Cloning and characterization of a naturally occurring soluble form of TGF-beta type I receptor

Mary E. Choi

Section of Nephrology and Department of Internal Medicine, Yale University School of Medicine and the Veterans Affairs Connecticut Healthcare Systems, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated to play an important role both in the process of normal development and in the pathogenesis of a wide variety of disease processes, including those of the kidney. TGF-beta 1 regulates diverse cellular functions via a heteromeric signaling complex of two transmembrane serine/threonine kinase receptors (types I and II). Several distinct type I receptors have been described and are thought to determine specificity of the TGF-beta response and confer multifunctionality. This report reveals the cloning of a novel, naturally occurring soluble form of TGF-beta type I receptor, designated sTbeta R-I, from a rat kidney cDNA library. In vivo expression of a mRNA transcript encoding the sTbeta R-I, which lacks the transmembrane and cytoplasmic domains, is confirmed by RT-PCR followed by Southern blot analysis and by RNase protection assay. The sTbeta R-I mRNA abundance is greater in the neonatal rat kidney compared with the adult rat kidney. Furthermore, sTbeta R-I is a functional protein capable of binding TGF-beta 1 ligands in the presence of a TGF-beta type II receptor on the cell surface, as determined by affinity cross-linking with 125I-labeled TGF-beta 1. Studies using p3TP-Lux reporter construct reveal that this novel protein may function as a potentiator of TGF-beta signaling. The discovery of a sTbeta R-I provides an additional level of complexity to the TGF-beta receptor system.

variant activin receptor-like kinase-5; alternative splicing; signaling; renal development; transforming growth factor-beta 1

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

TRANSFORMING GROWTH factor-beta 1 (TGF-beta 1) is a multifunctional cytokine that regulates diverse cellular functions including cell proliferation and differentiation, as well as extracellular matrix protein synthesis. TGF-beta 1 has been implicated in both the process of normal development and morphogenesis (19) and in the pathogenesis of a wide variety of disease processes, including tissue fibrosis, inflammation, and tumorigenesis (16). In embryonic kidney tissues, both temporally and spatially regulated differential expression patterns of TGF-beta and its receptors have been demonstrated, suggesting an important role for TGF-beta in renal development (6, 19).

TGF-beta 1 exerts its multiple biological actions by the interaction with two transmembrane serine/threonine kinases (types I and II), which are coexpressed by most cells (3). Molecular cloning has revealed that a distinct type II receptor exists for TGF-beta 1 and for members of the TGF-beta superfamily, including activin and bone morphogenetic protein (BMP), and that the type II receptor is capable of binding its respective ligands directly and interacting with different type I receptors (2, 10, 15). To date, at least six distinct type I receptors of the TGF-beta superfamily, named activin receptor-like kinases (ALK), have been cloned. ALK-5 has been identified as the predominant TGF-beta type I receptor (Tbeta R-I) in most cell types and has been shown to mediate TGF-beta signaling (12). ALK-1 and ALK-2 are thought to be activin type I receptors, but both have also been demonstrated to bind TGF-beta (1, 9). ALK-4 also appears to be an activin type I receptor (21). ALK-3 and ALK-6 are thought to be type I receptors for BMP (22). Features common to all type I receptors include a cysteine-rich extracellular domain and a single transmembrane-spanning domain. Furthermore, besides the characteristic cytoplasmic serine/threonine kinase domain, type I receptors have a region, between the transmembrane and kinase domains, containing a conserved TSGSGSG motif denoted the GS domain. Mutational analyses have revealed that phosphorylation of serine and threonine residues in the GS domain of Tbeta R-I by the TGF-beta type II receptor (Tbeta R-II) is essential for TGF-beta signaling (20, 25).

