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WO1996018735A2 - RECEPTEUR DE TYPE II DE LA SUPERFAMILLE DE TGF-β PRESENTANT UNE AFFINITE DE LIAISON AVEC LA PROTEINE MORPHOGENETIQUE OSSEUSE - Google Patents

RECEPTEUR DE TYPE II DE LA SUPERFAMILLE DE TGF-β PRESENTANT UNE AFFINITE DE LIAISON AVEC LA PROTEINE MORPHOGENETIQUE OSSEUSE Download PDF

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Publication number
WO1996018735A2
WO1996018735A2 PCT/US1995/016412 US9516412W WO9618735A2 WO 1996018735 A2 WO1996018735 A2 WO 1996018735A2 US 9516412 W US9516412 W US 9516412W WO 9618735 A2 WO9618735 A2 WO 9618735A2
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Prior art keywords
receptor
nucleic acid
polypeptide
acid molecule
type
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PCT/US1995/016412
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WO1996018735A3 (fr
Inventor
George N. Cox
Bradley L. Rosenzweig
Kohei Miyazono
Carl-Henrik Heldin
Takeshi Imamura
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Amgen Boulder Inc.
Ludwig Institute For Cancer Research
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Priority to AU44249/96A priority Critical patent/AU4424996A/en
Publication of WO1996018735A2 publication Critical patent/WO1996018735A2/fr
Publication of WO1996018735A3 publication Critical patent/WO1996018735A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators

Definitions

  • the present invention generally relates to a novel TGF- ? superfamily type II receptor, particularly to a receptor for bone morphogenic protein, corresponding nucleic acid molecules and their use.
  • TGF-/S The transforming growth factor- ⁇ superfamily consists of a family of structurally-related proteins, including three different mammalian isoforms of TGF- ⁇ (TGF-/?1 , 2, and ⁇ 3), activins, inhibins, m ⁇ llerian-inhibiting substance and bone morphogenic proteins.
  • TGF-/?1 , 2, and ⁇ 3 Three different mammalian isoforms of TGF- ⁇
  • activins activins
  • inhibins m ⁇ llerian-inhibiting substance
  • bone morphogenic proteins For reviews of these TGF- proteins, see Roberts & Sporn, Peptide Growth Factors and Their Receptors. Pt.1 , pp. 41 9-472 (Berlin:Springer- Verlag, 1 990); and Moses et al., £eH 63 * r 2 5-247 ( 1 990).
  • TGF- ? Glial cell line derived neurotrophic factor
  • the proteins of the TGF- ? superfamily have a wide variety of biological activities.
  • TGF- ? acts as a growth inhibitor for many cell types and appears to play a central role in the regulation of embryonic development, tissue regeneration, immuno-regulation, as well as in " fibrosis and carcinogenesis (Roberts & Sporn, supra.)
  • TGF- ⁇ s and activins transduce their signals through the formation of heteromeric complexes of two different types of serine/threonine kinase receptors, i.e.
  • Type II receptors of about 50-55 kDa and type II receptors of about 70-80 kDa (Massague et al.. Trends Cell Biol. 4, 172- 1 78, 1 994).
  • Type II receptors bind ligands in the absence of type I receptors, but they require their respective type I receptors for signaling, whereas type I receptors require their respective type II receptors for ligand binding.
  • TGF- ⁇ type I receptor T ⁇ R-l
  • Act and ActR-IB activin type I receptors
  • BMPR-IA bone morphogenic proteins type I receptors
  • Bone morphogenic proteins also known as osteogenic proteins, a a family of proteins that induce ectopic bone formation at extraskeletal sites in vi (reviewed in Reddi, Curr. Opinion Genet. Develop. 4, 737-744, 1 994; Wozne Prog. Growth Factor Res. 1, 267-280, 1 989).
  • BMPs act on osteoblasts a chondrocytes as well as other cell types, and they play important roles in embryon development (reviewed in Harland, Proc. Natl. Acad. Sci. U.S.A. 91, 10243- 1 024 1 994).
  • BMP-2 t 6, osteogenic protein (OP)-1 also termed BMP-7
  • OP-2 osteogenic protein
  • growth/differentiati factor-5 growth/differentiati factor-5 to -7
  • BMP-4 binds to BMPR-IA and BMPB- efficiently (ten Diike et al.. J. Biol. Chem. 269. 16985-16988, 1 994; oenig et a Mol. Cell. Biol.
