(Received for publication, August 15, 1994; and in revised form, November 1, 1994)
From the
Transforming growth factors (TGF-
s) and activins
induce and inhibins block secretion of follicle-stimulating hormone by
rat GH3 pituitary tumor cells. Cheifetz et al. (Cheifetz, S.,
Ling, N., Guillemin, R., and Massagué, J.(1988) J. Biol. Chem. 263, 17225-17228) reported that GH3 cells
express a
50-kDa surface protein, termed the type IV TGF-
receptor, that directly binds all of these peptide hormones. Here we
show that GH3 cells express the previously identified type I and type
II receptors for TGF-
and activin-A. Immunoprecipitation of
affinity-labeled surface binding proteins with antisera specific to
known receptors demonstrated independent heteromeric complexes of
TGF-
types I and II receptors and of activin types I and II
receptors. As judged by ligand-binding and cross-linking analysis,
TGF-
binding to the TGF-
receptors is not inhibited by
activin-A and activin-A binding to its receptors is not inhibited by
TGF-
. Screening of a cDNA library from GH3 cells for potential
receptor serine-threonine kinases yielded the known types I and II
TGF-
and activin receptors. The presumed common intracellular
signaling pathway for TGF-
and activin in GH3 cells appears to be
mediated by distinct cell-surface receptors.
The transforming growth factors- (TGF-
s), (
)activins and inhibins are structurally similar dimeric
polypeptides that regulate cell growth, differentiation, and
development (reviewed in (1, 2, 3) ). These
polypeptides belong to a large superfamily of growth factors which also
includes mammalian bone morphogenetic proteins and
Müllerian inhibiting substance(4) .
Inhibins are heterodimers of
and
chains while activins are
dimers of
chains(1) . Mammals have three homodimeric
isoforms of TGF-
, termed TGF-
1, -
2, and -
3, and at
least two heterodimeric isoforms, TGF-
1.2 and
TGF-
2.3(2, 3) . Activins induce synthesis and
release of follicle-stimulating hormone by pituitary
cells(5, 6) , stimulate steroidogenesis in granulosa
cells (7, 8) , stimulate erythroid
differentiation(9) , and induce mesodermal tissues during
amphibian development(10, 11) . In general, inhibins
have opposing effects to those of activins(1, 12) .
TGF-
s generally mimic the function of activins in the
above-mentioned systems(12, 13) and exhibit a number
of distinct regulatory functions, including stimulation of
extracellular matrix deposition (3) and suppression of immune
cell growth(14) . Both activins/inhibins and TGF-
s inhibit
growth of many cells(15, 16, 17) .
Specific cell-surface receptors mediate the physiological effects of
activins and TGF-s (reviewed in (18, 19, 20, 21) ). Three distinct
receptors for the TGF-
s and activin-A have been identified through
their ability to bind and be chemically cross-linked to radioiodinated
ligands: types I (TGF-
RI and ActRI), II (TGF-
RII and ActRII),
and III receptors (TGF-
RIII and ActRIII), of molecular mass 55,
80, and >100 kDa, respectively. The types I and II receptors for
TGF-
s and activin-A are transmembrane serine-threonine
kinases(16, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) which
are essential for signal transduction, while the type III receptors
present ligands to the types I and II receptors. The type II receptors
require their corresponding type I receptors for signaling, while
binding of TGF-
s or activin-A to the respective type I receptors
requires coexpression of the corresponding type II
receptor(16, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46) .
In most cells, activins/inhibins and TGF-s bind to distinct
sets of receptors. It has been reported that GH3 rat pituitary tumor
cells express a single high affinity receptor for both
activins/inhibins and TGF-
s(47) , reflecting the similar
physiological effects of these hormones on pituitary cells. This
55-60-kDa surface protein could be affinity-labeled by
I-TGF-
1; labeling was inhibited by TGF-
1,
TGF-
2, activin-AB, and inhibin-B at concentrations in the high
picomolar to low nanomolar range(47) . Thus, GH3 cells
apparently express a novel type of cell-surface TGF-
receptor
(type IV) capable of recognizing several members of the TGF-
superfamily.