Tbeta R-I is thought to determine the specificity of the cellular response to TGF-beta 1, whereas Tbeta R-II determines the ligand specificity. Tbeta R-I alone is unable to bind TGF-beta 1, on the basis of 125I-labeled TGF-beta 1 cross-linking studies, and Tbeta R-II is unable to signal without Tbeta R-I (27). Thus interaction of Tbeta R-II with different type I receptors may be a mechanism that confers multifunctionality of TGF-beta 1. This report shows that there are variant forms of the Tbeta R-I. Three unique cDNA clones encoding multiple receptor forms of ALK-5 have been isolated from a neonatal rat kidney cDNA library. Two of the rat cDNA clones encode membrane-spanning receptors that differ in the COOH-terminal region of the extracellular domain. The third clone encodes a previously undescribed soluble form of Tbeta R-I (sTbeta R-I). The present studies clearly demonstrate, for the first time, the existence of a naturally occurring sTbeta R-I mRNA expressed in greater abundance in the neonatal rat kidney compared with the adult rat kidney. Furthermore, the sTbeta R-I is a functional protein capable of binding TGF-beta 1 ligands in the presence of Tbeta R-II. This novel protein may function as a potentiator of TGF-beta signaling.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cloning and sequencing of rat Tbeta R-I cDNA clones. A 333-bp cDNA probe for ALK-5 (12) was generated by RT-PCR of total RNA from rat kidneys using sense primer A (nt 88-117 of human ALK-5, 5'-GGGGCGACGGCGTTACAGTGTTTCTGCCAC-3') and antisense primer B (nt 391-420 of human ALK-5, 5'-TGAGATGCAGACGAAGCACACTGGTCCAGC-3'). Using this PCR-amplified cDNA probe, a rat neonatal kidney cDNA library was screened by methods previously described (6). Three positive clones were identified. Partially overlapping nested deletion clones were prepared from the cDNAs with the Erase-a-Base kit (Promega), and then the sense and antisense strands were sequenced using the dideoxy chain termination method with 35S-dATP and Sequenase version 2.0 (Amersham).

RT-PCR. Total RNA (3 µg) from Sprague-Dawley rat neonatal and adult kidneys (Charles River, Wilmington, MA) was reverse-transcribed using M-MLV reverse transcriptase (GIBCO BRL), and the first-strand cDNA was used as a template for amplification. Each template cDNA was combined with 1 µM of each set of PCR primers, 200 µM dNTPs, and 2 U of AmpliTaq DNA polymerase in 1× PCR buffer (Perkin-Elmer); placed in the GeneAmp PCR System 9600 (Perkin-Elmer), and heated to 95°C for 1 min. This was followed by 30 cycles consisting of denaturing for 15 s at 95°C, annealing for 15 s at 50°C, and extending for 15 s at 72°C. The sequences of primers A and B were as described above; the sequence of primer C (nt 480-505 of rat cDNA clone 29-1, 3'-untranslated region) was 5'-GAGTAGCCAGGAGCCTGACTCCTGGG-3'. All experiments were performed at least three times, using RNA isolated from at least three separate neonatal rats and three adult rats.

Southern blot analysis. After RT-PCR, the amplified products were separated on a 1.5% agarose gel, incubated for 15 min in 0.25 N HCl, placed in denaturing solution (0.5 N NaOH and 1.5 M NaCl), and finally placed in neutralizing solution (1.5 M NaCl and 0.5 M Tris · HCl, pH 7.4) and then transferred and UV-linked to a nylon membrane (Nytran, Schleicher & Schuell). The blot was prehybridized and then hybridized in the presence of a random primer [32P]dCTP-labeled cDNA probe, as recommended by the manufacturer (GIBCO BRL). The two Tbeta R-I cDNA probes were Hind III/Sph I and Sph I fragments of clone 29-1 cDNA in pSPORT1 (GIBCO BRL), produced by restriction digests with the respective enzymes.

Solution hybridization/RNase protection. RNase protection analysis was done using the RPA II kit (Ambion) according to the manufacturer's instructions. The 32P-labeled antisense riboprobe was prepared from the linearized plasmid containing the Hind III/Sma I fragment of clone 29-1 cDNA using Sp6 RNA polymerase, yielding a probe 337 nt long. One, ten, and twenty micrograms of total RNA from neonatal and adult rat kidneys, or 10 µg of total RNA from wild-type and transfected COS-7 cells, were hybridized with the 32P-labeled probe for 16-18 h at 42°C, then digested with RNase A/T1 and resolved on a 6% acrylamide/7.7 M urea sequencing gel. A sample of 32P-labeled, 1-kb ladder DNA was loaded in an adjacent lane as the molecular size marker. The beta -actin mRNA was used as an internal standard to determine equivalent amounts of RNA used in parallel reactions. All experiments were repeated at least three times, using RNA isolated from at least three different neonatal and adult rats.

Cell culture and stable transfection of COS-7 cells. To generate clones that stably expressed Tbeta R-II and sTbeta R-I, COS-7 cells (ATCC CRL-1651) were transfected using Lipofectin (GIBCO BRL) as follows. Cells grown to ~40% confluency on six-well plates were incubated in OPTI-MEM (GIBCO BRL) in the presence of 1-5 µg full-length, wild-type Tbeta R-II cDNA (5) or sTbeta R-I cDNA ligated in pcDNA3 (Invitrogen) and 5-10 µl Lipofectin suspension for 5 h at 37°C in a 5% CO2 atmosphere. Control cells were incubated with pcDNA3 vector (without Tbeta R-II or sTbeta R-I) and Lipofectin. After a 5-h incubation, DMEM containing 20% fetal bovine serum (FBS) was added to make a final concentration of 10% FBS. After 48 h, the medium was changed to 10% FBS in DMEM and incubated for another 24 h. To select for stable transfectants, cells were treated with 400 µg/ml Geneticin (GIBCO BRL) in DMEM containing 10% FBS, and the medium was changed every 2-3 days. Clones emerging ~10 days after lipofection were subcloned using ring cylinders, expanded, and maintained in DMEM containing 10% FBS, 200 µg/ml Geneticin, 5 U/ml penicillin, and 5 µg/ml streptomycin. Four independent, stably transfected clones containing Tbeta R-II or cotransfected with Tbeta R-II and sTbeta R-I, and three clones containing empty vector were expanded. Nontransfected cells similarly treated with Lipofectin served as additional controls.