  • Type II receptors for activin ActR-ll and ActR-IIB
  • T ⁇ R- TGF- ⁇
  • the only BMP type II receptor identifi to date is DAF-4 isolated from Caenorhabditis eleqans (Estevez et al.. Nature 36 644-649, 1 993).
  • the present invention relates to the discovery of a novel mammalian TGF superfamily type II serine/threonine kinase receptor, referred to herein as U2
  • the invention is directed to an isolated, mammalian polypeptide capable of binding a bone morphogenic protein and having at least the extracellular amino acid sequence of the U2 receptor as shown in SEQ.ID.NO.1 or a functional derivative thereof.
  • the novel U2 receptor has a serine/threonine kinase domain followed by a long serine and threonine-rich C-terminus. The long serine/threonine- rich region in the C-terminal end spans residues 513 to 1038 of SEQ.ID.NO.1 .
  • the invention further relates to isolated nucleic acid molecules encoding the novel polypeptides.
  • the serine/threonine-rich C-terminus is encoded by nucleotides 1918 to 3495 of the nucleotide sequence encoding the novel U2 receptor as shown in SEQ.ID.NO.2.
  • Complementary nucleic acids are also provided that can hybridize under stringent conditions to the isolated nucleic acid molecules encoding the novel polypeptides.
  • Vectors containing the nucleic acids of the present invention and host cells containing such vectors are also provided. Such vectors can also contain operational elements to express the polypeptides of the present invention.
  • the invention further provides methods for the recombinant production of the novel polypeptides by using DNA encoding polypeptides having binding affinity for a bone morphogenic protein such as OP-1 .
  • Complexes containing the novel type II receptor of the present invention and a type I receptor of the TGF-/? superfamily are also provided, where the type I receptor is ActR-1 or BMPR-1 B.
  • the complexes in the presence of 30-100 ng/ml of a bone morphogenic protein, such as OP-1 can transduce an intracellular signal.
  • the present invention relates to a novel type II receptor (U2 or BMPR-II) having greater binding affinity for bone morphogenic proteins, particularly OP-1 , than activin.
  • This novel receptor is a member of the TGF- ? superfamily of receptors based on characteristics shared with known members of the TGF- ? superfamily of receptors.
  • the U2 receptor has a hydrophobic signal peptide, an extracellular domain with a cysteine pattern typical of other serine/threonine kinase receptors of the TGF-/?
  • the novel U2 receptor is distinguishable from other known TGF superfamily type II receptors.
  • the amino acid sequence of the huma U2 receptor differs from other known receptors of the TGF- ? superfamily.
  • Th human receptor also contains an extraordinarily long serine and threonine rich regio in the C-terminal end that spans about 500 amino acids (residues 513 to 1038 SEQ.ID.NO.1 ) in length compared with other known mammalian TGF-/?
  • the U2 receptor has substantiall greater binding affinity for OP-1 , a bone morphogenic protein, compared wit activin.
  • the present invention is accordingly directed to isolated, mammalia polypeptides having binding activity with a bone morphogenic protein and contai the amino acid sequence of the full length U2 receptor or a functional derivativ thereoff
  • polypeptide is used synonymously wit
  • FIGEDTRLNI NSSPDEHEPL LRREQQAGHD EGVLDRLVDR RERPLEGGRT NSNNNNSNPC
  • the corresponding rat U2 receptor has the following amino acid sequence:
  • a functional derivative of the U2 receptor is also provided.
  • the term "functional derivative” means any biologically active modified form of the polypeptides. Such modifications can be ( 1 ) substitutions or additions in the amino acid sequence, and/or (2) the addition of a functional group to be used as a cross-linking agent or to improve certain pharmacokinetic or immunologic properties.
  • a functional derivative of the human U2 receptor can be the extracellular domain (residues 1 to 150 of SEQ.ID.NO.1 ) or biologically active fragments thereof that contains the active site for binding with OP-1.
  • a functional derivative was prepared in which the serine/threonine kirase domain and the following serine/threonine-rich C-terminal region were deleted.
  • This functional derivative was found to be-biologically active in that it bound to OP-1.
  • modifications should not substantially decrease the binding affinity of the unmodified polypeptide for OP-1 .
  • the term "functional derivative” can mean an active fragment, an analog or a derivative that substantially retains the biological activity of the unmodified polypeptide.
  • such modified polypeptides preferable have an amino acid homology of greater than about 40% compared to the U2 amino acid sequence or its extracelluar domain, more preferably in excess of 50%, and most preferably in excess of 90%.
  • a molecule having an amino acid homology of about 99% is particularly useful.
