(Received for publication, August 10, 1994; and in revised form, November 2, 1994)
From the
A rat pituitary tumor cell line (GH) has been
reported to express transforming growth factor-
(TGF-
)
binding components of 70-74 kDa (ligand included), denoted
TGF-
type IV receptor. We investigated whether the type IV
receptor corresponds to any of the recently cloned type I receptors for
proteins in the TGF-
superfamily. TGF-
type I receptor
(T
R-I) complexes of 69-72 kDa formed a heteromeric complex
with T
R-II in GH
cells, as detected by
immunoprecipitation. In addition, TGF-
formed complexes of
72-74 kDa, which were different from T
R-I and the other
known type I receptors, and were not dependent on T
R-II for
binding. The GH
cells were resistant to the growth
inhibitory activity of TGF-
, but a transcriptional response was
activated by TGF-
in this cell line, presumably through the
T
R-II and T
R-I complex. These results indicate that GH
cells have T
R-I and T
R-II and, in addition, other
binding protein(s) which form 72-74-kDa complexes with TGF-
;
the function of the latter component(s) remains to be elucidated.
Transforming growth factor- (TGF-
) (
)is a
family of 25-kDa dimeric proteins which regulate cell proliferation and
differentiation, accelerate extracellular matrix production, and
modulate immune functions(1) . TGF-
s belong to a larger
protein family denoted the TGF-
superfamily which also includes
activins and inhibins and bone morphogenic proteins (BMPs)(2) .
These proteins have sequence similarities to each other, and in
particular 7 cysteine residues are conserved in most of the members in
the family. The active forms of most of the members are dimers, which
are derived from larger precursor proteins. Inhibins are heterodimers
of one
chain and one
chain (
or
), whereas activins are homodimers composed of two
inhibin
chains (activin A) or two inhibin
chains (activin B), or a heterodimer of one
and
one
chain (activin AB)(3) . Activins
stimulate the secretion of follicle-stimulating hormone from the
pituitary gland and the differentiation of hematopoietic cells.
Activins are also known to induce the mesoderm formation in Xenopus embryo. Inhibins, on the other hand, inhibit the secretion of
follicle-stimulating hormone from the pituitary gland.
TGF-s
exert their functions through the interaction with various receptors
and binding proteins on the cell
surface(4, 5, 6, 7, 8) .
These include TGF-
type I receptor (T
R-I, 53 kDa), type II
receptor (T
R-II, 75 kDa), betaglycan or type III receptor (more
than 200 kDa), endoglin (180 kDa), type IV receptor (57-62 kDa),
type V receptor (400 kDa), type VI receptor (180 kDa), and
phosphatidylinositol-anchored membrane proteins. T
R-I and
T
R-II are widely expressed on many different cell types (9, 10, 11) and form a heteromeric complex on
the cell surface after ligand
binding(12, 13, 14, 15, 16) .
Betaglycan (17, 18) and endoglin (19) are also
expressed on several different cell types. They have short
intracellular domains, which are highly similar to each other in their
amino acid sequences. Betaglycan and endoglin may present the ligands
to T
R-II and/or T
R-I, which are the receptors primarily
involved in
signaling(18, 20, 21, 22) . The
functions for the other receptors and binding proteins remain to be
elucidated.
cDNA cloning of TR-II revealed that it has an
intracellular serine/threonine kinase domain(9) . Type II
receptors for activin (ActR-II and ActR-IIB) (23, 24, 25) and BMPs (DAF-4 from Caenorhabditis elegans) (26) were also shown to be
transmembrane serine/threonine kinases. A series of novel
serine/threonine kinase receptors have also been identified and denoted
activin receptor-like kinases (ALK)
1-6(10, 27, 28) , and have been shown
to be type I receptors for the members in the TGF-
superfamily.
ALK-5 is a signaling TGF-
type I receptor (T
R-I) (10, 11, 28, 29) , ALK-2 and ALK-4
are activin type I receptors (ActR-I and ActR-IB,
respectively)(28, 30, 31) , whereas ALK-3 and
ALK-6 are type I receptors for osteogenic protein-1 and BMP-4 (BMPR-IA
and BMPR-IB, respectively)(32) .
The TGF- type IV
receptor has been reported to be expressed only on GH
rat
pituitary tumor cells(33) . Binding competition studies
revealed that this receptor binds not only TGF-
s, but also activin
AB and inhibin B. The molecular mass of the type IV receptor complex
has been reported to be 70-74 kDa including ligand, which is
similar to the sizes of type I receptor complexes. In the present
study, we have studied whether the type IV receptor complex on GH
pituitary tumor cells contains any of the known type I receptors.
