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Centre de Recherche en Reproduction Animale (CRRA), Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Québec, Canada
Address all correspondence and requests for reprints to: David W. Silversides, Centre de Recherche en Reproduction Animale (CRRA), Department of Veterinary Biomedicine, Faculty of Veterinary Medicine, University of Montreal, P.O. Box 5000, St-Hyacinthe, Québec, Canada, J2S 7C6. E-mail: silverdw{at}ere.umontreal.ca
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Abstract |
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Introduction |
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Appreciable progress recently has been made in the elucidation of the activin signaling mechanism by functional characterization of activin cell surface receptors. To date, four types of activin receptors have been identified by virtue of their capacity to bind activins and be chemically cross-linked to 125I-labeled activin. They were named activin receptor type I (ActRI) (5, 6, 7), activin receptor type IB (ActRIB) (8), activin receptor type II (ActRII) (9, 10, 11, 12), and activin receptor type IIB (ActRIIB) (13, 14, 15, 16), and their corresponding complementary DNA (cDNA) have been cloned from several species. In addition, several other type I receptors (activin receptor-like kinase1 (ALK-1), also named TGF-ß superfamily receptor type 1 (TSR1), ALK-5 and ALK-6) have been cloned that could bind either activin or TGF-ß in presence of the respective type II receptor (5, 17).
Analysis of the amino acid sequences showed that all four types of activin receptors cloned to date belong to the new family of serine/threonine kinase membrane bound receptor, which also includes receptors for other TGF-ß related factors such as bone morphogenetic proteins (BMP), Müllerian inhibiting substance, and TGF-ß (18).
Several studies suggest that activin signal transduction requires the formation of a heteromeric complex between type I and type II receptors (reviewed in 19 and possibly a type III receptor (20). Similar to what was shown in the TGF-ß receptor system (18), type II activin receptors bind activin with high affinity when expressed alone in CV-1, origin of SV40 (COS) cells, but type I activin receptors bind activin only when one of the activin type II receptors is coexpressed (5, 7, 21). Furthermore, studies on the ActRIB/ActRIIB complex suggest that activin type II receptor kinase activity is necessary to phosphorylate type I receptors on activin binding. It was further shown that an ActRIB mutant with a constitutively active kinase expressed alone is sufficient to generate intracellular signaling in absence of activin, suggesting that ActRIB acts downstream of type II receptors in the activin signal transduction (22, 23).
It has been shown by several reports that some serine/threonine kinase receptors are somewhat promiscuous with respect to their ligand partner. Until recently, it was thought that the specificity of ligand binding was dependent on the type II receptor present in the heteromeric receptor complex because type I receptors could be shared by different type II serine/threonine kinase receptors. For example, ActRI can bind activin, TGF-ß, or BMP-7 in presence of the respective type II receptor (17, 21, 24). However, it now appears that ligand binding to type II receptors is not stringently specific because it has been demonstrated that ActRIIB is also a functional BMP-2 (25) and BMP-7 (26) type II receptor, whereas ActRII can be a BMP-7 type II receptor as well as a growth/differentiation factor-5 type II receptor (27).
A breakthrough in the characterization of the signaling pathway of the
TGF-ß related proteins have been made by the identification of
cytoplasmic proteins potentially involved in signaling. Some reports
suggest that p21ras could be involved in the activin and
TGF-ß signaling pathways (28, 29). In correlation with this, the
subunit of p21rasfarnesyltransferase (FNTA) was shown
to interact directly with ligand-free ActRIB as well as with the
TGF-ß type I receptor (TßRI) (30, 31). FNTA is released on
ligand-induced phosphorylation of the type I receptor by the type II
receptor (30). However, its function in the signal transduction is
still unknown. Another intracellular factor, the immunophilin FKBP-12,
is a common binding protein of type I receptors and seems to function
in the inhibition of the signaling pathways of the TGF-ß family
ligands (32). Although no direct evidence was provided, it was further
suggested that calcineurin, which is a serine/threonine phosphatase
known to bind FK binding protein-12 (FKBP-12), could be the direct
cytoplasmic inhibitor of type I receptors by keeping them or their
bound substrates hypophosphorylated (32).