Here we show that GH3 cells express heteromeric type
I-type II receptor complexes for the TGF-s and for activin-A that
have been reported in other cultured cell systems. We find no evidence
for a type IV receptor. We hypothesize that in GH3 cells the TGF-
s
and activins use different receptor complexes to induce similar
physiological effects, presumably by activating common downstream
intracellular effectors.
Cross-linking of I-TGF-
1 to GH3 cells
resulted in a single predominant protein species of 70 kDa (Fig. 1, lane2), confirming the result in
reference(47) , where the type IV receptor was described. This
protein species is equivalent to 57 kDa, the approximate molecular mass
of the type I TGF-
and activin receptors, if the molecular mass of
the cross-linked monomeric TGF-
1 is subtracted; it has the same
size as the cross-linked type I receptors in many other cell lines that
express endogenous receptors or transfected recombinant type I
receptors. Fig. 1(lane 1) shows that prior treatment
of the cells with DTT resulted in partial inhibition of labeling of the
70-kDa species, suggesting that this species might consist of a
heterogeneous population of receptors, some of which exhibit the
typical DTT sensitivity of TGF-
type I receptors. Importantly,
cross-linking of 0.05 nM
I-TGF-
1 to the
57-kDa receptor was efficiently competed by a 100-fold excess of
unlabeled TGF-
1 (Fig. 1, lane6) and by a
500-fold excess of TGF-
3 (lane 12) but not by a 500-fold
excess of TGF-
2 (lane 9) or activin-A (lane15).
Thus, this 70-kDa species resembled in its binding properties TGF-
receptors characterized in other cell
lines(35, 37, 49) . In contrast to results in (47) , up to 25 nM activin-A could not inhibit binding
of TGF-
1 (lanes 13-15) or TGF-
2 (data not
shown) to this surface receptor.
Figure 1:
Chemical
cross-linking of I-TGF-
1 to GH3 cells. Confluent GH3
monolayers were incubated with 0.05 nM
TGF-
1 alone (lanes 1 and 2)
or together with increasing concentrations of unlabeled TGF-
1
(0.5, 1, 5, and 25 nM in lanes 3-6,
respectively), TGF-
2 (1, 5, and 25 nM in lanes
7-9, respectively), TGF-
3 (1, 5, and 25 nM in lanes10-12, respectively) or activin-A (1, 5,
and 25 nM in lanes13-15,
respectively). Cells in lane1 were treated with 1
mM DTT for 5 min at 37 °C prior to ligand binding. After
cross-linking with DSS, detergent extracts were analyzed by 5-10%
SDS-PAGE. The positions of molecular mass markers (in kDa) are
indicated on the left margin and that of the type I receptor
on the right margin of the panel. Only the relevant portion of
the autoradiogram is shown.
Binding of the TGF-s to the
type I receptor requires co-expression of the type II TGF-
receptor(20, 21) , but in GH3 cells affinity-labeling
experiments revealed the presence only of type I-like receptors; type
II receptors were undetectable ( Fig. 1and (47) ). Type
II receptors were visualized by immunoprecipitation analyses of GH3
proteins affinity-labeled with TGF-
1 using specific anti-receptor
antisera (Fig. 2). Antibody
(ALK-5), raised against the
type I TGF-
receptor, specifically immunoprecipitated both the
70-kDa TGF-
1 and the TGF-
2-affinity-labeled type I receptors
together with small amounts of an affinity-labeled protein of the
expected size of the type II receptor (Fig. 2, lanes 8 and 14). Importantly, antibody
(TGF-
RII),
specific for the type II TGF-
receptor, specifically
immunoprecipitated the 70-kDa TGF-
1 and the
TGF-
2-affinity-labeled type I receptors together with large
amounts of the type II receptor (lanes 9 and 16).