Covalent labeling of TGF-beta receptors on cell surface. Cells on 100-mm plates were affinity labeled with 400 pM 125I-TGF-beta 1 (Amersham) in the presence or absence of 100 nM unlabeled TGF-beta 1 (Collaborative Biomedical Products) and covalently cross-linked using disuccinimidyl suberate (Pierce), as previously described (5). The cells were subsequently lysed with 100 µl of 1% Triton X-100, 10 mM Tris 7.4, 1 mM EDTA, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, and 10 µg/ml pepstatin. Sample loading buffer (sucrose, 0.01% bromophenol blue, 2% beta -mercaptoethanol, and 5 mM EDTA) was added (1:1, vol/vol) and boiled for 5 min, followed by 12% SDS-PAGE. A sample of rainbow-colored protein molecular weight markers (Amersham) was loaded in an adjacent lane. The gel was stained with Coomassie brilliant blue (Bio-Rad) to allow visualization of equivalence in protein loading and was destained before autoradiography.

Luciferase assay. Luciferase reporter plasmid, p3TP-Lux (kindly provided by J. Massagué), was used to assay TGF-beta 1-induced transcription of plasminogen activator inhibitor-1 (PAI-1). The p3TP-Lux contains elements from the PAI-1 promoter and drives the expression of a luciferase reporter gene in response to TGF-beta 1 (26). COS-7 cells were transiently transfected with p3TP-Lux and the indicated receptor constructs, or with empty vector pcDNA3 (see Fig. 5). A truncated Tbeta R-I construct (Tbeta R-IM) that lacks the cytoplasmic GS and kinase domains but contains the transmembrane and extracellular domains was generated by PCR using rat Tbeta R-I cDNA (6) as a template, using a similar strategy as previously described for the kinase-deleted Tbeta R-II construct (Tbeta R-IIM) (5), and cloned in pcDNA3. Primer sequences used to amplify the Tbeta R-IM construct were as follows: sense primer, 5'-ACGG<UNL>GGTACC</UNL>CCATGGAGGCGGCGTCGGCTGCTTT-3'; antisense primer, 5'-GCGC<UNL>TCTAGA</UNL>GCGCCTATGGCACGCGGTGGTGAATGACA-3'. They contained the sequences for the restriction enzymes Kpn I and Xba I, respectively (underlined), for directional cloning, and a stop codon in the antisense primer. Control reporter vector pRL-CMV (Promega) was included in all transfections as an internal control to normalize transfection efficiency. Forty-eight hours after transfection using Lipofectin, cells were incubated for 20 h in the absence or presence of TGF-beta 1 (1 ng/ml). Luciferase activities in cell lysates were measured using the dual-Luciferase reporter assay system (Promega) in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). All experiments were performed at least three times.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Identification of a soluble Tbeta R-I. A 333-bp rat cDNA fragment obtained by RT-PCR, containing nucleotides of the extracellular region and homologous to the human ALK-5 cDNA (12), was used as a probe to screen a neonatal rat kidney library to identify cDNAs encoding variant forms of Tbeta R-I that differ in their extracellular ligand binding domains. A schematic representation of the three cDNA clones isolated is shown in Fig. 1A. All three rat cDNA clones were identical in the extracellular ligand binding domain with the exception of the COOH-terminal region. Clone 12-1 was found to be more than 95% homologous with the human ALK-5 at the amino acid level. In clone 21-1, an insert of 12 nt corresponding to amino acid residues GPFS was noted just upstream to the transmembrane domain. The remainder of the sequence was identical to clone 12-1, including the single hydrophobic transmembrane spanning domain. Interestingly, in clone 29-1, the divergent region in the extracellular domain also differed. It contained an insert of 15 nt, corresponding to amino acid residues GKLLY, followed by an in-frame stop codon. The remainder of the sequence downstream from the stop codon contained no homologous regions with the other two clones, and no sequence that would predict a transmembrane or a serine/threonine kinase domain was found in clone 29-1. The nucleotide sequence, including the flanking untranslated regions, and the deduced amino acid sequence are shown in Fig. 1B. Like the membrane-anchored form of ALK-5, clone 29-1 contained an NH2-terminal signal peptide domain. Based on the nucleotide sequence of clone 29-1, the deduced amino acid sequence predicted a soluble form of Tbeta R-I.