Until now, the only cytoplasmic factor known to interact with a type II serine/threonine kinase receptor is a Trp-Asp (WD)-domain protein, named TGF-ß-receptor interacting protein-1 (TRIP1), which associates with the TGF-ß type II receptor (TßRII) (33). Finally, several MAD (mothers against decapentaplegic)-related proteins have been identified as a new class of signaling molecules with nuclear functions that mediate responses to members of the TGF-ß family (25, 34, 35, 36, 37).
The present study was carried out to better understand the role of ActRIIB in the activin signal transduction mechanism in the reproductive axis. It has been demonstrated that activins are involved in many aspects of reproduction (4, 38). Until now, it has been shown that ActRIIB messenger RNA (mRNA) can be alternatively spliced to give four different isoforms in the mouse (13) but not in other species, such as rat and human (14, 16). Here we report that the bovine ActRIIB mRNA can be processed differently from its mouse homolog, and that an intracellular sequence coding for a 14-amino acid stretch very rich in proline residues can be alternatively spliced. We demonstrate that this proline-rich sequence, which resembles that of Src-homology 3 domain (SH3) binding sites, interacts specifically with two proteins of 69 kDa and 71 kDa from an uterine endometrial cell line. This suggests a potential role in activin signal transduction for these proteins and a protein-protein interaction with bAct-RIIB via an SH3 domain interaction. It also suggests that this interaction could be regulated by alternative splicing of bAct-RIIB mRNA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First-strand cDNA synthesis
All cDNAs used were made from corpus lutea RNA except for the
amplified fragment F-dT, which was from pituitary RNA. First-strand
cDNA was primed with an oligo-(dT)17 or a specific primer
based on mouse and rat ActRIIB sequences, and synthesized using
Superscript II (Life Technologies, Burlington, Ontario, Canada)
according to the manufacturer’s instructions.
RT-PCR amplification of bActRIIB cDNAs
The cloning strategy used four PCR amplifications of different
overlapping products and one 5'rapid amplification of cDNA ends
(RACE) described below. The following sense primers: bActRIIB.A
(5'-GGGAATTCTTTGTGGCTGTGAAGATCTT), bActRIIB.E
(5'-GAATTCTACT ACAACGCCAACTGG) and antisense primers:
bActRIIB.1 (5'-GGGAATTCATTCT TGCTTTTGAAGTCCCT), bActRIIB.3
(5'-GAATTCAGATCCACTGAGTCTGG) were designed based on sequence homologies
between mouse and rat ActRIIB sequences. The sense primers bActRIIB.B
(5'-GAATTCACGTGGCAGAGACGATG- TCTA) and bActRIIB.F
(5'-TCCAGAGAGACGCCTTTCTG), and the antisense primer bActRIIB.2
(5'-GAATTCTGGAGCCTCGCTTCTCAGCG) were bovine specific. As indicated in
the primer sequences, EcoRI restriction sites were added at
the 5'end of primers to aid in subsequent cloning steps. Polymerization
reactions were performed in an Ericomp thermal cycler (San Diego, CA)
in the presence of 2.5 U Taq polymerase (Perkin Elmer,
Norwalk, CT). The amplification conditions with primers pairs
bActRIIB.A and bActRIIB.1, bActRIIB.E and bActRIIB.2, bActRIIB.B,
and bActRIIB.3 were one cycle of 95 C for 3 min, 58 C for 2 min, 72 C
for 3 min and 45 cycles of 95 C for 55 sec, 58 C for 55 sec, 72 C for 1
min. The amplification of bActRIIB cDNA 3'end was done in a three-step
procedure. First of all, amplification with bActRIIB.A and
poly(dT)17 was performed (one cycle of 94 C for 45 sec, 40
C for 45 sec, 72 C for 90 sec and 35 cycles of 94 C for 45 sec, 52 C
for 45 sec, 72 C for 90 sec) followed by two nested PCR amplifications
(40 cycles of 94 C for 45 sec, 56 C for 45 sec, 72 C for 90 sec), using
primers bActRIIB.B and bActRIIB.F successively in conjunction with
poly(dT)17 to amplify the specific 3'end.