Immunoprecipitation was inhibited by incubation of the antibodies with
their corresponding immunogenic peptide (lanes13 and 15). Neither antibody precipitated receptors affinity-labeled
with
I-activin-A (lanes4 and 5; see below). Antibodies
(ALK-4) and
(ALK-2) also
immunoprecipitated a small amount of the 70-kDa TGF-
1 and
TGF-
2-affinity-labeled type I receptors (lanes 6, 7, 10, and 12); this was surprising since
ALK-4 and ALK-2 are thought to be type I receptors specific for
activin-A. However, TGF-
will bind to these receptors if
co-expressed in COS cells with large amounts of the type II TGF-
receptor (
)(35) or, in the case of ALK-2, when
overexpressed in murine epithelial cells(28) . Thus, GH3 cells
express the conventional type I (ALK-5, ALK-4, and ALK-2) and type II
TGF-
receptors.
Figure 2:
Immunoprecipitation with anti-receptor
antibodies of GH3 proteins cross-linked to I-TGF-
1,
-
2, and activin-A. GH3 cells were chemically cross-linked to 0.6
nM
I-labeled activin-A (lanes
1-5), TGF-
2 (lanes 6-9), or TGF-
1 (lanes 10-16). Detergent extracts were
immunoprecipitated with antibodies
(ALK-2) (lanes 1, 6, and 10),
(ALK-4) (lanes 2, 3, 7, 11, and 12),
(ALK-5) (lanes 4, 8, 13, and 14), or
(TGF-
RII) (lanes5, 9, 15, and 16) alone or in the presence of equimolar
amounts of the respective immunogenic peptides (lanes 2 and 11 for
(ALK-4), lane13 for
(ALK-5), and lane15 for
(TGF-
RII)).
The resulting immunocomplexes were analyzed by 5-10% SDS-PAGE.
The positions of molecular mass markers (in kDa) are indicated on the leftmargin and of the type I and II receptors on the rightmargin of the panel. Only the relevant portion
of the autoradiogram is shown.
Fig. 3shows that cross-linking of I-activin-A to GH3 cells resulted in a single predominant
protein species of 74 kDa (lane 2), equivalent to 61 kDa if
the molecular mass of the cross-linked monomeric activin-A is
subtracted. This species migrates slower than the TGF-
1
affinity-labeled type I receptor of 70 kDa (compare lanes 3, 9, and 14 of Fig. 2), and has the molecular
size expected for type I activin receptors(16, 35) . Fig. 3(lane 1) shows that labeling of this receptor
with
I-activin-A was somewhat affected by prior treatment
of cells with DTT, similar to results obtained with the TGF-
type
I receptor (Fig. 1). Importantly, binding of 0.5 nM
I-activin-A to this GH3 cell receptor was inhibited
by as little as a 10-fold excess of unlabeled activin-A (lanes
3-6) but not at all by as much as 250 nM (500-fold)
of any TGF-
isoform (lanes 7-15). Thus, in contrast
to the results in (47) , GH3 cells express distinct type I
receptors for TGF-
and for activin-A. We find no evidence for a
receptor that can directly bind to both ligands.
Figure 3:
Chemical cross-linking of I-activin-A to GH3 cells. Confluent GH3 monolayers were
incubated with 0.5 nM
I-activin-A alone (lanes 1 and 2) or together with increasing
concentrations of unlabeled activin-A (5, 10, 50, and 250 nM in lanes 3-6, respectively), TGF-
1 (10, 50,
and 250 nM in lanes 7-9, respectively),
TGF-
2 (10, 50, and 250 nM in lanes 10-12,
respectively), and TGF-
3 (10, 50, and 250 nM in lanes
13-15, respectively). Cells in lane 1 were treated
with 1 mM DTT for 5 min at 37 °C prior to ligand binding
(marked as +), whereas cells in lane2 were not
(marked as -). After cross-linking with DSS detergent, extracts
were analyzed by 5-10% SDS-PAGE. The positions of molecular mass
markers (in kDa) are indicated on the left margin and of the
type I receptor on the right margin of the panel. Only the
relevant portion of the autoradiogram is
shown.
The GH3 receptor
affinity-labeled by activin can be immunoprecipitated by (ALK-4),
specific for the type I activin receptor, together with small amounts
of an affinity-labeled protein of the expected size of the type II
activin receptor (Fig. 2, lane 3). Since we do not have
antisera specific for the type II activin receptor, we are unable to
confirm the identity of this species by specific immunoprecipitation.
