169) may be
important binding sites. However, it is possible that the differences in the competition binding studies may be due to the use of relatively small peptides, which would lack the potentially important secondary or
tertiary structure needed for optimal binding. Despite these limitations, more than one region of IGFBP-5 is likely to be important for binding and possible stimulation of receptor activity. Thus it will
be important to identify all potential receptor binding sites of
IGFBP-5, because cell-specific proteolysis (19) likely plays a major
role in the regulation of IGFBP-5 function and receptor activation.
The identity of the IGFBP-5 receptor is unknown. A review of the
literature has revealed the existence of only one other large molecular
weight membrane protein that contains serine/threonine kinase activity,
the 400-kDa type V TGF- receptor (14). This receptor has structural
similarities to the type II TGF-
receptor (11), the activin receptor
(12), and daf-1 gene products (7). Although the IGFBP-5 receptor and
the type V TGF-
receptor are functionally similar in being able to
phosphorylate casein on serine residues, their ligand binding
characteristics appear to be different, because TGF-
was not able to
stimulate IGFBP-5 receptor phosphorylation (data not shown). It is
still possible that structural similarities exist between the IGFBP-5
receptor and this class of membrane receptors within their
membrane-spanning or cytoplasmic domains. It is also possible that the
IGFBP-5 receptor may be structurally related to the IGFBP-3-specific
membrane-associated proteins, which were recently shown to mediate
IGF-independent growth inhibition of epithelial-like carcinoma cells
(16). However, those membrane acceptor proteins (20-50 kDa) are
smaller than the IGFBP-5 receptor, and they have not yet been shown to
internalize on ligand binding or to possess phosphorylation capability,
which would classify them as signaling receptors.
The functional role of enhanced serine/threonine kinase activity in
response to IGFBP-5 remains speculative at this time. In
osteoblast-like cells, IGFBP-5-(1169) (4) and intact IGFBP-5 (13)
have both been shown to stimulate
[3H]thymidine
incorporation into DNA by IGF-independent mechanisms. Moreover, IGFBP-5
stimulates GH receptor synthesis in cultured rat osteoblasts (18), and
IGFBP-5-(201
218) induces mesangial cell cytoskeletal reorganization
and migration independent of IGF-I stimulation (1). These seemingly
diverse physiological functions most likely relate to cell-specific
differences in IGFBP-5-induced stimulation and presumptive signaling.
However, because of the lack of a unifying pattern of cell
responsiveness (proliferative vs. differentiated function), it is
unclear whether all of these IGFBP-5-inducible events are secondary to
IGFBP-5 receptor signaling or are mediated by nonsignaling pathways.
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ACKNOWLEDGEMENTS |
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The technical assistance of Dawn Moran is gratefully acknowledged.
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FOOTNOTES |
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This work was supported by funds provided by the Research Service of the Department of Veterans Affairs.
Address for reprint requests: D. L. Andress, VA Medical Center (111A), 1660 South Columbian Way, Seattle, WA 98108.
Received 28 August 1997; accepted in final form 9 January 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abrass, C. K.,
A. Berfield,
and
D. L. Andress.
Heparin binding domain of insulin-like growth factor binding protein-5 stimulates mesangial cell migration.
Am. J. Physiol.
273 (Renal Physiol. 42):
F899-F906,
1997
2.
Andress, D. L.
Heparin modulates the binding of insulin-like growth factor (IGF) binding protein-5 to a membrane protein in osteoblastic cells.
J. Biol. Chem.
270:
28289-28296,
1995
3.
Andress, D. L.,
and
R. S. Birnbaum.
Human osteoblast-derived insulin-like growth factor (IGF) binding protein-5 stimulates osteoblast mitogenesis and potentiates IGF action.
J. Biol. Chem.
267:
22467-22472,
1992
4.
Andress, D. L.,
S. M. Loop,
J. Zapf,
and
M. C. Kiefer.
Carboxy-truncated insulin-like growth factor binding protein-5 stimulates mitogenesis in osteoblast-like cells.
Biochem. Biophys. Res. Commun.
195:
25-30,
1993[Medline].
