A Widely Expressed Transmembrane Serine/Threonine Kinase That Does Not Bind Activin, Inhibin, Transforming Growth Factor @, or Bone Morphogenic Factor*

Molecular cloning of complementary DNAs (cDNA) whose expression products bind activin and transforming growth factor @ (TGF-@l and -@) suggests that transmembrane serinelthreonine kinases constitute a new class of signaling molecules. A human liver cell cDNA which codes for a new serinelthreonine kinase receptor (SKR1) was identified using degenerate oligonucleotide primers complementary to coding se- quence for mouse activin and Caenorhabditis elegans daf- 1 serinetthreonine receptor kinase subdomains VI and VI11 in the polymerase chain reaction. The deduced 600-amino acid product consisted of a cysteine-rich extracellular domain and a cytoplasmic serinelthreo- nine kinase domain which are 10-20 and 40% homologous to the respective domains in the activin and transforming growth factor j3 receptor kinases. Cells overexpressing SKRl exhibited no increase in binding of activin, inhibin, TGF-81, TGF-82, or bone morphogenic factor type 2B. Except for its absence in bone and spleen, SKRl exhibits a tissue expression pattern similar to the TGF-@ receptor I1 gene. Similarly, SKRl is expressed in normal parenchymal cells, endothelial cells, fibroblasts,

The nucleotide sequence(s) reported in this paper hes been submitted to the GenBankTM/EMBL Data Bank with accession number($ LO291 I . 3 To whom correspondence should be addressed W. Alton Jones Cell Science Center, Inc., 10 Old Barn Rd., Lake Placid, NY 12946. Tel.: 518-523-1273; Fax: 518-523-1849. The abbreviations used are: TGF-@, transforming growth factor type 8; cDNA, complementary DNA; SKRl, serine/threonine kinase receptor type 1; PCR, polymerase chain reaction; TGFpRII, transforming growth factor-@ receptor type 11; ActRII, activin receptor type 11; BMP, bone morphogenic factor; RACE, rapid amplification of cDNA ends; kb, kilobase pair(s); bp, base pair(s). P superfamily of regulatory polypeptides are transmembrane serine (Ser)/threonine (Thr) protein kinases (1)(2)(3)(4)(5). Although the 120-160-residue cysteine-rich extracellular domains of the two receptors exhibit limited homology, both have conserved clusters of residues in the intracellular domain that define subdomains of the Ser/Thr kinase superfamily (6, 7). To characterize Ser/Thr kinase receptors in human hepatoma cells, we exploited the polymerase chain reaction (PCR) to generate cDNAs to kinase subdomains VI and VI11 in the activin receptor kinase and a homolog from Caenorhubditis ekgalzs (daf-1) (8). In addition to cDNA fragments coding for the human homolog of mouse activin receptor IIB (ActRIIB) (1)(2)(3)(4) and the human ActRII (9), we identified a novel cDNA (SKR1) that is homologous to, but distinct from, the activin receptor isoforms (1)(2)(3)(4) and the TGF-Pl and -P2 receptor I1 kinases (TGFPRII) (5). Here we report the deduced structure of SKRl and show that the product of SKRl cDNA binds neither activin, inhibin, TGF-Pl and $2 nor bone morphogenic factor type 2B (BMP-2B). SKR1, which is widely expressed in both normal and tumor tissues and cells similar to the TGFPRII, may be a receptor for a novel member of the TGF-8 branch of the superfamily of ligands.

EXPERIMENTAL PROCEDURES
RNA Extraction and Analysis-RNA isolation and Northern hybridization analysis were performed as described previously (10,11) except that the hybridization for SKRl was carried out in 40% formamide, 6 X SSC, 0.1% SDS, 100 pg/ml salmon sperm DNA, and 0.1% each of bovine serum albumin, Ficoll, and polyvinylpyrrolide. Complementary 32P-labeled cDNA probes were a 1.3-kb PstIIXbaI fragment, a 1.2-kb EcoRIIBglII fragment, a 0.5-kb KpnI fragment coding for the extracellular domains of SKR1, TGFpRII, and ActRII, respectively.
PCR and Analysis of Products-Random-primed cDNA was synthesized from poly(A+) RNA prepared from the human hepatoma cell, HepG2, and subjected to 40 cycles of amplification for 1 min at 94 "C, 2 min at 45 "C, and 3 min at 72 'C with primer mixtures (5'-
The positive 180-base pair (bp) band was excised from the gel, digested with EcoRI/XhoI, and cDNA clones were prepared in bacterial SK vector (Stratagene, La Jolla, CAI. Nucleotide sequence of activin and SKRl cDNA clones was determined as described (11).