5'-RACE
Amplification of the 5'-end was done using the
single-strand ligation to ss-cDNA strategy (39) in conjunction with the
use of the nucleotide analog 7-deaza-2'deoxy-guanosine
(c7dGTP; Pharmacia Biotech, Piscataway, NJ) in the PCR
reaction (40). Briefly, first-strand cDNA primed with a bActRIIB
specific biotinylated primer (bActRIIB.5(Bt): 5'-TTCTTGAC
GAGCTCGATGGTG) was synthesized. RNA was then hydrolyzed with 0.4 N NaOH
at 65 C for 30 min. After neutralization with 0.4 N HCl, the
first-strand cDNA was purified using Geno-bind resin (CLONTECH, Palo
Alto, CA) to eliminate nonincorporated biotinylated primers. The
biotinylated cDNA was further purified by capturing it on magnetic
beads coupled with streptavidin (Dynabeads M-280, Dynal). Eight
picomole of a modified oligonucleotide (Anchor seq.V3;
5'-GCAGGATCCTGAAGCTTGAATTC, with a phosphate group added to the 5'-end,
as well as a 3'-end blocked with an amino group) was incubated 20
h at room temperature with 2.5 µl single-stranded cDNA and 10 U T4
RNA ligase (New England Biolabs, Mississauga, Ontario, Canada) in the
hexamine cobalt chloride buffer as previously described (39). PCR
amplification (1 cycle of 95 C for 5 min, 56 C for 2 min, and 72 C for
3 min followed by 45 cycles of 95 C for 55 sec, 56 C for 55 sec, and 72
C for 1 min) was performed using a bActRIIB specific nested primer
(bActRIIB.5, 5; 5'-GAATTCCGATGGTGCCCGAGCTGTT) and a primer
complementary to the ligated oligonucleotide (Anchor Primer V3;
5'-GAATTCAAGCTTCAGGATCCTGC) in the presence of 2.5 U Taq
polymerase and 2 µl of a 3:1 c7dGTP/dGTP mixture (50
µM each deoxycytidine ATP, deoxycytidine cytidine
5'-triphosphate, deoxycytidine ribothymidine 5'-triphosphate and 12.5
µM dGTP and 37.5 µM c7dGTP). To
reduce the probability of nonspecific amplifications, a nested-PCR was
performed with bActRIIB.6 (5'-GAATTCTAGCAGTGCAGCCGCTTGT) as follows: 35
cycles of 95 C for 55 sec, 57 C for 55 sec, and 72 C for 1 min.
Cloning and sequencing
PCR cDNA products were cloned into pGEM-T vector (Promega).
Double stranded DNA were sequenced by the di-deoxy chain termination
method using the T7 sequencing kit (Pharmacia, Baie d’ Urfé,
Québec, Canada). The G + C-rich clone bActRIIB.6-V3 was sequenced
by cycle sequencing for G + C-rich template (UCDNA sequencing services,
University of Calgary) to resolve band compression. Three independent
clones were sequenced for each amplification to overcome the lack of
fidelity inherent to Taq polymerase.
PCR amplification of genomic bActRIIB sequence
Bovine genomic DNA was isolated from bull white blood
cells by standard methods (41). PCR amplification of the genomic
sequence corresponding to the intracellular spliced region was
performed using sense primer H (5'-GGATCCCGACATCGCAAGCCCCCCTAC; with a
5' BamHI restriction site) and antisense primer 4
(5'-GAATTCTCGTGCTTCATGCCAGGC; with a 5' EcoRI restriction
site). Primer H was located just 3' to the transmembrane region,
whereas primer 4 was located in the first part of the kinase domain
(Fig. 5A
). Fragment H-4 PCR amplification was carried out on 100 ng
genomic DNA with 2.5 U plaque-forming units polymerase (Stratagene, La
Jolla, CA), and the resulting fragment was cloned into pCR-Script SK
(Stratagene) and sequenced.