  • the present invention further relates to nucleic acid molecules encoding th novel receptor or functional equivalents thereof.
  • nucleic acid molecule whic encodes the novel human U2 receptor has the nucleotide sequence of SEQ.ID.NO.3 i.e.:
  • the nucleic acid sequence encoding the rat U2 receptor has the sequence of SEQ.ID.NO.4, i.e.:
  • the term "functional equivalent” means a modified nucleotid sequence having one or more additions, deletions, or substitutions to the abov sequence that do not substantially affect the ability of the sequence to encode polypeptide having OP- 1 binding activity.
  • modified sequences can b produced by means known in the art, including, for example, site directe mutagenesis.
  • sequences can be obtained from natural sources, such as the natur DNA sequences encoding the extracellular domain of the U2 receptor protein Alternatively, the sequences can be produced synthetically according to method known in the art. Additionally, such DNA sequences can be derived from combination of synthetic and natural sources.
  • the natural sequences furthe include cDNA and genomic DNA segments. Methods of obtaining the synthetic an natural DNA sequences are described in PCT Publication No. WO 93/00431 published on January 7, 1 993, which is incorporated herein by reference. Nucleic acids thgt are complementary to the nucleic acids encoding the U receptor or its functional derivatives are also contemplated.
  • complementar nucleic acids can be used, for example, as probes to hybridize under stringen conditions and, therefore, detect the U2 nucleic acids according to methods know to those skilled in the art.
  • stringent conditions refers t parameters with which the art is familiar. More specifically, stringent conditions as used herein, refers to hybridization in 1 M NaCI, 1 % SDS, and 1 0% dextra sulfate. This hybridization is followed by two washes of the filter at roo temperature for 5 minutes in 2xSSC, and one wash for 30 minutes in 2xSSC, 0.1 SDS. There are other conditions, reagents and so forth that can be used, whic result in the same or higher degree of stringency. One skilled in the art will b familar with such conditions.
  • the present invention further relates to methods of recombinantly producin the U2 receptor and its functional derivatives.
  • the methods are accomplished b obtaining a DNA sequence encoding the desired U2 polypeptide, inserting the DN sequence into a vector having operational elements for expression of the DNA transferring the vector into a host cell capable of expressing the polypeptide culturing the host cell under conditions suitable for expression of the polypeptide, and harvesting the polypeptide.
  • the desired nucleic acid sequence can be inserted into a variety of vectors known to those skilled in the art using conventional methods.
  • the nucleic acid sequence can be inserted and linked into a vector with any desired operational elements to effect its expression.
  • the vectors can contain one or more of the following operational elements: ( 1 ) a promoter; (2) a Shine-Dalgarno sequence and initiator codon; (3) a terminator codon; (4) an operator; (5) sequence encoding a leader sequence to facilitate transportation out of the host cell; (6) a gene for a regulator protein; (7) a Kozak sequence preceding the initiator codon; and (8) any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vectors.
  • 90 1 1 3 673.9 discloses several useful vectors and desirable operational elements.
  • the vectors can be transferred into suitable host cells by various methods known in the art, including transfection and transformation procedures. Various transfer methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference.
  • host cells can be either eucaryotic or procaryotic cells. Examples of such host cells include Chinese hamster ovary (CHO) cells, monkey kidney COS-1 cells, yeast, E. Coli and baculovirus infected insect cells.
  • the host cells described in EP Application No. 90 1 1 3 673.9, which is incorporated herein by reference, are also useful in the present methods.
  • the host cells of the present invention can be cultured under conditions appropriate for the expression of the U2 polypeptide. These conditions are generally specific for the host cell and are readily determined by one of ordinary skill in the art in light of the published literature regarding the growth conditions for such cells. For example, Beroev's Manual of Determinative Bacteriology, 8th ed. , Williams & Wilkins Co., Baltimore, Maryland, which is incorporated herein by reference, contains information relating to appropriate conditions for culturing bacteria. Similar information relating to culturing yeast and mammalian cells are described in R. Pollack, Mammalian Cell Culture. Cold Spring Harbor Laboratori ( 1975), incorporated herein by reference.
  • the invention is further directed to complexes comprising the U2 type receptor and a TGF- ? superfamily type I receptor, such as ActR-1 or BMPR-IB.
  • a TGF- ? superfamily type I receptor such as ActR-1 or BMPR-IB.
  • the complex can further contain a bone morphogen protein, such as OP-1 .
  • the polypeptides of the present invention have a number of in vitro and vivo uses.