Deduction of the Full-length SKRl Sequence-Ten groups of IO6 clones from a HepG2 cDNA phage library (CloneTech, Inc., San Diego, CA) were screened by PCR amplification of a specific 101-bp SKRl sequence between the coding sequences for kinase subdomains VI and VI11 ( Gly-77 in the extracellular domain of SKR1. A sense primer based on the 5'-most sequence of the above cDNA (5"GGGAAAGAT-GACCTGTAAG-3') and antisense primer 5"CAGCTGGGCCGT-GATGTTCCTGTTA-3' was used to further screen the HepG2 library as described above and resulted in a 1.8-kb cDNA that contained 330 bp of apparent 5"noncoding sequence followed by an open reading frame extending through coding sequence for residue Ile-491 of SKR1. Transfection, Ligand-Binding, and Covalent Affinity Cross-linking to Monkey Kidney (COS) Cells-Transfection and cross-linking experiments were carried out as described (11,13) except that input ligand was 1.6 X lo6 cpm (1.1 ng/ml), 7.8 X lo5 cpm (1.3 ng/ml), and 3.0 X lo6 cpm (10.7 ng/ml) for activin, TGF-P1, and BMP-2B, respectively. '251-Labeled activin, inhibin, TGF-01, and TGF-02 were prepared as described (14,15). Recombinant viral plaques were identified by a combination of visual screening and Southern dot blot hybridization. Sf9 cells were prepared, infected with virus (13), and tested for binding of lZ5I-labeled TGF-01 (3.2 X lo5 cpm, 1.3 ng/ml) and BMP-2B (2.3 X lo6 cpm, 10.7 ng/ml). Cells were washed three times with binding buffer, extracted with 1% Triton X-100 in phosphate-buffered saline, and the extract was counted using a y-counter.
Immunochemical Analysis of Infected Sf9 Cells for Expression of SKRl Surface Antigen-Immunochemical analysis were carried out as described previously (17) except that the presence of cell surface SKRl antigen was analyzed with a rabbit antiserum prepared against the synthetic peptide sequence, EDEKPKVNPKLYMCV, from the NH, terminus of SKR1. Sf9 cells were incubated with a 1:lOO dilution of antiserum in 500 p1 and then incubated with lZ5I-labeled donkey anti-rabbit IgG (4 X lo5 cpm, 16 ng/ml).

RESULTS AND DISCUSSION
Identification and Characterization of SKRl -To identify activin-related and potentially novel Ser/Thr kinase receptor cDNAs, we designed degenerate sense (1024 sequences) and antisense (256 sequences) oligonucleotides complementary to the amino acid sequences of kinase subdomains VI and VI11 (6,7) which are conserved between the mouse ActRII gene (1) and the C. ekguns duf-1 gene (8) (Fig. lA). human hepatoma cell line HepG2 (11). Hybridization of PCRgenerated fragments at low stringency with mouse ActRII cDNA indicated the presence of a band at about 180 bp, consistent with the presence of human activin or related cDNA fragments. The 180-bp band was excised, cloned, and 37 clones subjected to sequence analysis. Twenty cDNA clones exhibited nucleotide coding sequences for amino acid residue landmarks for Ser/Thr kinase subdomain VI1 which is between subdomains VI and VI11 (1,6,7). Six clones exhibited nucleotide sequences identical to human ActRII (9), amino acid sequences deduced from four cDNAs were identical to mouse ActRIIB (2,3), and the rest coded for a Ser/Thr kinase (SKR1) that was significantly different from cDNAs coding for human Ser/Thr receptor kinases ActRII and TGFPRII (5, 9). Similar to the C. eleguns duf-1 gene (8), the SKRl cDNA coded for a DLG sequence instead of the DFG tripeptide sequence in human ActRII and TGFPRII which is highly conserved and defines subdomain VI1 throughout the Ser/ Thr kinase superfamily (6,7). The complete nucleotide sequence of SKRl was determined by isolation of cDNA clones using a combination of PCR-based screening of a HepG2 cDNA phage library and RACE-PCR. The complete nucleotide sequence of the SKRl cDNA extended for 3000 nucleotides and exhibited a single long open reading frame of 1527 nucleotides which encodes a putative 58-kDa primary translation product of 509 amino acids (Fig. 1B). The predicted product of the SKRl gene meets the criteria for a transmembrane Ser/Thr kinase receptor. The first 21 amino acids are hydrophobic and likely serve as a signal sequence for membrane transport. Hydropathy analysis revealed an additional stretch of 23 hydrophobic residues followed by a cluster of basic amino acids that probably serve as the