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Fig. 1.   A: schematic illustration of human activin receptor-like kinase (ALK)-5 and 3 variant forms of rat ALK-5 cDNAs. Human ALK-5 cDNA and rat cDNA clones 12-1 and 21-1 are membrane-anchored forms of transforming growth factor-beta type I receptor (Tbeta R-I). Clone 29-1 represents soluble form of Tbeta R-I, lacking transmembrane and cytoplasmic domains. Note divergent region in COOH-terminal region of extracellular domain of 3 cDNA clones. Horizontal arrows denote primers A and B, used to generate a cDNA probe to screen the library, and primer C, the additional primer used to perform RT-PCR. Horizontal solid bars denote 2 cDNA probes (Hind III/Sph I and Sph I fragments of clone 29-1 used for Southern analysis) and the antisense riboprobe containing the Hind III/Sma I segment of clone 29-1 used for RNase protection assay. B: nucleotide and deduced amino acid sequences of soluble form of Tbeta R-I (sTbeta R-I) cDNA from rat. Nucleotides and amino acids are numbered at beginning and end of each line, respectively. The protein coding sequence is indicated by uppercase letters, with +1 position nucleotide denoting the start codon (ATG). NH2-terminal hydrophobic signal sequence is enclosed by arrows, and potential N-glycosylation site is underlined. Divergent amino acid residues are in boldface letters, followed by in-frame stop codon (TAG). Extracellular cysteine residues are circled. Flanking untranslated nucleotide sequences are shown by lowercase letters.

In vivo expression of sTbeta R-I mRNA. To determine whether mRNA corresponding to the sTbeta R-I is endogenously expressed in vivo, RT-PCR followed by Southern blot analysis were performed with rat neonatal and adult kidney tissue samples. As illustrated in Fig. 2, A and B (top), primer pairs A and C should only amplify sequences homologous to the soluble form of ALK-5, a predicted product 430 bp in length. Primers A and B are expected to amplify sequences corresponding to the membrane-anchored form of ALK-5, with a predicted product 333 bp long. As controls, 29-1 and 21-1 cDNAs were used as templates. Primers A and C would only amplify the 430-bp product with clone 29-1 and not clone 21-1. Conversely, primers A and B would amplify the 333 bp with clone 21-1 cDNA and not clone 29-1. 


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Fig. 2.   RT-PCR and Southern blot analysis to detect Tbeta R-I and sTbeta R-I mRNA expression. Total RNA isolated from neonatal (Neo) and adult rat kidneys was reverse-transcribed using primers A and C or primers A and B as indicated. As controls, 29-1 and 12-1 cDNAs were used as templates for PCR amplification. A and B, top: schematic diagram of expected RT-PCR-amplified products separated on a 1.5% agarose gel. Solid bands, products predicted to hybridize with 32P-labeled cDNA probes from 29-1 Hind III/Sph I fragment (A) and 29-1 Sph I fragment (B). A and B, bottom: Southern analysis. As predicted, 29-1 Hind III/Sph I probe specifically hybridized only with 430-bp products (A), corresponding to the sTbeta R-I, whereas the 29-1 Sph I probe hybridized both 333- and 430-bp products (B).

To determine specificity of the RT-PCR products, Southern blot analysis was performed after RT-PCR with cDNA probes as indicated by the horizontal bars in Fig. 1A. Note that the 32P-labeled probe from Hind III/Sph I fragment of clone 29-1 cDNA is specific for the soluble form of ALK-5 and would hybridize only with the amplified products 430 bp in length corresponding to the sTbeta R-I, as illustrated by solid bands in Fig. 2A, top, and not with the 333-bp products (open bands). The second probe generated from Sph I fragment of clone 29-1 cDNA, which overlaps the extracellular domains of both membrane-anchored and soluble forms of ALK-5, would be predicted to hybridize with both the 333-bp and 430-bp amplified products (Fig. 2B, top). As predicted, the probe 29-1 Hind III/Sph I specifically hybridized only with the 430-bp products, corresponding to the sTbeta R-I (Fig. 2A, bottom), whereas the 29-1 Sph I probe hybridized both 430-bp and 333-bp products (Fig. 2B, bottom).