5.
Bar, R. S.,
B. A. Booth,
M. Boes,
and
B. L. Dake.
Insulin-like growth factor-binding proteins from vascular endothelial cells: purification, characterization, and intrinsic biological activities.
Endocrinology
125:
1910-1920,
1989[Abstract].
6.
Duclos, B.,
S. Marcandier,
and
A. J. Cozzone.
Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis.
Methods Enzymol.
201:
10-21,
1991[Medline].
7.
Georgi, L. L.,
P. S. Albert,
and
D. L. Riddle.
Daf-1, a C. elegans gene controlling Dauer larva development, encodes a novel receptor protein kinase.
Cell
61:
635-645,
1990[Medline].
8.
Hemmings, H. C.,
A. C. Nairn,
and
P. Greengard.
DARPP-32, a dopamine-and adenosine 3': 5'-monophosphate-regulated neuronal phosphoprotein. Comparison of the kinetics of phosphorylation of DARPP-32 and phosphatase inhibitor 1.
J. Biol. Chem.
259:
14491-14497,
1984
9.
Jones, J. I.,
and
D. R. Clemmons.
Insulin-like growth factors and their binding proteins: biological actions.
Endocr. Rev.
16:
3-34,
1995[Medline].
10.
Jones, J. I.,
A. Gockerman,
W. H. Busby,
G. Wright,
and
D. R. Clemmons.
Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the 5
1 integrin by means of its Arg-Gly-Asp sequence.
Proc. Natl. Acad. Sci. USA
90:
10553-10557,
1993[Abstract].
11.
Lin, H. Y.,
X. F. Wang,
E. Ng-Eaton,
R. A. Weinberg,
and
H. F. Lodish.
Expression cloning of the TGF- type II receptor, a functional transmembrane serine/threonine kinase.
Cell
68:
775-785,
1992[Medline].
12.
Matthews, L. S.,
and
W. W. Vale.
Expression cloning of an activin receptor, a predicted transmembrane serine kinase.
Cell
65:
973-982,
1991[Medline].
13.
Mohan, S.,
Y. Nakao,
Y. Honda,
E. Landale,
U. Leser,
C. Dony,
K. Lang,
and
D. J. Baylink.
Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells.
J. Biol. Chem.
270:
20424-20431,
1995
14.
O'Grady, P.,
Q. Liu,
S. S. Huang,
and
J. S. Huang.
Transforming growth factor (TGF-
) type V receptor has a TGF-
-stimulated serine/threonine-specific autophosphorylation activity.
J. Biol. Chem.
67:
21033-21037,
1992.
15.
Oh, Y.,
H. L. Muller,
G. Lamson,
and
R. G. Rosenfeld.
Insulin-like growth factor (IGF)-independent action of IGF-binding protein-3 in Hs578T human breast cancer cells.
J. Biol. Chem.
268:
14964-14971,
1993
16.
Oh, Y.,
H. L. Muller,
H. Pham,
G. Lamson,
and
R. G. Rosenfeld.
Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells.
J. Biol. Chem.
268:
26045-26048,
1993
17.
Rajah, R.,
B. Valentinis,
and
P. Cohen.
Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor -1 on programmed cell death through a p53- and IGF-independent mechanism.
J. Biol. Chem.
272:
12181-12188,
1997
18.
Slootweg, M. C.,
C. Ohlsson,
E. J. van Elk,
J. C. Netelenbos,
and
D. L. Andress.
Growth hormone (GH) receptor activity is stimulated by insulin-like growth factor binding protein (IGFBP)-5 in rat osteosarcoma cells.
Growth Reg.
6:
238-246,
1996[Medline].
19.
Thrailkill, K. M.,
L. D. Quarles,
H. Nagase,
K. Suzuki,
D. M. Serra,
and
J. L. Fowlkes.
Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation.
Endocrinology
136:
3527-3533,
1995[Abstract].
20.
Walton, G. M.,
J. Spiess,
and
G. N. Gill.
Phosphorylation of high mobility group protein 14 by casein kinase II.
J. Biol. Chem.
260:
4745-4750,
1985[Abstract].