transmembrane domain and stop-transfer signal. The extracellular domain includes 10 cysteines and one potential N-linked glycosylation site (Fig. 1B). Except for a similarity in spatial distribution of the cysteine residues, SKRl exhibited only a 12 and 20% amino acid sequence homology, respectively, with human ActRII and TGFPRII Ser/Thr receptors in the extracellular domain (Fig. 1B). Although there is less than 5% homology in the juxtamembrane sequences, the intracellular domain of SKR1, which has consensus ATP-binding and a kinase domains as well as a COOH-terminal tail, exhibited a 40% homology with both human ActRII and TGFPRII receptors. Although there is yet no context consensus sequences for Ser/ Thr kinase receptor substrate phosphorylation sites, SKRl exhibits seven Ser/Thr sites in the intracellular domain that are conserved in all three Ser/Thr receptor kinases that may be potential autophosphorylation sites (Fig. 1B) Ligand Binding to Recombinant SKRl-To determine whether SKRl was an isoform of the activin or TGFPRII Ser/Thr kinase receptors, we transfected monkey kidney cells (COS) with a mammalian expression vector bearing an SKRl cDNA and tested for an increase in 1251-labeled activin and TGF-B1 binding. In contrast to cells transfected with vector bearing ActRII and TGFBRII cDNAs, cells transfected with SKRl cDNA exhibited no increase in binding of the two ligands (Fig. 2, A and B ) , although analysis of mRNA confirmed that SKRl mRNA was overexpressed. Separate experiments in which labeled inhibin and TGF-PB was substituted for activin and TGF-P1 revealed no increase in ligand binding to cells transfected with SKRl cDNA. In addition no increase in 1251-labeled bone morphogenic factor type 2B (BMP-2B) could be detected in SKR1-transfected cells (Fig. 2C) relative to mouse fibroblast cells which exhibit BMP-2B binding sites (16). The lack of binding of TGF-Dl and BMP-2B to insect cells infected with recombinant baculovirus bearing the SKRl cDNA and which express cell surface SKRl antigen confirmed the negative binding results in transiently transfected mammalian cells (Fig. 3).
Expression of SKRl Tissues and Celk-The expression of SKRl in cells and tissues was compared with that of the ActRII and TGFBRII receptor kinases. Northern hybridization using cDNAs coding for the extracellular domain of the three genes was performed to avoid potential cross-hybridization between homologous intracellular domains (Fig. 4). Human cells exhibited a major 3.6 and two minor 5.8-and 8.6-kb mRNAs, whereas rat and mouse cells and tissues exhibited a major species at 4.2 kb. The tissue expression pattern of SKRl is more similar to TGFBRII than that of ActRII. SKRl was most abundant in heart, liver, intestine, and kidney, low in brain and lung, and undetectable in spleen and bone. In contrast, TGFPRII mRNA was undetectable in brain, higher in lung and liver, lower in heart and intestine, but detectable in spleen and bone. Similar to the TGFPRII gene, SKRl is expressed in both normal parenchymal cells (skin keratinocytes and hepatocytes) and endothelial cells and fibroblasts (Fig. 4). Of the cells examined, the hepatoma cells (HepG2) from which SKRl was cloned and endothelial cells exhibited the highest levels of expression of both SKRl and TGFPRII. Our results suggest that SKRl is a receptor for an unidentified member of the TGF-/3 ligand superfamily (1)(2)(3)(4)(5). SKRl may be a specific receptor for one or more of the cloned, but less well characterized members or a completely new member, of the superfamily (1)(2)(3)(4)(5). The lack of binding to a prototypic member (BMP-2B also called BMP-4) of the BMP-2, -3, and -4 branch of the superfamily and absence of SKRl in bone suggests that SKRl is unlikely a receptor for a ligand within the subgroup. The similarity of the cell and tissue expression pattern of SKRl to TGFBRII further suggests that the ligand for SKRl is likely to be within the TGF-Dl-5 branch of the superfamily (5). However, the BMP-5, -6, and -7 subgroup which is expressed significantly in non-skeletal tissues as lung, liver, and kidney (22, 23) cannot be eliminated. Identification and characterization of the ligand for SKRl is essential to understanding its function in the large number of tissues and cells in which it is expressed.