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Synthesis of complementary RNA probes
Fragment K-11, containing both the proline-rich sequence and the
following exon, was amplified by PCR from the genomic clone H-4 (Fig. 5A
) and cloned into pCR-Script. After linearization of the DNA
template, antisense riboprobe was transcribed from the flanking T7
promoter in the presence of 0.5 mM each of ATP, cytidine
5'-triphosphate, and GTP; 5 µM uridine triphosphate; 50
µCi [-32P]uridine triphosphate; 20 U T7 RNA
polymerase; 16.5 U RNase inhibitor; 10 mM dithiothreitol;
and 1x transcription buffer (Promega) in a total volume of 20 µl.
After 1 h at 37 C, 20 U RNase-free DNase I (Pharmacia) were added.
The resulting riboprobe was not gel-purified and was diluted in 0.5
M ammonium acetate; 1 mM EDTA; 0.2% SDS before
hybridization.
The resulting 232-nucleotide (nt) antisense riboprobe was complementary to the alternative proline-rich coding sequence (42 nt) and to the next exon (102 nt). Therefore, the predicted size of the riboprobe protected by bActRIIB mRNA containing the alternative sequence would be of 144 nt, whereas a bActRIIB mRNA without this alternative sequence should give a 102-nt protected fragment.
RNase protection assay (RPA)
RNase protection assay was carried out using the RPA II kit
(Ambion, Austin, TX) with slight modifications to the manufacturer’s
protocol. Antisense riboprobe at 1 x 105 cpm and 10
µg poly(A)+ RNA were ethanol coprecipitated and dissolved
in 20 µl hybridization buffer (80% formamide; 100 mM
sodium citrate, pH 6.4; 300 mM sodium acetate pH 6.4; 1
mM EDTA). The samples were heated at 95 C for 5 min and
incubated at 43 C overnight. Digestion of unhybridized RNA was
performed with 2.5 U RNase A and 100 U RNase T1 for 30 min at 37 C.
RNA-RNA hybrids were precipitated with 200 µl ethanol and 300 µl
RNase inactivation/precipitation mixture supplied by the manufacturer
and dissolved in 8 µl nondenaturing gel loading buffer (8% sucrose;
0.025% bromophenol blue; 0.025% xylene cyanol). Protected fragments
were directly loaded, without previous heating, on a nondenaturing 5%
polyacrylamide gel and separated for 3 h at 100 V. The gel was
dried and exposed to x-ray film for 2 weeks.
Expression and purification of fusion proteins
The fusion protein pGEX-IIBSH3b.s. was constructed by using two
complementary oligonucleotides representing the short alternative
bAct-RIIB sequence of 42 bp, with BamHI and
EcoRI cohesive ends at the 5'ends of the sense and antisense
strands, respectively. Complementary oligonucleotides (5 ng) were mixed
and boiled for 2 min and then cooled slowly at room temperature to
allow them to anneal. The DNA fragments were ligated directly into
pGEX-4T-1 digested with EcoRI and BamHI, which
allows the expression of fusion proteins with the glutathione
S-transferase (GST). The recombinant plasmid was sequenced to verify
sequence integrity.
The GST and GST-IIBSH3b.s. fusion protein were expressed in 200-ml cultures of E. coli BL-21 induced with 0.1 mM isopropyl-1-thio-ß-galactopyranoside when the culture had reached an A600 nm of 1. After a 4-h incubation at 37 C, the cells were centrifuged and dispersed in 10 ml PBS 1x (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). The cells were sonicated and Triton X-100 was added to a final concentration of 1%. The solution was then agitated gently for 30 min. The GST and the fusion protein were purified on glutathione-Sepharose 4B and eluted in elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0).
