(Received for publication, December 20, 1994; and in revised form, January 18, 1995)
From the
The transforming growth factor- (TGF-
) superfamily
comprises a number of molecules that are involved in a wide variety of
biological processes. Specific receptors for several members of this
family have been molecularly identified, forming a new category of
transmembrane serine/threonine kinase receptors. The type I and type II
receptor interact both physically and functionally, thereby cooperating
to generate intracellular signals. The yeast two-hybrid system was used
to identify proteins that can interact with the cytoplasmic region of
the type I TGF-
receptor. One of the proteins identified encodes a
novel putative serine/threonine kinase receptor. Sequence analysis
suggests that this molecule belongs to the type II receptor class. This
receptor, however, is distinct from other type II receptors in having
an extraordinarily long C-terminal tail region. The pattern of
expression in adult tissues is different from that of other known type
II receptors; it is highly expressed in heart and liver. In the yeast
system, the cytoplasmic regions of different combinations of type I and
type II receptors heterodimerize, providing a new cloning strategy for
the large number of serine/threonine kinase receptors likely to exist
for the many ligands of the TGF-
superfamily.
Transforming growth factor-s (TGF-
s) (
)belong to a family of multifunctional cytokines that
regulate cell proliferation, differentiation, extracellular matrix
formation, and immunosuppression (1, 2, 3, 4) . TGF-
s exert
their pleiotropic effects through binding to specific cell surface
receptors. Three major classes of receptors for TGF-
s have been
identified by chemical cross-linking to
I-TGF-
s.
Those are type I (T
R-I, 55 kDa), type II (T
R-II, 75 kDa), and
type III (T
R-III, 280 kDa). Previous studies on chemically
mutagenized mink lung epithelial cell lines suggested that the type I
and type II receptors interact with each other and both receptor types
are required for mediation of biological responses to
TGF-
1(5) .
Recently a number of receptors for different
members of the TGF- superfamily have been molecularly
cloned(3, 4, 6, 7) . T
R-III has
a short cytoplasmic region and probably facilitates TGF-
effects
by presenting ligand to the signaling receptors(8) . T
R-I
and T
R-II are serine/threonine kinases directly involved in
TGF-
signaling. The function and role of each type of the
receptors, however, are distinct. T
R-II binds ligand without
T
R-I whereas T
R-I requires T
R-II to bind
ligand(9) . T
R-II determines ligand
specificity(10) , and T
R-I specifies cellular
responses(11) . A model of ligand-induced activation of the
receptors has been proposed (12) . T
R-II kinase is
constitutively active and autophosphorylated. Upon ligand binding,
T
R-II associates with and phosphorylates T
R-I. This
transphosphorylation presumably activates T
R-I kinase resulting in
the phosphorylation of downstream substrates that are still unknown.
It has been shown that the TGF- receptors interact both
physically and functionally. In
I-TGF-
1 chemically
cross-linked cells, antibodies specific for type I or type II receptor
precipitated both type I and type II
receptors(13, 14) . Anti-type II receptor antibodies
also precipitated type III receptor(14) , showing that
T
R-I, T
R-II, and T
R-III form heterooligomeric complexes
in the presence of ligand. Evidence suggests that T
R-II and
T
R-III can exist as homooligomers both in the absence and presence
of TGF-
1 (15, 16) . Two-dimensional gel analysis
of ligand-bound receptor complex indicates that T
R-I and
T
R-II may exist in a tetramer containing two molecules of
T
R-I and T
R-II(17) . More recent studies indicate
that the association of T
R-I with T
R-II is ligand-dependent;
however, the possibility that these receptors may have a low intrinsic
affinity for each other was not excluded(12) .
The TGF-
superfamily comprises over 20 different members. The type II receptors
for two family members, activin (ActR-IIA) and TGF-
1, were
initially cloned through expression
screening(18, 19) . Another mammalian type II receptor
for activin (ActR-IIB)(20) , the Drosophila activin
type II receptor (Atr-II)(21) , and the
Müllerian-inhibiting substance (MIS) type II
receptor(22, 23) were subsequently isolated. The
protein product of the Caenorhabditis elegansdaf-4 gene, responsible for the inhibition of dauer larva formation, was
shown to be a distinct type II receptor that binds human BMP-2 and
-4(24) . However, type II receptors for other ligands in the
TGF-
superfamily have not been identified as yet.