  • the polypeptides can be used to affinity purify OP- 1 fro a number of sources, including serum from patients, cell culture supernatants recombinantly produced OP-1 , according to well known affinity purificatio procedures.
  • methods of purifying OP-1 from a sample are accomplished b (a) contacting polypeptides of the present invention with the sample; (b) allowin the polypeptides to bind to OP-1 ; (c) dissociating the OP-1 from the polypeptide and (d) collecting the dissociated OP-1 .
  • the polypeptides of the present invention can also be used as diagnosti reagents to detect or quantify OP-1 according to diagnostic methods well know in the art.
  • diagnostic methods well know in the art.
  • methods of detecting or quantifying OP-1 in a sample accomplished by (a) contacting polypeptides of the present invention with th sample suspected of containing OP-1 ; (b) allowing the polypeptides to bind to OP-1 and (c) detecting or quantifying OP-1 bound to the polypeptides.
  • the polypeptides of the present invention can further be used in combinatio with type I receptors, particularly ActR-l or BMPR-1 B, to detect or quntify OP- 1 i samples, such as biological fluids for example.
  • methods of detecting quntifying OP-1 in a sample can be accomplished by (a) co-transfecting mammalian cell line, such as monkey kidney COS-1 cells or Chinese hamster ovar cells (CHO), with plasmids capable of expressing U2 and ActR-l or BMPR-1 B, an a suitable reporter plasmid such as p3TP-Lux capable of expressing a quantifiable enzyme activity, such as luciferase; (b) contacting the transfected cells with a sample suspected of containing Op-1 ; (c) allowing the transfected cells to bind OP- 1 ; and (d) detecting or quantitating the amount of enzyme activity produced in the cultures.
  • Commercially available detection kits can be used in these methods.
  • polypeptides can also be used as immunogens to produce polyclonal and monoclonal antibodies according to procedures well known in the art and as described, for example, in Harlow & Lane, Antibodies: A Laboratory Manual (1 988), incorporated herein by reference.
  • Such antibodies can, in turn, be used to detect the U2 receptors or receptor complexes for in vitro as described above and in vivo uses such as imaging according to procedures known in the art.
  • TGF-;.?) type II receptor (Lin et al., Cell 68:775-785, 1 992)
  • murine activin type II receptor Mothews and Vale, Cell 65:973-982, 1 991
  • murine TGF- ⁇ type I receptor (Ebner et al.) revealed conserved peptide regions within the kinase domain. conserveed regions with specificity to membrane bound serine /threonine kinases were selected for the design of degenerate oligonucleotide primers.
  • H C,A or T
  • W A or T
  • D A, or T
  • M A or C
  • S G or C
  • N A,C,G, or T
  • R A or G
  • Y C or T
  • a i lnosine.
  • RNA was isolated from 25 mg of adult rat substantia nigra tiss using a Quick Prep mRNA Purification Kit (Pharmacia, Ippsala, Sweden) .
  • Sing strand cDNA was synthesized as follows; 1 ⁇ g polyA" RNA and 0.5 ⁇ g oligo d( (BRL, Gaithesburg, MD) in 10 ⁇ l H 2 0 were heated to 70°C for 1 0min, then put ice.
  • the cDNA synthesis reaction was performed in 50mM Tris-HCI pH 8.3, 75m KCI, 3mM MgCl 2 , 10mM dithiothreitol, 0.2mM each of dATP, dCTP, dGTP, a dTTP, 1 unit Human Placental Ribonuclease Inhibitor (BRL), and 200 units Molon Murine Leukemia Virus reverse transciptase (BRL) in 20 I total volume at 42° C f one hour. The reaction was then diluted to 40 ⁇ l with H 2 O and stored at -70°
  • PCR was performed in 1 0mM Tris pH 8.3, 50 mM KCI, 0.001 %, bovine seru albumin (BSA), 1 .5mM or 2.5mM MgCI 2 , 50pmol of the TR-D and TR-ER primer 0.2mM each of dATP, dCTP, dGTP, and dTTP, 2.5 units AmpliTaq DN Polymerase (Roche Molecular Systems, Branchburg, N.J.), and 3 ⁇ of single stra cDNA template at a volume of 40 ⁇ l which was then used in the PCR Gem hot sta method as set forth herein.
  • BSA bovine seru albumin
  • the reaction mixtures were heated to 95 ° C for minutes and 35 amplification cycles were carried out ( 95°C 1 min, 45 °C 1 min, 72°C 1 min), followed by a final 10 minute extention reaction at 72 °C.