Preparation of metabolically radiolabeled proteins
Endo 8.3 cells, which is a bovine uterine endometrial cell line
immortalized with SV40 large T antigen sequences (42), were incubated
in a 75 cm2 tissue culture dishes to 90% confluency in
DMEM + 10% FCS. Cells were washed twice with 10 ml long-term labeling
medium (9 vol methionine and cysteine-free DMEM; 1 vol complete DMEM;
10% FCS). The cells were then incubated overnight in presence of 100
µCi 35S-methionine and 35S-cysteine
(Expre35S35S, Dupont, Canada Inc., Mississauga,
Ontario, Canada/ml long-term labeling medium. One milliliter RIPA
buffer (58 mM NaCl; 5 mM EDTA; 10
mM Tris-HCl, pH 7.2; 0.1% SDS; 1% sodium deoxycholate;
1% Triton X-100) was used to lyse the cells at 4 C for 30 min. After
pelleting the cells at 28,000 x g, the protein
concentration of the lysate was estimated by a Bradford test [Bio-Rad
Labs., (Canada) Ltd., Mississauga, Ontario, Canada].
Coprecipitation
Preclearing of 0.5 mg radiolabeled proteins was done for each
reaction by incubation with 50 µg GST and 50 µl of a 50% slurry of
glutathione Sepharose 4B at 4 C for 2 h. After centrifugation at
500 x g for 5 min, the supernatant was mixed with 30
µg GST or GST-IIBSH3b.s. and a 50% slurry of glutathione Sepharose
4B at 4 C for 4 h. The Sepharose-bound proteins were washed four
times with 500 µl RIPA buffer, and the coprecipitated proteins were
eluted by an overnight thrombin cleavage of the GST moiety at room
temperature. The samples were fractionated on 10% SDS-PAGE, and the
gel was dried and exposed overnight to autoradiographic film.
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Results |
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. Clones bActRIIB.E-2, bActRIIB.A-1, and
bActRIIB.B-3 represent the majority of the coding region and part of
the 3'untranslated region (3'UTR). The complete 3'UTR is represented by
clone bActRIIB.F-dT. The bActRIIB cDNA consensus sequence and its
predicted amino acid sequence (Fig. 2) were determined
from these overlapping clones. The region around the ATG initiation
codon was found to be rich in guanosine and cytosine bases, and this
reduced the efficiency of amplification of the Taq
polymerase in a standard PCR reaction. 5'-RACE amplification
incorporating the structure-destabilizing base analog
c7dGTP proved useful during the PCR step in resolving this
sequence.
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Expression of activin type IIB receptor mRNA in bovine reproductive
tissues
Northern analysis of bActRIIB gene expression showed two major
transcripts of 2 kb and 2.3 kb in all reproductive tissues analyzed
(Fig. 3). A very weak 10-kb hybridization signal was
also observed. A differential ratio of expression of the 2- and 2.3-kb
species can be seen within these tissues. The 2-kb species was the most
abundant in the majority of the tissues, except for the prepubertal
testicles in which both species were present in equal intensity, and
the cotyledons in which the 2.3-kb is the most abundant bActRIIB mRNA
species. The 2-kb transcript correlated well with the 1833 bp of cloned
cDNA, suggesting that the sequence shown in Fig. 2 represents a nearly
full-length cDNA. The biological significance of the 2.3-kb transcript
is not understood, but it may simply represent an intermediate stage of
RNA processing or a more distal 3' site of polyadenylation.
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). However, the corresponding region is always present in the ActRIIB
cDNA cloned from other species (14, 15, 16), and even in the mouse in which
the ActRIIB gene can generate four different activin receptor isoforms
through mRNA alternative splicing (13). The deleted segment was
contiguous to the alternatively spliced segment located in the
cytoplasmic juxtamembrane domain reported in the mouse but ended before
the kinase domain. This deletion caused the loss of a 14-amino acid
stretch very rich in proline without altering the reading frame coding
for the bActRIIB deduced amino acid sequence. Analysis of the deleted
sequence indicated that this event could have been the result of
splicing because the characteristic AG dinucleotide of a 3' acceptor
splice site was present. The 5'donor splice site was not observed in
the sequence examined because it was probably in an upstream segment
not observed in the bovine bActRIIB cDNA sequence but observed in the
mouse isoforms ActRIIB1 and ActRIIB3.
Therefore, it is conceivable that this segment could be present in the
bovine molecule, and that splicing in this region could occur by using
one or the other of the putative alternative 3' acceptor splice sites.