The used oligonucleotide primers are as
follows: 5R-3 (sense), nucleotides 96-113 of TR-I (5`-GCTCT
AGATT TCTGC CACCT CTGTAC-3`); 5R-2 (antisense), nucleotides,
441-424 of T
R-I (5`-GCGAA TTCGA CAGTG CGGTT ATGGC A-3`);
2R-1 (sense), nucleotides, -58 to -41 of T
R-II
(5`-GCTCT AGACC CGAGG CTCGT TCGCG G-3`); 2R-2 (antisense), nucleotides
585-568 of T
R-II (5`-GCGAA TTCCT GCTGC CGGTG GACAC G-3`).
The numbering of nucleotides start from the first nucleotide
(+1) of the open reading frames of rat homologue of TR-I, R4 (11) or the rat T
R-II(36) . The sense primers have
an XbaI site (TCTAGA) in the 5` end regions, and the antisense
primers have an EcoRI site (GAATTC) in the 5` end regions.
Identification of TR-I and T
R-II in
GH
Cells-Cross-linking studies using
I-TGF-
1 revealed that GH
cells expressed
a triplet of receptor complexes of molecular masses of 69-74 kDa,
including the TGF-
1 monomer (Fig. 1, lane 1);
these properties are identical to those reported for the TGF-
type
IV receptor(33) . In order to examine whether the complexes of
69-74 kDa contain any of known type I receptors for members in
the TGF-
superfamily, immunoprecipitation using antisera against
the type I receptors was performed. The antiserum against T
R-I
immunoprecipitated components which appeared to correspond to the lower
one or two bands of the triplet band of 69-72 kDa observed by
cross-linking without immunoprecipitation (Fig. 1). A component
of 86 kDa, i.e. a size similar to that of a T
R-II
complex, which could not be seen without immunoprecipitation because of
its low abundance, was coimmunoprecipitated with T
R-I. Antisera to
the other type I receptors did not efficiently immunoprecipitate any
cross-linked complexes in GH
cells.
Figure 1:
Analysis
of cross-linked TGF- receptor complexes in GH
cells by
immunoprecipitation using antisera against type I receptors. GH
cells were incubated with
I-TGF-
1, followed by
cross-linking with DSS. Aliquots of cell lysates were analyzed directly
by SDS-gel electrophoresis, followed by autoradiography (lane
1), or first subjected to immunoprecipitation (IP) using
antisera against type I receptors (lanes
2-7).
The
immunoprecipitated TR-I and T
R-II complexes in GH
cells were compared to those in another rat cell line, NRK, which
is known to express T
R-I, T
R-II, and betaglycan and to
respond to TGF-
(34, 37) . Although the binding
efficiencies observed by cross-linking (Fig. 2, lanes 1 and 2) were different between these cell types, the sizes
of the T
R-I and T
R-II complexes immunoprecipitated from
GH
cells corresponded to those of NRK cells (Fig. 2, lanes 3, 5, 7, and 9). Using both
GH
cells and NRK cells, T
R-II complexes were
coimmunoprecipitated with T
R-I complexes and vice versa.
The fact that similar amounts of T
R-I were brought down from
GH
cells with antisera against either T
R-I or
T
R-II, indicates that T
R-II is present in GH
cells at an amount equal to or higher than that of T
R-I. The
reason for the poor efficiency in cross-linking to TGF-
receptors
on GH
cells, compared to NRK cells, is not known. The
T
R-I complexes immunoprecipitated by both the T
R-I and
T
R-II antisera had molecular masses of 69-72 kDa, which
appear to correspond to the lower components of the triplet of bands
observed without immunoprecipitation.
Figure 2:
Comparison of cross-linked TGF-
receptor complexes in GH
cells and NRK cells by
immunoprecipitation using antisera against T
R-I and T
R-II.
GH
cells and NRK cells were incubated with
I-TGF-
1, followed by cross-linking with DSS.
Aliquots of the cell lysates were analyzed directly by SDS-gel
electrophoresis (lanes 1 and 2), or first subjected
to immunoprecipitation (IP) using antisera against T
R-I
or T
R-II (lanes 3-10). Blocking of the antisera (Block) was performed with 10 µg each of the corresponding
peptides (lanes 4, 6, 8, and 10).
The gel was analyzed by autoradiography.