  • the product band of the TR-D and TR-ER primed PCR reaction was in the predicted size range of 300 - 350 bp. This band was size selected on a 1 % agarose, 1 .5% Nuseive (FMC, Rockland, ME) gel in TAE buffer. The product band was excised and
  • DNA was extracted with a Qiaex Gel Purification Kit (Qiagen, Chatsworth, CA) following the manufacturer's instructions. The DNA fragment was then digested with EcoRI, and ligated to EcoRI-digested and phosphatase treated pBluescriptll SK plasmid DNA (Stratagene, San Diego, CA). A sample of the ligation product was used to transform E. coli strain XLI Blue (Stratagene) by electroporation.
  • Transformed colonies were selected by plating the bacteria with 10 ⁇ l l OOmM isopropylthio-yff-galactoside, and 100//I 2% 5-bromo-4-chloro-/?-D-galactoside on LB pH 7 agar plates containing 50 ⁇ g/ml ampicillin. Recombinant colonies were analyzed for inserts by PCR using T3 + , T7* ⁇ SK + , or KS* primers flanking the polylinker. The following oligonucleotide primers were synthesized on an ABI 360
  • ABS DNA synthesizer
  • a Dye-Deoxy Terminator Sequencing Kit (ABI) and T3 + , T7 + , SK + , or KS + primer outside the insert were used to determine the DNA sequence of the PCR products
  • SEQ.ID.NO.4 SEQ.ID.NO.4
  • TGF-/? type II and activin type receptors were found.
  • a huma substantia nigra cDNA library in ⁇ gt10 (Clontech, Palo Alto, CA) was hybridize with a 32 P-labeled rat U2 probe.
  • the PCR fragment from one rat U2 containin plasmid was agarose gel purified with a Qiaex Gel Extraction Kit (Qiagen).
  • Qiagen Qiaex Gel Extraction Kit
  • rat U2 fragment Three ng of rat U2 fragment was used in a reaction with 60pmol SK + and KS * primers; 0.2mM dATP, dGTP, an dTTP; 0.0125mM dCTP; 50pmol ⁇ 32 P-dCTP - 3000Ci/mmol; 5 units AmlpiTaq DN polymerase (Perkin Elmer Cetus); containing 10mM Tris pH 8.3, 50mM KCI, 2. mM MgCI 2 , 0.001 % BSA; in 50 ⁇ l total reaction volume. The reaction was heate to 95 C for 5 min then 25 cycles at (95°C 1 min, 62°C 1 min, and 72°C 1 min were carried out.
  • the filters were hybridized in 40 formamide, 0.90 M NaCI, 0.05M NaPO 4 , 5mM EDTA (pH 7.4), 0.1 % Ficoll, 0.1 % polyvinylpyrrolidone, 0.1 % bovine serum albumin, 0.1 % SDS, 100 ⁇ g/ml yeast tRNA, with the rat U2 probe at 1 x10 6 cpm/ml hybrididation solution at 42 C overnight.
  • the insert sizes of five of the cDNA clones ranged from 1 .0 kb to 4.0 kb.
  • U2A gave a major PCR product band in combination with only one flanking primer and artifact bands with the other flanking primer.
  • the insert in ⁇ 14-1 was excised by EcoRI digestion, electrophoresed on a 0.8% agarose gel in TAE buffer, and the 4kb cDNA insert isolated using a Qiaex Gel Extraction Kit (Qiagen) .
  • This 4kb EcoRI DNA fragment was ligated into EcoRI digested, dephosphorylated pBluescriptll SK ' plasmid DNA (Stratagene) .
  • O transformant, pSK/U2-27, with the 4kb insert was identified by PCR methods detailed above. Plasmid DNA of pSK/U2-27 was prepared using a Plasmid Mini (Qiagen).
  • the DNA sequence of the 4kb U2 cDNA was determined using a seri of oligonucleotide primers in Dye-Deoxy Terminator Sequencing Kit (ABI) reaction
  • the full-length coding sequence of U2 and the translation for the polypeptide encodes, along with 5' and 3' flanking DNA sequences are shown in SEQ. ID. NO.
  • the human U2 polypeptide has typical features of TGF-,5 super-family memb receptors; a hydrophobic signal peptide, residues 1 to 26, with a presum cleavage site after residue 26; an extracellular domain, residues 27 to 1 50, with cysteine pattern typical of other serine/threonine kinase receptors; three potenti N-linked glycosylation sites (Asn-Xaa-Ser/Thr, where Xaa can be any amino acid a hydrophobic transmembrane domain of 22 residues; and an intracellul serine/threonine kinase domain of approximately 300 residues.