To test this hypothesis, a PCR was carried out on bovine genomic DNA
using a sense primer complementary to the beginning of the
intracellular region (bActRIIB.H) and an antisense primer (bActRIIB.4)
located in the kinase domain. A fragment of 842 bp was amplified,
i.e. 593 bp longer than the expected 249-bp
fragment that should amplify from RNA. Sequencing of the genomic
fragment revealed that it contained two introns; one of 78 bp, located
after nucleotide 522 of the coding sequence, and a second one of 515 bp
located after nucleotide 666 of the coding sequence. The first intron
was the bovine counterpart of the murine intracellular alternative
splicing region as observed in murine isoforms ActRIIB1 and
ActRIIB3 (13). However, the bovine sequence did not retain
high homology with the murine sequence (data not shown). Furthermore,
the bovine sequence was 6 bp longer (78 vs. 72 bp), and the
last nucleotide triplet was a stop codon in frame with the bActRIIB
cDNA open reading frame. For this reason, this bovine region cannot be
considered an alternative exon as reported in the mouse. Of note are
the putative 5'-donor splice site and the 3' acceptor splice site
typical of introns. Thus, the contiguous proline-rich sequence
could be alternatively spliced by using one of the two alternative
3'-acceptor splice site (Fig. 4).
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). As shown in Fig. 5B, a fragment of 144 nt can be
protected in the presence of bovine cotyledon and uterus RNA. This
fragment was comparable in size to the proline-rich coding sequence
attached to the next exon. Furthermore, the splicing event described
above predict the protection of a 102-nt fragment in this assay
corresponding to the last exon without the proline-rich coding
sequence. Although a fragment of this length can be seen with the RPA
analysis, it is difficult to ensure that it is a genuine bActRIIB
protected fragment because the signal intensity is near the background
level. Nevertheless, these results clearly indicate that the majority
of the bActRIIB mRNA conserve the proline-rich coding sequence of 42
bp.
Comparison of the alternatively spliced segment with SH3 binding
site sequences
Comparison of the amino acid stretch encoded by the
alternatively spliced segment with other known proline-rich sequences
revealed homologies with SH3 binding motifs (43, 44). The minimal SH3
binding motif, PXXP, common to all SH3 binding sites known to date, is
found three times in the bActRIIB alternatively spliced segment (Fig. 6). Diverse studies (43, 45) suggest that SH3 binding
sites can be divided into two classes according to the position of an
arginine or lysine residue at the N- or C-terminus, which determines
the N-terminal to C-terminal ligand binding orientation. Class I sites
usually have an arginine at the N-terminus, whereas in class II sites
the arginine residue is located at the C-terminus and can be
substituted by a lysine. Following this classification, the putative
bActRIIB SH3 binding site would belong to class II ligands because a
lysine residue is present at the C-terminus after the three PXXP
motifs. Although the lysine residue is not juxtaposed to the PXXP
motif, flexibility in the distance between them may exist in members of
the class II ligands (43).
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).
Xenopus sequences diverge by only one amino acid, but still
retain homologies with SH3 binding site sequences. All of the activin
receptors described to date, including type I, type IB, type II, and
type IIB are considered serine/threonine kinases showing relative
homologies between each other, with the highest homology being between
receptors type II and type IIB. Comparison between all the different
activin receptor types in the bovine species shows that the putative
SH3 binding site can be found in the activin type II receptor at the
same relative location as in the type IIB receptor,
i.e. in the cytoplasmic juxtamembrane region just
before the kinase domain (Fig. 7). The nucleotide sequences are 70%
homologous, but the amino acid sequences are identical except for one
amino acid residue. In contrast, the bovine activin type I receptor has
no SH3 binding site-like sequence in the cytoplasmic juxtamembrane
region (our unpublished observations, Genbank accession no. U58095).
Moreover, we failed to find a SH3 binding site-like within other type
II serine/threonine kinase receptor sequences.