Further confirmation of in vivo expression of the sTbeta R-I mRNA was provided by RNase protection assay. The 337-nt riboprobe contained 304 nt of authentic rat sTbeta R-I sequence and included the extracellular ligand-binding domain that overlapped with 252 nt of the membrane-anchored receptor and a portion of the 3'-untranslated region (Fig. 3, lane 1). The predicted 304-bp-long protected fragments were detected in both the neonatal and adult kidneys, corresponding to sTbeta R-I (Fig. 3, lanes 3-8). A second, slightly smaller protected fragment was also observed in lanes 3-8 and may represent an alternatively spliced variant. The 252-bp-long protected fragments, corresponding to the membrane-anchored Tbeta R-I, were seen in both the neonatal and adult kidneys. In addition, note that there was greater mRNA abundance of both Tbeta R-I and sTbeta R-I in the neonatal kidneys compared with the adult kidneys.


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Fig. 3.   Detection of Tbeta R-I and sTbeta R-I mRNA expression by RNase protection assay. Lanes 1 and 2, 337-nt riboprobe, incubated without (-) and with (+) RNase, respectively. Lanes 3, 4, and 5: total RNA from neonatal rat kidneys (1, 10, and 20 µg, respectively); lanes 6, 7, and 8: adult rat kidney total RNA (1, 10, and 20 µg, respectively). In both neonatal and adult kidneys, the predicted 304-bp protected fragments are detected, corresponding to the sTbeta R-I. The second, slightly smaller protected fragment may represent an alternatively spliced variant. The 252-bp protected fragments correspond to the membrane-anchored Tbeta R-I. Lanes 9, 10, and 11: total RNA (10 µg) from COS-7 cells transfected with empty vector pcDNA3 (C) and cells transfected with a construct containing the coding sequence for the rat sTbeta R-I ligated in pcDNA3, either alone (TX) or cotransfected with a full-length rat TGF-beta type II receptor (Tbeta R-II) cDNA (coTX), respectively. As expected, no protected fragments were observed in control cells (lane 9). Predicted 268-bp protected fragments are observed in cells transfected with sTbeta R-I construct alone (lane 10) and in cells cotransfected with sTbeta R-I and Tbeta R-II (lane 11).

Overexpression of sTbeta R-I in COS-7 cells. COS-7 cells were stably transfected with a construct containing the coding sequence for the rat sTbeta R-I ligated in pcDNA3, either alone or cotransfected with a full-length, wild-type rat Tbeta R-II cDNA. Wild-type COS-7 cells and cells transfected with empty vector pcDNA3 were used as controls. mRNA expression of the sTbeta R-I construct was confirmed by RNase protection assay using the same riboprobe complementary to clone 29-1 from Hind III to Sma I sites, which contained 268 nt of the coding sequence of sTbeta R-I. As expected, no protected fragments were observed in control wild-type COS-7 cells (data not shown) and in cells transfected with empty vector alone (Fig. 3, lane 9). The predicted 268-bp protected fragments were observed in COS-7 cells transfected with sTbeta R-I construct alone (Fig. 3, lane 10) and in cells cotransfected with sTbeta R-I and Tbeta R-II constructs (Fig. 3, lane 11).

sTbeta R-I binds TGF-beta 1 in the presence of Tbeta R-II. Cell-surface ligand binding is demonstrated by affinity cross-linking with 125I-labeled TGF-beta 1. Two distinct bands, approximately 97 and 70 kDa in size, corresponding to Tbeta R-II and Tbeta R-I, respectively, are detected in wild-type COS-7 cells (data not shown) and in cells transfected with empty vector (Fig. 4, lane 2). In COS-7 cells transfected with full-length Tbeta R-II cDNA, a more intensely labeled 97-kDa band is observed (Fig. 4, lane 4). Because Tbeta R-I alone did not exhibit significant binding of TGF-beta 1 ligand when assessed by cross-linking analysis (27), COS-7 cells were cotransfected with full-length Tbeta R-II and sTbeta R-I cDNAs. In these cells, additional specifically labeled bands of lower molecular weight are seen (Fig. 4, lanes 6 and 8), corresponding to sTbeta R-I. Preincubation with unlabeled TGF-beta 1 demonstrates specificity of ligand binding. Based on the deduced amino acid sequence of clone 29-1, the sTbeta R-I protein has a predicted molecular mass of ~12 kDa. It also contains a potential N-glycosylation site, as indicated in Fig. 1B. Hence, the 45-kDa bands observed by affinity cross-linking are interpreted to represent glycosylated sTbeta R-I protein cross-linked to TGF-beta dimer. Evidence for glycosylation of the ALK-5 protein, which also contains the same potential site for N-glycosylation in the extracellular domain, has been previously shown by enzymatic deglycosylation of the affinity cross-linked complexes using endoglycosidase F (12).