Here we
report the identification of a novel putative type II serine/threonine
kinase receptor through its interaction with the cytoplasmic region of
TR-I. We further show that the cytoplasmic regions of several of
the type I and type II receptors heterodimerize in the yeast two-hybrid
system while type I-type I and type II-type II interaction were not
observed.
Proteins that interact with TR-I appear to play an
important role in TGF-
signaling. We used a modified version of
the two-hybrid system, the interaction trap screen developed by Brent
and co-workers(25, 26) , to identify proteins that
interact with the cytoplasmic region of the rat T
R-I,
R4(27) . EGY48, the yeast host strain, was first transformed
with the
-galactosidase reporter gene, pSH18-34, and the
T
R-I bait, pEGR4, plasmids followed by introduction of the HeLa
cell expression cDNA library as a prey. One million five-hundred
thousand transformants were screened. Primary yeast colonies that grew
on selection media were tested for leucine auxotrophy and
-galactosidase activity, and 17 positive clones were isolated. One
class of the final clones encodes FKBP12, a binding protein for FK506
and rapamycin. (
)One of the other clones, CL130, had an
insert of 1.5 kb, and partial sequencing revealed that the cDNA encoded
a putative serine/threonine kinase receptor. Rescued CL130 plasmid was
reintroduced into EGY48 with the reporter gene and the T
R-I bait,
and the interaction was reproduced (data not shown).
The sequence of CL130 was most homologous to the cytoplasmic region of the activin receptor type IIA (data not shown) but lacked corresponding transmembrane and extracellular regions. To obtain the full coding region, human kidney cortex and human placenta libraries were screened with CL130 as a probe. Twelve clones were isolated and one of them, CL4-1, with an insert of 3.3 kb was found to contain the entire coding region. Nucleotide sequencing of CL4-1 revealed an open reading frame of 3114 base pairs, encoding 1038 amino acids. The nucleotide and deduced amino acid sequence of CL4-1 are shown in Fig. 1A. An in-frame stop codon was found at -21 to -19 in the 5`-untranslated region. The predicted starting ATG codon is followed by a stretch of hydrophobic amino acids that is assumed to be a signal peptide. Another hydrophobic region, a putative transmembrane region, was identified between amino acids 151 and 174. Three potential N-glycosylation sites were found in the extracellular region. The calculated molecular mass of the protein is 115,317 daltons.
Figure 1:
DNA sequence and protein structure of
T-ALK. A, nucleotide and deduced amino acid sequence of T-ALK.
Two hydrophobic regions (signal peptide and transmembrane region) are underlined. Three potential glycosylation sites are indicated
with asterisks. The putative kinase domain is shown between brackets. Protein sequence shared by CL130 and CL4-1 is
shown between arrowheads. B, peptide sequence
alignment of type II receptors. hActR-IIA, human type
IIA activin receptor(18) ; mActR-IIB, mouse type IIB
activin receptor(20) ; rMISR-II, rabbit
Müllerian-inhibiting substance
receptor(23) ; hTR-II, human type II
TGF-
receptor(19) ; dAtr-II, Drosophila type II activin receptor(21) ; daf-4, C.
elegansdaf-4 (24). Conserved amino acids are shown in uppercase. Part of the tail region (amino acids 699-1038) of
T-ALK is omitted. Romannumerals indicate the
subdomains of the conserved kinase region. C, schematic
representation of T-ALK, type II activin receptor, and type I TGF-
receptor. Cysteine residues are shown with verticalbars. TM represents transmembrane region. The GS
domain is a region with a series of serine and glycine residues, which
is characteristic of type I receptors(11) . D,
comparison of CL130 and CL4-1. DNA and peptide sequences are
shown. The DNA sequence between 1586 and 2867 (1280 bases) of
CL4-1, shown in lowercase, is missing in
CL130.