Figure 3:
Biochemical characterization of the
69-74 kDa complexes affinity cross-linked with I-TGF-
1. A, binding of
I-TGF-
1 to GH
cells treated with
dithiothreitol. GH
cells were treated with or without 1
mM dithiothreitol (DTT) in the binding buffer without
bovine serum albumin at 37 °C for 8 min, and then incubated with
I-TGF-
1, followed by cross-linking with DSS. The
cross-linked complexes were directly analyzed by SDS-gel
electrophoresis (lanes 1 and 2) or first subjected to
immunoprecipitation (IP) using the T
R-I antiserum (lanes 3 and 4). The gel was analyzed by
autoradiography. B, deglycosylation with endoglycosidase F (Endo F) of the complexes affinity cross-linked with
I-TGF-
1. GH
cells were incubated with
I-TGF-
1 and followed by cross-linking with DSS. The
samples were treated with 0.5 unit of endglycosidase F and directly
analyzed by SDS-gel electrophoresis (lanes 1 and 2)
or first subjected to immunoprecipitation (IP) with the
T
R-I antiserum (lanes 3 and 4). The gel was
analyzed by autoradiography.
TR-I has been reported to have an N-linked carbohydrate chain and can be deglycosylated by
endoglycosidase F(10) . When the cross-linked complexes of
GH
cells were treated with endoglycosidase F, the upper
bands of the cross-linked complexes (72-74 kDa) did not shift (Fig. 3B, lanes 1 and 2). However,
when the 69-72-kDa complexes immunoprecipitated by the T
R-I
antiserum were subjected to deglycosylation, they shifted to 68 kDa (Fig. 3B, lanes 3 and 4). The
T
R-II component that coimmunoprecipitated with T
R-I also
shifted from 86 to 76 kDa (Fig. 3B, lanes 3 and 4), consistent with a previous report(10) .
These results indicate that the 69-72-kDa components represent
TR-I, which forms a heteromeric complex with T
R-II. In
contrast, the 72-74-kDa complexes are distinct from T
R-I.
Since the 72-74-kDa components were not immunoprecipitated by the
T
R-II antiserum (see Fig. 2, lane 5), they do not
form a complex with T
R-II.
Figure 4:
Competition of the binding of I-TGF-
1 to the 69-74-kDa components in
GH
cells by unlabeled ligands. GH
cells were
incubated with
I-TGF-
1 in the presence or absence of
200-fold excesses of unlabeled TGF-
1 or activin A (Cold
ligand), followed by cross-linking with DSS. Aliquots of cell
lysates were directly analyzed by SDS-gel electrophoresis (lanes
1-3) or first subjected to immunoprecipitation (IP)
using the antiserum against T
R-I (lanes 4-6). The
gel was analyzed by autoradiography.
Figure 5:
RT-PCR analysis of expression of TR-I
and T
R-II mRNAs in GH
cells and NRK cells. Total RNAs
from GH
cells and NRK cells were reverse transcribed into
cDNAs using oligonucleotide primers specific for the rat T
R-I or
T
R-II. cDNAs were amplified by PCR using specific primers for
T
R-I or T
R-II and analyzed by 1% agarose gel electrophoresis,
followed by ethidium bromide staining.
The signaling activity of TGF-1 was also investigated by a
transcriptional assay using the p3TP-Lux promoter-reporter construct (13, 30) . In GH
cells transfected with
the p3TP-Lux plasmid, transcriptional activation of p3TP-Lux was
observed by the addition of TGF-
1 (Fig. 6), which indicates
that TGF-
1 can transduce signals in this cell line.
Figure 6:
Transcriptional activation by TGF-1
in GH
cells transfected with p3TP-Lux promoter-reporter
construct. GH
cells were transfected with p3TP-Lux and
stimulated by various concentrations of TGF-
1 for 24 h. The
cellular proteins were extracted and luciferase activity was measured
using the luciferase assay system (Promega). Luciferase activity is
expressed relative to a control value without
stimulation.
The present study shows that TR-I and T
R-II are
expressed in GH
cells and form a heteromeric complex in the
presence of TGF-
. Affinity labeled complexes of 69-72 kDa
including
I-TGF-
1 were immunoprecipitated by an
antiserum against T
R-I, but not by antisera against the other type
I receptors. The T
R-II antiserum immunoprecipitated the
69-72-kDa T
R-I components together with T
R-II. Binding
of TGF-
1 to the components of 69-72 kDa was abolished by
dithiothreitol treatment of GH
cells before incubation with
I-TGF-
1, and the complexes shifted to 68 kDa after
deglycosylation. RT-PCR analysis revealed that GH
cells
express mRNAs for T
R-I and T
R-II. These results indicate that
the 69-72-kDa components on GH
cells represent
T
R-I.