  • the cDNA of U2 encodes a protein of 1038 amino ac residues containing an N-terminal hydrophobic leader sequence, followed by extracellular domain, a single transmembrane domain, and an intracellular doma with a serine/threonine kinase region.
  • the U2 protein lacks a glycine-and serine-ri sequence in the juxtamembrane domain, which is typical for type I recepto (Massague et al., Trends Cell Biol., 4, 172-178, 1 994; Kingsley, Genes and De
  • the 4 kb PCR fragment of human U2 in pSK/U2-27 was gel isolated and labeled with a 32 ? dCTP by random primer synthesis with a Quick Prime Kit
  • 1 % SDS 100 ⁇ g/ml yeast tRNA; or in 50% formamide, 1 .08M NaCI, 0.06M NaPO 4 , 6mM EDTA (pH 7.4), 0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin, 0.1 % SDS, 100 ⁇ g/ml yeast tRNA; with 2x10 6 cpm/ml U2 probe at 42°C overnight.
  • U2 transcripts were also detected in heart, kidne lung, placenta, testis, pancreas, ovary, prostate, and small intestine.
  • the 1 1 k transcript was the most prominent band in each tissue; the 10kb and 5. transcripts were detected only in the highest expressing tissues.
  • RNAs from human adult caudate nucleus, hippocampu substantia nigra, whole brain, kidney, lung and human fetal whole brain, fet kidney and fetal lung (Clontech) were electrophoresed on agarose-f ormaldehyde ge and tansferred to Hybond N. membranes (Amersham, Arlington Heights, IL) .
  • portion of the human U2 cDNA from 93 bp upstream of the ATG condon to b 251 8 was PCR amplified and 32 P-labeled using a Quick Prime Kit (Pharmacia Hybridization conditions were 0.5 M.
  • U2 is a single copy human gene
  • a Southern blot of human genomic DNA (Clontech) restriction endonucleas disgested with BamHI was prepared.
  • the blot was hybridized with the 32 P-labele rat U2 PCR probe, and the same hybridization and wash conditions, described f the cDNA library screen.
  • Autoradiographic exposures of the washed filter gave onl one band greater than 20kb, indicating U2 is most likely a single copy gene.
  • ligands for U2 binding studies using 25 l-labeled membe of the TGF- ⁇ superfamily were performed.
  • cDNAs for U2 and various type receptors were transfected singly or together into COS-1 cells; cells were then incubated with various 125 l-labeled ligands, washed and subjected to cross-linking with a homobifunctional cross-linker. Samples were then analyzed by SDS-gel electrophoresis after immunoprecipitation using antisera to type II or type I receptors. The methods used are described below.
  • cDNAs for type I receptors ten Dijke et al. , Oncogene ⁇ , 2879-2887, 1 993; ten Diike et al.. Science 264. 101 -104, 1 994
  • type II receptors DAF-4 obtained from D.L. Riddle, University of Missouri, Missouri, or U2
  • pSV7d Truett et al.. DNA 4, 333-349, 1985
  • pcDNA3 Invitrogen, San Diego, CA
  • pCMV5 Andersson et al.., J. Biol. Chem. 264,
  • the cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 50 ⁇ g/ml streptomycin) in a 5% CO 2 atmosphere at 37°C.
  • the transfected cells were incubated on ice for 2-3 h with 0.2-0.5 nM of 125 l-labeled ligands in the presence or absence of unlabeled ligands in the binding buffer (phosphate-buffered saline containing 0.9 mM CaCI 2 , 0.49 mM MgCI 2 and 1 mg/ml bovine serum albumin).
  • the cells were washed with the binding buffer without bovine serum albumin and cross-linking was done in the same buffer containing 1 mM bis(sulfosuccinimidyl) suberate (BS 3 ) (Pierce Chemical Co. , Rockford, IL) for 1 5 min on ice.
  • the cells were washed once with 1 0 mM Tris-HCI, pH 7.4, 1 mM EDTA, 10% glycerol and 0.3 mM phenylmethylsulphonyl fluoride.
  • solubilization buffer 1 50 mM NaCI, 20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.3 mM phenylmethylsulphonyl fluoride, 1 .5% Trasylol, 1 % Triton X-100 and 1 % deoxycholate
  • solubilization buffer 1 50 mM NaCI, 20 mM Tris-HCI, pH 7.4, 1 mM EDTA, 0.3 mM phenylmethylsulphonyl fluoride, 1 .5% Trasylol, 1 % Triton X-100 and 1 % deoxycholate
  • the immune complexes were separated by boiling 3 min in SDS-sample buffer with 1 0 m dithiothreitol, and subjected to SDS-gel electrophoresis, followed by analysis usi a Phosphorlmager (Fuji film).