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suggesting a complete signaling pathway in
these cells. Two proteins of 69 and 71 kDa from the Endo 8.3 cell line
coprecipitated specifically with the GST-IIBSH3b.s. fusion protein
(Fig. 8). The 69-kDa protein coprecipitated more
abundantly than the 71-kDa protein. Attempts to verify the specificity
of interaction by expressing two other GST fusion proteins consisting
of the entire bAct-RIIB intracellular domain, with or without the
putative SH3 binding sites, have been hampered by inclusion body
formation within E. coli cells. Nevertheless, these results
suggest that the short alternative sequence of bActRIIB encoding a
proline-rich sequence can be involved in protein-protein interactions
with intracellular proteins.
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Discussion |
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The two bActRIIB mRNA species of 2 and 2.3 kb expressed in bovine tissues correlate well with the length of ActRIIB mRNA species found in the rat reproductive tissues, which were 1.7 and 2.25 kb (11). In human fetal nonreproductive tissues, two major hActRIIB mRNA species of 2.5 and 10 kb are found, whereas an additional minor 2.1-kb transcript is observed in K562 erythroleukemia cells (16). This is consistent with the results reported in this study because a 10-kb transcript is also observed in bovine tissues, although it is a minor mRNA species. Based on a previous study (12), it can be deduced that the bActRIIB mRNA has a lower relative steady state level in these tissues than the bActRII mRNA because it took 7 days of exposure for the bActRIIB hybridized Northern blot to reach a comparable intensity of a 1-day exposure of bActRII hybridized Northern blot. This is consistent with previous reports in which it was demonstrated by in situ hybridization and Northern analysis that in rat brain and reproductive tissues (47, 11) the steady state level of ActRIIB mRNA is lower than that of ActRII mRNA. Furthermore, we found in the same array of tissues that the bovine activin type I receptor mRNA has a higher steady state level than the bActRII and bActRIIB mRNAs (data not shown).
In parallel to activin receptor expressions, several reports have
demonstrated the expression of the inhibin -, ßA-, and
ßB-subunits in bovine reproductive tissues (48, 49, 50). The
activin receptor complex in the bovine could thus in theory respond to
three types of activin and possibly to other undiscovered inhibin ß
homodimers because a putative human inhibin ßC-subunit
cDNA was recently cloned (51) augmenting the theoretical number of
activin forms. Moreover, it is also possible that bovine activin
receptor complexes serve in the signal transduction of other ligands of
the TGF-ß family, e.g. BMP2 or BMP7, because it
has been shown in other species that the activin receptors show laxity
in their ligand specificity.
The recent structural descriptions and functional characterizations of the activin receptors have opened the door to a better comprehension of the activin signal transduction, but the mechanism is not completely understood. As previously noted, the activin receptors might signal through their serine/threonine kinase activity. The bovine activin type IIB receptor, described in this study, should act the same way because it is highly homologous with activin type IIB receptor from other species. However, the finding of a novel mRNA isoform for the bovine activin type IIB receptor allows speculations on the biochemical pathway that might be used for activin signal transduction.
Before this report, ActRIIB mRNA alternative spicing was observed only
in the mouse (13) because RT-PCR examination on human mRNA failed to
detect any evidences of alternative splicing in the juxtamembrane
region of the Act-RIIB mRNA in the pituitary (52) and fetal brain
(16). Our results suggest that the bovine ActRIIB alternative splicing
events seem to diverge from the ones described in the mouse. The
bActRIIB isoforms were designated bActRIIB2 and
bAct-RIIB5 to keep the mouse nomenclature (Fig. 9). The ActRIIB2 isoform, as already
described, is the predominant mRNA isoform in the mouse (13, 53), and
the only one reported in other species until now (14, 16, 52). It
possess the extracellular alternatively spliced segment but not the
cytoplasmic alternatively spliced segment found in mouse. The alternate
extracellular sequence seems to affect ligand-binding affinity, but no
function has been assigned to the alternate intracellular sequence. The
genomic clone generated for the RPA analysis encompassed the bovine
homolog of the murine intracellular alternative exon, and analysis of
the bovine genomic sequence showed that it was not highly homologous to
the murine sequence. The bovine sequence was longer and finished with a
stop codon in frame with the rest of the bActRIIB sequence. This
sequence is then considered as an intron for the bovine species and not
as an alternative exon as in the mouse. The bActRIIB isoforms 1 and 3
are thus improbable in the bovine species.