The coding region of CL4-1 was used to search
a data base (the BLAST network service at NCBI). The most highly
related molecule was the activin receptor type IIA, followed by the
activin receptor type IIB, anti-Müllerian hormone
receptor, and TR-II (data not shown). The type I receptors had
less similarity. These results suggest that CL4-1 encodes a novel
type II receptor, and we named this receptor T-ALK (type II activin
receptor-like kinase). The amino acid sequence of T-ALK was compared
with other type II receptors using the combination of the MACAW program (28) and manual alignment (Fig. 1B). T-ALK has
6 single cysteines and one stretch of 4 cysteines (cysteine
box(11) ) between the signal peptide and the transmembrane
region. Most of these cysteines are conserved among the type II
receptors whereas their arrangement is different from that of the type
I receptors. T-ALK has a spacer region between the transmembrane region
and the kinase region. The spacer region is variable among the type II
receptors. T-ALK lacks the GS domain that is characteristic to the
spacer region of the type I receptors(11) . The kinase region
is highly conserved with other type II receptors. One of the most
distinct features of T-ALK is its long tail region following the kinase
region. Most of the type II receptors have a short tail region that
ranges from 20 to 50 amino acids. Only daf-4 has a
relatively long tail with approximately 140 amino acids. In contrast,
the tail of T-ALK has about 430 amino acids that comprises
approximately 40% of the entire coding region (Fig. 1C). Data base search with the tail sequence did
not give any significant homologous proteins. Comparison between CL130
and CL4-1 revealed that CL130 contains the entire kinase region
and lacks most of the tail region (Fig. 1, A and D), probably due to alternative splicing. The coding frame of
CL130 after the deletion is different from that of CL4-1,
resulting in a premature stop.
To determine the tissue-specific
expression of T-ALK, Northern blot analysis of poly(A) RNA from different tissues was performed. Three distinct messages
of 11, 8, and 5 kb were detected in every tissue examined. The 11-kb
message was highly expressed in heart and liver (Fig. 2). The
relative abundance of the three transcripts varied from tissue to
tissue. This may reflect tissue-specific processing of mRNA.
Figure 2: Northern blot of T-ALK mRNA. A blot with mRNA (2 µg/lane) from various human tissues (Clontech) was probed with T-ALK cDNA. The sizes of molecular mass markers are indicated in kb.
From
the amino acid sequence alignment, T-ALK is most likely to be a novel
type II serine/threonine kinase receptor. We cloned T-ALK through its
interaction with TR-I. Tsk 7L(29) , the mouse homolog of
ALK-2/SKR-1/ActR-IA, and T
R-II interact with each other at their
cytoplasmic regions in the yeast two-hybrid assay as well as in
immunoprecipitation with mammalian cells (data not shown). These two
independent results indicate that the heterodimerization of the
cytoplasmic regions may be more universal and apply to other
combinations of the type I and type II receptors. To address this
hypothesis, we tested the interaction between the type I and type II
receptors more thoroughly. The bait and prey plasmids that express the
cytoplasmic region of Tsk 7L, T
R-I, T
R-II, and T-ALK (CL130)
were constructed. All possible combinations of these plasmids were
tested in the yeast assay. A unique feature of the interaction trap
system is that the expression of the prey protein is under the control
of a derivative of the GAL1 promoter(25, 26) .
Therefore the interaction detected in this assay is significant only
when it is galactose-dependent, which, in turn, indicates that the
observed interaction is explicitly dependent on the prey proteins. Any
combination between the type I and type II receptor showed
galactose-dependent interaction both in the
-galactosidase and
leucine auxotrophy assay (Table 1). In contrast, neither
combination of type I with type I nor type II with type II gave
positive results. Thus, the data indicate that the cytoplasmic regions
of the serine/threonine kinase receptors do heterodimerize but not
homodimerize.