  • Antisera to type I receptors were made against synthetic peptid corresponding to the intracellular juxtamembrane parts of type I receptors previously reported (ten Dijke et al... Science 264, 101 -104, 1 994). Antise against U2 (referred to as SMN and NRR) were generated against peptid corresponding to amino acid residues 1 85-202 and 534-556, respectively. Peptid were coupled to keyhole limpet hemocyanin and injected into rabbits as describ (ten Diike et al... Science 264. 101 -1 04, 1 994). SMN and NRR antisera recogniz
  • Recombinant human OP-1 (Sampath et al.. J. Biol. Chem. 267, 2035 20362, 1 992), TGF-B1 and activin A, were obtained from T. Kuber Sampa (Creative BioMolecules, Hopkington, MA), H. Ohashi (Kirin Brewery Compan
  • the other components may represent oligomer(s) of U2 and type I receptors, or receptors cross-linked to the OP-1 dimer. Similar multiple bands have also been identified when the COS-1 cells were transfected with DAF-4 cDNA together with the corresponding type I receptors (ten Dijke et al.. J.Biol. Chem. 269, 1 6985-1 6988, 1 994). The binding of • 25 l-OP-1 was completed by excess amounts of unlabeled OP-1 , but not by TGF- ⁇ 1 , activin A or GDNF.
  • 125 l-labeled BMPs bind BMPR-IA, BMPR-IB or ActR-l in certain cultured cell lines, including mink lung epithelial (Mvl Lu) cells and U-1 240MG glioblastoma cells (ten Dijke et al. , J. Biol. Chem. 269, 1 6985- 16988, 1 994) .
  • Mvl Lu mink lung epithelial
  • U-1 240MG glioblastoma cells ten Dijke et al. , J. Biol. Chem. 269, 1 6985- 16988, 1 994.
  • the binding of 125 l-OP-1 to receptors endogenously expressed in these cultured cells were studied using antiserum to U2 (NRR), the preparation of which is described above.
  • the U-1 240MG glioblastoma cells (Nister et al.. Cancer Res.
  • Mvl Lu cells (catalogue #CCL-64) were obtained from the American Type Culture Collection (Rockville, MD) . The cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics (100 units/ml penicillin and 50 ug/ml streptomycin) in a 5% CO 2 atmosphere at 37°C.
  • the cells were incubated with 12s l-OP- 1 and treated with a cross-linking reagent as described above. Immunoprecipitation of the cross-linked complexes followed by SDS-gel electrophoresis revealed that type II receptor ⁇ complexes of 1 30 kDa, as well as other components, which may represent co-immunoprecipitated type I receptors and oligomer(s) of type I and/or type II receptors, could b observed in R mutant Mvl Lu cells, which are described in Example 8, and U 1 240MG glioblastoma cells. Experiments with wild type Mvl Lu cells gave simila results as with R mutant cells.
  • EXAMPLE 8 Signaling Activity of U2 The signaling activity of U2 was investigated using a p3TP-Lux promoter reporter construct (Wrana et al.. Cell 7_1_, 1003-1014, 1 992; Attisano et al.. Ce 75. 671 -680, 1 993).
  • R mutant Mvl Lu cells which are highly transfectable an suitable for the p3TP-Lux assay, were used.
  • the R mutant cell line (clone 4- ;Laiho et al.. J. Biol. Chem. 265. 1 851 8-1 8524, 1 990) was created by chemic mutagenesis of the Mvl Lu cell line and was a gift from M.
  • Laiho Universality o
  • the cells were cultured in Dulbecco' modified Eagle's medium containing 10% fetal bovine serum and antibiotics ( 100 units/ml penicillin and 50 ug/ml streptomycin) in 5% CO 2 atmosphere at 37°C.
  • the R mutant Mvl Lu cells were co-transfected with the p3TP-Lux promoter-reporter construct and plasmids containing type II or type I receptor cDNAs as described above. Cells were washed with phosphate-buffered saline on the following day.
  • the cells were starved in Dulbecco's modified Eagle's medium containing 0.1 % fetal bovine serum and antibiotics ( 100 units/ml penicillin and 50 ⁇ g/ml streptomycin) for 6 h and then exposed to various concentrations of OP-1 for 24 h.