Neither GH3 receptor affinity-labeled by activin-A can be
immunoprecipitated by
(ALK-5), specific for the type I TGF-
receptor, or by
(ALK-2) (Fig. 2, lanes 4 and 1, respectively).
We extended our analysis of type I and
type II receptors expressed in GH3 cells by screening mRNA and a cDNA
library made from these cells by means of the polymerase chain
reaction(48) . We used sets of degenerate primers corresponding
to the most conserved amino acid sequences in the kinase domain of the
known members of the TGF- receptor family.
Of a total
of 27 PCR clones analyzed, one was 99% identical to the corresponding
region of the human type II TGF-
receptor and encoded its rat
counterpart(26) . Three other types of cDNA clones were
isolated; nine corresponded to ALK-2, also called ActRI or
tsk-7L(28, 29, 30, 35) . Twelve
corresponded to ALK-4(16, 35) and one to ALK-5, the
type I TGF-
receptor(31) . The immunoprecipitation studies
described here showed that the protein products of all these clones
were expressed in functional form on the plasma membrane of GH3 cells.
Taken together, our PCR cloning and
affinity-labeling/immunoprecipitation studies show that GH3 cells
express ``conventional'' types I and II receptors for activin
and TGF-
and provide no evidence for a novel type IV TGF-
receptor.
Here we report that GH3 pituitary tumor cells express
conventional heteromeric type I and type II receptor complexes for
TGF-s and activin-A, rather than a type IV receptor (47) that binds all of these growth factors. Since TGF-
s
and activins bind to distinct GH3 cell-surface receptors yet induce
common physiological responses, certain intracellular signal
transduction molecules may interact with receptors for both hormones.
Since virtually nothing is known of intracellular proteins that mediate
signaling by the TGF-
and activin receptors, it is difficult to
speculate what these common molecules might be.
Our
affinity-labeling experiments ( Fig. 1and Fig. 3)
demonstrate two distinct type I receptors on the surface of GH3 cells,
one specific for the TGF-s and one for activins. Whether inhibins
might utilize the same receptor complex as activins remains an open
question and was not addressed here. The type I TGF-
receptor
migrates slightly faster (70 kDa) than the activin receptor (74 kDa)
upon SDS-PAGE. Using the same SDS-PAGE system, we have consistently
observed this difference in mobility when we compared the
affinity-labeled recombinant type I TGF-
receptor ALK-5 and the
type I activin receptor ALK-4 expressed in transfected COS cells
together with the corresponding type II receptors.
Our
immunoprecipitation experiments established that in GH3 cells at least
some of these type I receptors are in heteromeric complexes with type
II receptors. The principal TGF- receptor is a heteromer of ALK-5
and TGF-
RII, while the principal activin receptor is a heteromer
of ALK-4 and the type II activin receptor. Taken together, our results
argue that the major type I TGF-
and activin receptors expressed
in GH3 cells are ALK-5 and ALK-4, respectively, the predominant type I
receptors found in other cells(16, 31, 35) .
Our PCR cloning studies are consistent with this conclusion. However,
the possibility remains that GH3 cells may also express additional type
I-like receptor proteins, which may have escaped detection by our
experimental approaches. Importantly, the competition experiments in Fig. 1and Fig. 3indicate that any GH3 protein that
directly binds TGF-
(i.e. in the absence of any other
receptor protein) cannot bind activin with measurable affinity, nor can
any activin-binding protein also bind TGF-
.