The 69-74 kDa components observed by cross-linking
studies contained at least three bands, and the lower one or two bands
corresponded to TR-I. The upper components of the 72-74-kDa
multiple components in GH
cells were not immunoprecipitated
by antisera to T
R-I, T
R-II, or the other type I receptors ( Fig. 1and Fig. 2) and were resistant to treatments with
dithiothreitol or endoglycosidase F. Similarly multiple components with
molecular masses of 70-77 kDa were observed in other cell types, e.g. human foreskin fibroblasts (AG1518), human lung
adenocarcinoma cells (A549), and human oral squamous cell carcinoma
cells (HSC-2)(10) . Also in these cell lines, only the 70-kDa
affinity labeled complex was immunoprecipitated by an antiserum against
T
R-I. The 69-74-kDa-cross-linked components in GH
cells were reported to bind activin AB and inhibin B in addition
to TGF-
s and have been suggested to be a novel type of cell
surface binding protein, denoted type IV receptor(33) . In the
present study, the binding of
I-TGF-
1 to the
72-74-kDa component was only slightly competed with unlabeled
excess amounts of activin A, whereas the 69-72-kDa T
R-I
complexes were not at all competed by activin A. Taken together, the
present data revealed that GH
cells express unidentified
components capable of forming a complex of 72-74 kDa with
I-TGF-
1; the protein(s) are different from T
R-I
and the other known type I receptors, and do not form a complex with
T
R-II. It remains to be elucidated whether the novel binding
protein is a serine/threonine kinase receptor, like T
R-I and
T
R-II; alternatively, it may serve as an accessory molecule, which
indirectly regulates the signal transduction by T
R-I and
T
R-II.
Type II receptor-like components were not detected by
the cross-linking studies using I-TGF-
1; however,
immunoprecipitation of the cross-linked complexes by an antiserum
against T
R-II revealed that T
R-II is present in GH
cells and is able to form a heteromeric complex with T
R-I.
That GH
cells express the canonical T
R-II is also
supported by the demonstration of mRNA for T
R-II by RT-PCR
analysis. Similar observations were reported using A549 cells and HSC-2
cells(10) , where cross-linked T
R-II complexes occurred at
very low abundance; immunoprecipitation of the cross-linked complexes
using a specific antiserum against T
R-II revealed the expression
of T
R-II protein. It appears that the efficiency in cross-linking
to T
R-II can vary (see Fig. 2). Thus, in order to study the
expression of T
R-II by affinity cross-linking, it is important to
immunoprecipitate the cross-linked complexes to avoid that the binding
to T
R-II is hidden by a nonspecific background.
Activins and
inhibins regulate the secretion of follicle-stimulating hormone from
rat pituitary cells. Since activin A did not efficiently compete with I-TGF-
1 for binding to the 72-74-kDa complex,
it is unlikely that this putative receptor transduces activin responses
in GH
cells. We have recently found that rat pituitary
cells in primary culture express ActR-II and ActR-IB. (
)
The signaling activity of TGF- in GH
cells was investigated by growth inhibition and p3TP-Lux
transcriptional activation assays. GH
cells did not respond
to TGF-
1 with regard to the growth inhibition; analogously other
transformed cell lines, e.g. HSC-2 oral squamous cell
carcinoma cells (40) , PC-3 prostate carcinoma
cells(39) , EJ bladder carcinoma cells, and SW 480 colon
carcinoma cells (41) are also unresponsive to TGF-
even
though they express T
R-I and T
R-II on their cell surfaces. On
the other hand, TGF-
1 stimulated the transcription of p3TP-Lux.
Since the T
R-I and T
R-II complex has been shown to induce the
p3TP-Lux transcriptional activation by
TGF-
1(11, 13, 31) , T
R-I and
T
R-II expressed on GH
cells may be responsible for
this signal. Whether the 72-74-kDa components are involved in
TGF-
signal transduction remains to be elucidated. These data
indicated that the signaling pathway for the transcriptional activation
of p3TP-Lux is conserved, but the growth inhibitory signal is perturbed
in the GH
cell line. Escape from the growth inhibition by
TGF-
may be related to the loss of growth control of cancer cells.
Further studies of the signal transduction pathways for TGF-
are
important to elucidate the mechanisms for carcinogenesis.