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Fig. 4.   Cell-surface affinity cross-linking of 125I-labeled TGF-beta 1. Lanes 1 and 2: COS-7 cells transfected with empty vector pcDNA3, cross-linked in presence (+) or absence (-) of unlabeled TGF-beta 1, respectively. Lanes 3 and 4: cells transfected with full-length Tbeta R-II cDNA, in presence (+) or absence (-) of unlabeled TGF-beta 1, respectively. Two independent clones of cells cotransfected with Tbeta R-II and sTbeta R-I cDNAs (coTX1, lanes 5 and 6, and coTX2, lanes 7 and 8) are shown in presence (+) or absence (-) of unlabeled TGF-beta 1, respectively. Specifically labeled bands are observed at approximately 97 and 70 kDa, corresponding to Tbeta R-II and membrane-anchored Tbeta R-I, respectively, in all cells. In cells cotransfected with Tbeta R-II and sTbeta R-I cDNAs, additional specifically labeled bands of lower molecular weight are seen (lanes 6 and 8), corresponding to the transfected sTbeta R-I. The very high molecular weight TGF-beta type III receptors are observed in all cells.

TGF-beta 1 signaling. To explore the potential function of sTbeta R-I, the effects of its expression on TGF-beta 1 signaling were determined using the luciferase reporter plasmid, p3TP-Lux (Fig. 5). Transient transfection of p3TP-Lux and empty vector pcDNA3 (as controls) into COS-7 cells resulted in the induction of luciferase activity by exogenous TGF-beta 1, presumably due to endogenous expression of TGF-beta receptors in these cells. Remarkably, in cells transfected with sTbeta R-I and p3TP-Lux, further enhancement of TGF-beta 1 signaling was observed, with consistently higher levels of TGF-beta 1-induced luciferase activity observed in cells expressing sTbeta R-I compared with pcDNA3 controls. In cells transfected with full-length Tbeta R-II and p3TP-Lux, increased basal luciferase activity was observed, which increased little with exogenous TGF-beta 1 addition. Similarly, in cells cotransfected with full-length Tbeta R-II and sTbeta R-I, and p3TP-Lux, basal luciferase activity was increased, but in the presence of exogenous TGF-beta 1, further induction of luciferase activity above basal levels was noted. TGF-beta 1 responsiveness was completely abrogated when signaling was blocked by transfection of a dominant negative mutant Tbeta R-IM or Tbeta R-IIM.


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Fig. 5.   Transcriptional activation of p3TP-Lux reporter by TGF-beta 1. COS-7 cells were transiently transfected with luciferase reporter p3TP-Lux and expression plasmids for indicated receptors, or with parental vector pcDNA3. Forty-eight hours after transfection, cells were treated with (+) or without (-) exogenous TGF-beta 1 (1 ng/ml) for another 20 h. Luciferase activity in cell lysates was measured in a luminometer, and results are expressed in relative light units. Data represent means ± SD for triplicate determinations from a representative experiment. pcDNA3, empty vector control; sTbeta R-I, soluble type I receptor; Tbeta R-II, wild-type full-length type II receptor; Tbeta R-IM, kinase-deleted dominant negative mutant type I receptor; Tbeta R-IIM, kinase-deleted dominant negative mutant type II receptor.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

TGF-beta 1 exerts diverse biological activities through the interaction with a heteromeric signaling complex consisting of Tbeta R-I and Tbeta R-II (28). Given that Tbeta R-II is unable to mediate TGF-beta 1 signaling without Tbeta R-I, the specificity of cellular response to TGF-beta 1 is thought to be determined by Tbeta R-I. One plausible mechanism whereby different sets of cellular responses may be elicited by a single regulatory molecule is the interaction of the type II receptor with different type I receptors, or ALKs, to form the heteromeric signaling complex. Although ALK-5, ALK-1, and ALK-2 have all been demonstrated to complex with ligand-bound Tbeta R-II, ALK-5 is the predominant Tbeta R-I in most cell types (12).

Alternatively, diversity of TGF-beta actions may be mediated by Tbeta R-II interacting with variant forms of the signaling Tbeta R-I subtype. This study reports the identification of three variant forms of ALK-5. Two of the cDNA clones encoded membrane-anchored forms of Tbeta R-I but differed in the COOH-terminal region of the extracellular ligand-binding domain. One clone contained an in-frame insertion of four amino acid residues, GPFS, not present in the second clone. At precisely the same position in the extracellular domain, where the nucleotide sequences diverged in the two membrane-anchored forms of ALK-5, the third cDNA clone contained an insert of five amino acid residues, GKLLY, followed by an in-frame stop codon, and lacked the entire transmembrane and cytoplasmic serine/threonine domains. Based on the nucleotide sequence of this clone, the deduced amino acid sequence predicted a secreted form of ALK-5, named sTbeta R-I. In vivo expression of the sTbeta R-I mRNA was demonstrated in the neonatal and adult rat kidneys by RT-PCR followed by Southern analysis (Fig. 2), and confirmed by RNase protection assay (Fig. 3).