Our
immunoprecipitation experiments (Fig. 2) suggest the presence of
type II receptors for both TGF- and activin-A on the surface of
GH3 cells, although the identity of the activin-A type II receptor was
not confirmed by direct antibody precipitation. As we reported
previously (37) , the use of anti-receptor antibodies increases
the sensitivity of detection of minor affinity-labeled receptor
components. The type II receptors determine the ligand recognized by
the type I receptors(36, 37) . Thus, it is interesting
that in GH3 cells both activin-A and TGF-
ligands can be
cross-linked to the type I receptors with higher efficiency than to the
type II receptors ( Fig. 1and Fig. 3). This phenomenon
may reflect initial binding of activin and TGF-
ligands to their
respective type II receptors, followed by efficient shuttling, or
presentation, of the bound ligand to type I receptors. Thus, the cell
surface may contain solitary, affinity-labeled type I receptors as well
as type I receptors in complexes with type II receptors. An alternative
hypothesis is that, upon ligand binding to the type II receptor,
TGF-
s or activins undergo a conformational change that makes them
more accessible to the cross-linking agent and/or more able to bind to
the type I receptor.
An important unanswered question is the exact
stoichiometry of the heteromeric type I-type II receptor complex for
TGF- and activin, both in GH3 cells, where more type I receptor is
detected than type II by affinity labeling, and in Hep3B, Rat-1, and
Mv1Lu cells, in which approximately equal amounts of types I and II
receptors are detected by affinity-labeling. We have shown that the
type II receptor forms homo-oligomers, probably homodimers, both in the
absence and presence of TGF-
ligands(51) . We hypothesize
that the heteromeric complex contains two copies of the type I and two
of the type II receptors, but the exact composition may differ in
different types of cells.
Interestingly, the antibody specific for
ALK-4 (activin type I receptor-B) immunoprecipitated not only activin
affinitylabeled heteromeric type I-type II receptor complexes, but also
TGF- affinity-labeled heteromeric receptor complexes. This is in
disagreement with the assignment of activin-A as the sole ligand that
can specifically signal through the ALK-4 type I receptor (also called
ActRI-B; (16) and (35) ). Our results do agree with
the ligand-binding studies performed in COS cells co-expressing type I
and II receptors
(35) ; when we expressed
TGF-
RII together with ALK-4, the ligand binding specificity of
ALK-4 (and TGF-
RII) for all three TGF-
isoforms was
indistinguishable from that of the known functional type I TGF-
receptor ALK-5
. Despite the fact that ALK-5 but not ALK-4
was able to reconstitute functional TGF-
responses in mutant mink
lung epithelial cells defective in type I
receptors(16, 33, 35) , it is plausible that
ALK-4 serves as a type I TGF-
receptor in other cells, such as
pituitary cells. Possibly ALK-4 mediates cell responses different from
the growth inhibition and induction of the plasminogen activator
inhibitor type I gene that have been studied mainly in Mv1Lu cells.
ALK-2, like ALK-4, is able to bind TGF- when co-expressed in
COS and epithelial cells with the type II TGF-
receptor(28, 29, 30, 32, 35) .
ALK-2 was also thought to transduce signals by activin, but not by
TGF-
, but the ability of ALK-2 to mediate biological responses to
TGF-
has been uncovered recently. (
)We found that a
small amount of TGF-
can be affinity-cross-linked to ALK-2 on GH3
cells (Fig. 2, lanes 6 and 10). These results
cannot be attributed to receptor overexpression (such as occurs in COS
cells), since we studied endogenous receptors expressed in very low
numbers per cell.
Thus GH3 cells express type I receptors that are
capable of binding both TGF- and activin. It is difficult to
compare the properties of the type I receptors of GH3 cells we
described here with those of the GH3 cell type IV
receptor(47) . We do not detect inhibition of binding of
radiolabeled TGF-
1 by activins, and it is possible that the
competition detected in (47) was due to impurities in the
activin and inhibin preparations available then. The electrophoretic
mobility of the type IV receptor (47) resembles that of the
type I receptors. However, binding of either TGF-
or activin to a
type I receptor requires the presence of the appropriate type II
receptor; ligands apparently bind first to a type II receptor and then
are shuttled to a type I receptor. Thus, binding of TGF-
to any
type I receptor is not inhibited by the presence of activin, although
it is by other TGF-
isoforms, since activin cannot bind to the
type II TGF-
receptor. Similarly, TGF-
does not inhibit
binding of activin to its type I receptor. These results indicate the
necessity for careful analysis of specific receptors in various cell
types before the ligand specificity and function of the members of this
complex growth factor receptor family can be assigned conclusively.