  • Luciferase activity in the cell lysate was measured using the luciferase assay system (Promega, Madison, Wl) according to the manufacturer's protocol and a luminometer (model 1250; LKB Pharmacia, Piscataway,NJ).

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Abstract

La présente invention concerne un nouveau récepteur de type II de la superfamille des facteurs de croissance transformants (TGF)-β ayant une affinité de liaison avec une protéine osseuse morphogénétique, la protéine ostéogénique I (OP-1). Le nouveau récepteur de type II a un domaine des sérine/thréonine kinases suivi d'une longue région riche en sérine et en thréonine à l'extrémité C-terminale. L'invention concerne également des acides nucléiques assurant le codage du nouveau récepteur de type II ainsi que des vecteurs et des cellules hôtes exprimant les acides nucléiques et les méthodes de production recombinée qui font appel à ces acides. On décrit par ailleurs des complexes du nouveau récepteur de type II avec un récepteur de type I approprié de la superfamille des TGF-β.
PCT/US1995/016412 1994-12-14 1995-12-14 RECEPTEUR DE TYPE II DE LA SUPERFAMILLE DE TGF-β PRESENTANT UNE AFFINITE DE LIAISON AVEC LA PROTEINE MORPHOGENETIQUE OSSEUSE WO1996018735A2 (fr)

Priority Applications (1)

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AU44249/96A AU4424996A (en) 1994-12-14 1995-12-14 A tgf-beta superfamily type ii receptor having binding affinity

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US35600594A 1994-12-14 1994-12-14
US08/356,005 1994-12-14

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WO1996018735A2 true WO1996018735A2 (fr) 1996-06-20
WO1996018735A3 WO1996018735A3 (fr) 1996-08-29

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999012560A1 (fr) * 1997-09-09 1999-03-18 Creative Biomolecules, Inc. Effets synergiques de morphogenes op/bmp et de facteurs neurotropes gdnf/ngf
US5968752A (en) * 1995-08-14 1999-10-19 Creative Biomolecules, Inc. Method for identifying an OP-1 analog which binds an ALK-1 receptor
US6936582B1 (en) 1997-09-09 2005-08-30 Curis, Inc. Synergistic effects of OP/BMP morphogens and GDNF/NGF neurotrophic factors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014412A2 (fr) * 1994-11-04 1996-05-17 The Procter & Gamble Company Adn complementaire codant pour un recepteur de type ii de proteines morphogenetiques osseuses (bmp)
WO1996014579A1 (fr) * 1994-11-04 1996-05-17 The Procter & Gamble Company Utilisation d'un complexe de recepteurs de proteines bmp destine a cribler des agents actifs du metabolisme osseux, et cellules co-transfectees par un recepteur de type ii et de type i de bmp

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014412A2 (fr) * 1994-11-04 1996-05-17 The Procter & Gamble Company Adn complementaire codant pour un recepteur de type ii de proteines morphogenetiques osseuses (bmp)
WO1996014579A1 (fr) * 1994-11-04 1996-05-17 The Procter & Gamble Company Utilisation d'un complexe de recepteurs de proteines bmp destine a cribler des agents actifs du metabolisme osseux, et cellules co-transfectees par un recepteur de type ii et de type i de bmp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM. , vol. 269, no. 25, 1994, pages 16985-16988, XP002007952 P. TEN DIJKE ET AL.: "Identification of type 1 receptors for osteogenic protein 1 and bone morphogenetic protein 4" *
PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 7632-7636, XP002007953 B. ROSENZWEIG ET AL.: "Cloning and characterisation of a human type II receptor for bone morphogenetic proteins" *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5968752A (en) * 1995-08-14 1999-10-19 Creative Biomolecules, Inc. Method for identifying an OP-1 analog which binds an ALK-1 receptor
WO1999012560A1 (fr) * 1997-09-09 1999-03-18 Creative Biomolecules, Inc. Effets synergiques de morphogenes op/bmp et de facteurs neurotropes gdnf/ngf
US6936582B1 (en) 1997-09-09 2005-08-30 Curis, Inc. Synergistic effects of OP/BMP morphogens and GDNF/NGF neurotrophic factors
US7507709B2 (en) 1997-09-09 2009-03-24 Stryker Corporation Synergistic effects of OP/BMP morphogens and GDNF/NGF neurotrophic factors

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AU4424996A (en) 1996-07-03
WO1996018735A3 (fr) 1996-08-29

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