To date, a naturally occurring soluble form of TGF-beta receptor had not previously been reported. Curiously, similar variant receptor forms, including a naturally occurring soluble form, have been described for another cytokine, fibroblast growth factor (FGF) (8). The FGF receptor 1 (FGFR-1) gene contains three alternative exons encoding the COOH-terminal portion of the extracellular region (13). One of these alternative exons includes a stop codon and therefore encodes a secreted form of FGFR-1, and the other two exons encode membrane-anchored forms of FGFR-1 with the characteristic intracellular tyrosine kinase domain. As with the FGFR-1, the variant forms of Tbeta R-I described here may arise from three alternatively spliced exons at the position in the extracellular domain where the nucleotide sequences diverge. The published human Tbeta R-I gene consists of nine exons, and alignment with the nucleotide sequences for the three variant forms of ALK-5 described in this report reveals that the point of sequence divergence occurs precisely between exons 2 and 3, located just upstream of the transmembrane domain (24). Thus it is likely that the three variant receptor forms of ALK-5 are produced by major alternative splicing. Moreover, given the high degree of nucleotide sequence homology between the human and rat Tbeta R-I, indicating that the gene is highly conserved during evolution, it is likely that similar alternatively spliced forms of ALK-5 also occur in humans.

The significance for the existence of the variant forms of Tbeta R-I is yet unknown but may represent an endogenous mechanism to carefully and precisely control and modulate the activity of TGF-beta . Given that the multiple receptor forms vary in their extracellular ligand-binding domain, a potential physiological function may be to confer specificity for binding TGF-beta ligands. Alternative exons conferring ligand-binding specificity by altering ligand affinity have been demonstrated for the FGF receptors. For instance, an alternatively spliced variant of FGFR-2, named keratinocyte growth factor (KGF) receptor, bound acidic FGF with higher affinity than basic FGF (17), in contrast to the bek form of FGFR-2, which bound both basic and acidic FGF with high affinity (7). Moreover, a secreted form of FGFR-1 protein, when expressed in Chinese hamster ovary cells, oligomerized on ligand binding and preferentially bound basic FGF over acidic FGF (8).

In the case of TGF-beta receptors, although Tbeta R-II is required for initial binding of TGF-beta 1 and thus is said to possess ligand-binding specificity (27), additional ligand specificity may be determined by the particular form of Tbeta R-I involved in the heteromeric signaling complex with Tbeta R-II, perhaps by differing affinities for the three isoforms of TGF-beta . During embryonic development, all three isoforms, TGF-beta 1, -beta 2, and -beta 3, have been shown to be temporally and spatially regulated and differentially expressed in embryonic tissues including the kidneys (19). Both Tbeta R-I and Tbeta R-II have also been shown to be temporally and spatially regulated and differentially expressed in developing kidneys (6). It is possible that each of the multiple forms of TGF-beta receptors may possess specific roles and patterns of expression during embryonic development and may mediate different functions. Interestingly, the sTbeta R-I mRNA expression was observed to be greater in abundance in the rat neonatal kidneys compared with the adult kidneys, similarly to a previously reported (6) developmental study of Tbeta R-I mRNA abundance, thus implicating a role for sTbeta R-I in renal development.

Soluble receptors may potentially act as either antagonists or potentiators of cytokine activities. Inhibitory actions by soluble receptors, presumably through competition with membrane-anchored receptors for ligand binding, have been demonstrated for soluble receptors of interleukin (IL)-4, platelet-derived growth factor (PDGF)-alpha , and vascular endothelial cell growth factor (VEGF) (14, 18, 23). In contrast, potentiating action of IL-6 by its soluble receptor, IL-6sR, to markedly induce collagenase 3 expression in osteoblasts has also been demonstrated (11).