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The SH3 domain is present in a large variety of proteins including various signaling proteins and consists of a small protein domain of approximately 60 amino acids. In recent years, binding sites that associate with SH3 domains were identified in SH3 binding proteins (44) and in peptide fragments (43) as proline-rich sequences.
We hypothesize that the function of the bActRIIB proline-rich sequence could be the binding of cytoplasmic SH3-containing proteins, which could be implicated in propagation of the intracellular activin signal. Coprecipitation experiments have supported this hypothesis by the identification of 69- and 71-kDa proteins having a direct interaction with the bActRIIB putative SH3 binding site. Whether these interacting proteins are negative or positive regulators of the activin signaling is unknown at this point of time.
Some intracellular proteins having direct interactions with receptors
of the TGF-ß family have already been identified. However, it is
unlikely that the 69- and 71-kDa proteins represent the FKBP-12 or FNTA
because these proteins failed to interact with the cytoplasmic domain
of ActRII in a yeast two-hybrid system (30, 32). Because ActRII has a
nearly similar putative SH3 binding site as ActRIIB (Fig. 7), we can
extrapolate that ActRIIB does not have a direct interaction with
FKBP-12 or FNTA through this putative SH3 binding site. Until now, only
one cytoplasmic protein that associates with a type II serine/threonine
kinase receptor has been identified. This protein, TRIP-1, is a
WD-domain protein that associates with TßRII in a kinase-dependent
way (33). However, TRIP-1 is not a candidate for the 69- or 71-kDa
coprecipitated proteins because TRIP-1 is a 37-kDa protein, and it has
been shown that TRIP-1 does not interact with ActRII (33).
Because putative SH3 binding sites are not detected in any other serine/threonine kinase receptors, it is suggested that the 69- and 71-kDa proteins interact specifically with ActRII and ActRIIB. Although there is a generally accepted mechanism of action that seems to be common for all serine/threonine kinase receptors, there might be some subtle mechanisms specific to each receptor to transduce the diverse response generated by members of the TGF-ß superfamily. This could take the form of specifically interacting proteins, such as the 69- and 71-kDa proteins identified in this study. This is not the first time that a cytoplasmic factor has been isolated having a specific interaction restricted to some of the serine/threonine kinase receptors. FNTA was shown to associate with TßRI and ActRIB but not with other tested type I receptors, such as TSRI and ActRI (30). Interestingly, some signals generated by TGF-ß and activin seem to be specifically transduced by TßRI and ActRIB but not by TSRI and ActRI (8). Taken together, these observations suggest that FNTA could be involved in some specific TGF-ß and activin responses. In the same vein, TRIP-1 shows specific interactions with TßRII but not with ActRII (33). Without excluding that TRIP-1-related proteins could exist for ActRII and Act-RIIB, the specific interaction of TRIP-1 with TßRII could also suggest that some cytoplasmic interacting proteins could be different from one receptor type to another.
Finally, it is generally accepted that alternative splicing can serve to introduce functional diversity, such as modification of protein activity. Thus, the alternative splicing event shown in this study, giving rise to different activin type IIB receptor isoforms, may allow for the fine modulation of the receptor activity. It is well known that activins can induce diverse responses depending on the targeted cells. It will thus be interesting to look at the implication of the alternative SH3 binding site in the generation of multiple activin biological effects.
We are presently focusing on the cloning of the cDNAs encoding the 69- and 71-kDa proteins interacting with the bActRIIB putative SH3 binding site. A better understanding of the molecular mechanisms regulating the activin signal transduction should be achieved through isolation of such SH3-containing proteins.
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Received November 11, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit in
TGF-ß and activin signaling. Science 271:1120–1122
. J Biol Chem 270:29628–29631, ßA and ßB subunit
messenger ribonucleic acids following increases in size and during
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