To detect protein-protein interaction in the two-hybrid
assay, it is essential for both bait and prey proteins to localize in
the yeast nucleus(30) . This is one of the reasons we chose the
cytoplasmic region of TR-I as the bait. The extracellular and
transmembrane region might affect the nuclear localization of the bait
proteins. We have not found any other report of cloning of a
transmembrane receptor through the two-hybrid screening. Our results
suggest that the two-hybrid screening can be applicable to isolation of
transmembrane proteins as well as cytoplasmic proteins.
We cloned a
novel serine/threonine kinase receptor, T-ALK, through the two-hybrid
screening system with the cytoplasmic region of TR-I as a bait.
T-ALK has several features characteristic to type II receptors. First,
the activin, anti-Müllerian hormone, and TGF-
type II receptors showed high scores in a homology search of a data
base with the whole coding sequence of T-ALK while type I receptors
shared less homology. More specifically, cysteine residues in the
extracellular region of T-ALK have an arrangement common to type II
receptors. The spacer region does not have the GS box that is shared by
type I receptors. Therefore T-ALK is likely to be a novel type II
serine/threonine kinase receptor. T-ALK is highly expressed in heart
and liver. In contrast, mouse T
R-II is abundant in lung, uterus,
and skeletal muscle(31) . The expression of mouse ActR-IIA is
high in brain and kidney(32) . C14, a putative rat
Müllerian hormone type II receptor, is exclusively
expressed in testis and ovary(23) .
T-ALK was isolated
through its interaction with TR-I. Recently it was shown that
T
R-II can phosphorylate T
R-I(12) , suggesting that
the cytoplasmic region of T
R-II interacts with that of T
R-I.
Our results support this idea. The association in mammalian cells with
the full-length receptor, however, was ligand-dependent. In our system,
we used the cytoplasmic region of the receptors. One possibility is
that the cytoplasmic region of both receptors without the extracellular
and transmembrane regions could be constitutively active. The
cytoplasmic region of rat T
R-I fused to glutathione S-transferase was shown to have autophosphorylation activity in vitro(27) , whereas T
R-I was not
phosphorylated in the absence of TGF-
in
vivo(12) . The cytoplasmic region of T
R-II was also
shown to have autophosphorylation activity in
vitro(19) . This is similar to the protein product of the
v-erbB oncogene, which is the truncated form of the epidermal
growth factor receptor with a constitutively active
kinase(33) . A second possibility is that the interaction in
the yeast system may reflect the ligand-independent low intrinsic
affinity between the receptors (12) since the two-hybrid system
is a remarkably sensitive assay for protein-protein interaction as
compared with co-immunoprecipitation (34) . Interestingly,
dimerization within the same receptor class was not detected. Recently
homomeric interaction of T
R-II both in the presence (15, 17) and absence (15, 16) of
ligand was reported. The reason for the discrepancy between these
results and our results is not clear. The extracellular region without
ligand may be sufficient to mediate the T
R-II homodimerization. In
one of the reports, however, it was shown that the cytoplasmic regions
of T
R-II interact with each other(16) . In mammalian
cells, multimeric interaction such as type II binding type I-type II
heterodimer via endogenous receptors could happen. The existence of
tetramers of T
R-I and T
R-II has been suggested(17) .
In contrast, yeast cells do not have endogenous TGF-
receptors.
Activin and TGF- type II receptors were cloned by expression
cloning using COS cells(18, 19) . The daf-4 gene was isolated through a genetic approach(24) . C14,
the rat MIS receptor, was identified by differential screening between
testosterone-treated Sertoli cells and untreated cells(22) .
Most of the other receptors have been cloned by homology screening.
Cloning of type I receptors by expression screening seems to be
difficult since type I receptors require type II receptors to bind
ligand(9) . Here we present a novel strategy to isolate
serine/threonine kinase receptors. Type I and type II receptors
interact with each other in the yeast assay. Therefore the two-hybrid
screening would be another useful method to isolate both type I and
type II serine/threonine kinase receptors. Considering the large number
of ligands in the TGF-
superfamily(3, 4, 6, 7) , it is
likely that a large number of receptors in this class remains to be
identified.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U20165[GenBank].