In this report, the functional role of sTbeta R-I in TGF-beta signaling was explored using a TGF-beta -inducible luciferase reporter assay. First, the ability of sTbeta R-I to complex with ligand-bound Tbeta R-II on the cell surface was demonstrated by affinity cross-linking with 125I-labeled TGF-beta 1 in COS-7 cells expressing both the sTbeta R-I and Tbeta R-II. Next, the effects of its expression on TGF-beta 1 signaling were determined using the luciferase reporter plasmid, p3TP-Lux, to ascertain TGF-beta 1-induced transcriptional activation of the PAI-1 promoter. Transient transfection of sTbeta R-I with p3TP-Lux in COS-7 cells resulted in higher luciferase activity in response to exogenous TGF-beta 1 compared with control COS-7 cells transfected with empty vector pcDNA3 and p3TP-Lux. This increased TGF-beta 1 responsiveness demonstrates the ability of sTbeta R-I to potentiate signaling by the endogenously expressed wild-type TGF-beta receptors. To determine whether TGF-beta 1 responsiveness could be further enhanced by increased levels of Tbeta R-II and ligand binding, the effects on TGF-beta 1 signaling were assayed on transfection of Tbeta R-II and in combination with sTbeta R-I. Transient transfection of wild-type Tbeta R-II in COS-7 cells resulted in higher basal luciferase activity, indicating that high-level expression of the receptors can initiate TGF-beta signaling, due to either the presence of endogenous TGF-beta 1 or the inherent affinity of the receptors to interact and cooperate with each other to signal in a ligand-independent manner. Transfection and overexpression of wild-type Tbeta R-I have previously been shown to exhibit ligand-independent activation (4). The inability of exogenous TGF-beta 1 to further increase luciferase activity in Tbeta R-II-transfected cells suggests probable saturation of the signaling receptors with the ligand. However, exogenous TGF-beta 1 was able to induce increased luciferase activity upon expression of sTbeta R-I, by cotransfection of Tbeta R-II with sTbeta R-I, but not above the levels observed in the sTbeta R-I-transfected cells. Taken together, these data suggest that the signaling Tbeta R-I may be expressed in limiting levels and that the sTbeta R-I functions as a potentiator but not as a direct signaling receptor, as it lacks the cytoplasmic domain. Next, the effects of sTbeta R-I were compared with those of another truncated form, Tbeta R-IM, which is membrane anchored but lacks the cytoplasmic GS and kinase domains and inhibits TGF-beta 1 signaling. As predicted, complete inhibition of TGF-beta 1-induced luciferase activity was observed in cells expressing the truncated receptors, Tbeta R-IM. Thus the sTbeta R-I actions clearly differ from those of the kinase-deleted mutant Tbeta R-IM, which inhibits TGF-beta 1 signaling, presumably by functioning in a dominant negative fashion to compete with membrane-anchored Tbeta R-I for complexing with the ligand-bound Tbeta R-II. Similarly, transfection of a dominant negative Tbeta R-IIM also inhibited TGF-beta signaling.

The stimulatory action exhibited by a soluble type I receptor for TGF-beta is rather intriguing. In contrast to IL-6sR, which binds IL-6 directly to potentiate its action, sTbeta R-I and Tbeta R-I require the presence of Tbeta R-II for ligand binding. Thus the soluble form of Tbeta R-I presumably functions as an agonist to augment TGF-beta 1 actions through an entirely novel mechanism of action not yet understood. The secreted sTbeta R-I might serve as a chaperone, for instance, by recognizing and interacting with ligand-bound Tbeta R-II to stabilize the ligand-bound complex and prevent its dissociation and degradation, and may cooperate to facilitate the recruitment of the membrane-anchored Tbeta R-I to form the signaling heteromeric complex. The soluble form of receptor may then dissociate and allow the membrane-anchored Tbeta R-I to form a more stable complex with ligand-bound Tbeta R-II to allow transphosphorylation and initiation of the signaling cascade. The concept of agonist activity of a natural sTbeta R-I to potentiate TGF-beta 1 actions has important clinical implications for its potential role in a wide range of biological processes, such as growth and development, wound repair, and pathogenesis of fibrotic diseases. The present studies demonstrate upregulation of sTbeta R-I expression in the developing kidney, potentially to enhance TGF-beta signaling and thereby amplify TGF-beta actions during renal development.

In summary, three variant forms of Tbeta R-I were isolated from a rat neonatal kidney library, and data presented in this report demonstrate for the first time the existence of a naturally occurring variant Tbeta R-I transcript whose nucleotide sequence and deduced amino acid sequence predict a soluble form of ALK-5. Physiological functions of these variant receptor forms are not yet known, but their variable in vivo expression may represent a mechanism conferring multiple cellular responses to TGF-beta 1. Moreover, the biological importance of a soluble Tbeta R-I is yet to be determined, but the findings here suggest that it may serve as a natural potentiator of TGF-beta signaling. The molecular cloning and identification of a naturally occurring sTbeta R-I adds a new level of complexity to our present knowledge of the TGF-beta receptor system and will facilitate further investigations to expand our understanding of the mechanism of TGF-beta 1 actions.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Physician Scientist Award 5-K12-DK-0129809, a Grant-in-Aid (96015510) from the American Heart Association, and a U.S. Department of Veterans Affairs Career Development Award.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. E. Choi, Section of Nephrology, Yale University School of Medicine, 333 Cedar St., 2073 LMP, New Haven, CT 06520-8029.

Received 18 August 1998; accepted in final form 8 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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