JPH06500574A - Cloning and recombinant production of receptors of the activin/TGF-β superfamily - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Cloning and recombination of activin/TG-superfamily receptors Production announcement by This invention is made possible by grant numbers HD13527 and D This project was supported by the US government under K26741. The U.S. government has certain rights in this invention. do.

Good skin Danko ■ The present invention provides a receptor protein, a DNA sequence encoding this protein, and its various uses.

1 scallop 1 view Activins (BctivinS) are follicle-stimulating hormone (FS) produced by the pituitary gland. Activins are dimeric tannins that have the ability to stimulate the production of has a common subunit, t, with inhibins. Inhibins are part of FSH inhibit secretion.

Activins include inhibins, transforming growth factor-β (TGF- β), mycolar duct inhibitor, shojo noguko, dekaventaburisi, cupep polypeptides, including some bone morphogenetic proteins and Vg-related peptides. Member 1 of the peptide growth factor superfamily.

As a result of its wide anatomical distribution and multiple biological actions, polypeptides Members of this suit family of growth factors are involved in numerous biological processes. It is thought to be involved in the regulation of stress. For example, activin is found in many tumor cell lines. proliferation, secretion of anterior pituitary hormones (e.g. FSH, GH and ACTH) and regulation of expression, neuronal survival, hypothalamic oxytocin secretion, erythropoiesis, It is involved in steroid production in the placenta and gonads, and early embryonic development.

Activin/TGF-β superfamily of polypeptide growth factors members are involved in the regulation of cell function and cell proliferation in many adult and embryonic cell types. There is. For example, the polypeptide growth factor activin/TGF-β superfactor Cells that are regulated by one or more members of the MMM include mesenchymal cells, muscle cells, skeletal cells, immune cells, hematopoietic cells, steroidogenic cells, endothelial cells, liver cells There are cells, epithelial cells, etc.

Chemical cross-linking studies with many cell types have shown that multiple binding sites (i.e. receptors) suggests that it exists on the surface of cells. However, these receptors structure, or the second receptor signaling that these receptors Little is known about the system. Therefore, these well-characterized It is hoped that the unknown properties of receptor proteins can be better understood. Delicious.

14 days, 1 hours' time With the present invention, we have identified three distinct domains: binding domain, hydrophobic transmembrane domain, and intracellular serine kinase-like activity. A new suite of receptor proteins encompassing a sex-enhancing receptor domain Members of the Parr family have been identified and characterized.

In addition, DNA encoding the above receptor protein and its protein antibodies against such proteins, and bioassays, such proteins and/or antibodies. Body-involving therapeutic compositions and uses thereof are provided.

The DNA of the present invention is a member of the superfamily of receptor proteins of the present invention. As a probe for use in identifying other members and receptors of the invention Codins that can be used for recombinant expression of proteins or functional fragments thereof useful as a tag array. Receptor proteins of the present invention and Antibodies are used to treat disorders such as tumor development, wound healing, immune system deficiencies, reproductive system or central nervous system. Useful for diagnosis and therapeutic treatment of harm.

To 1! The next month FIG. 1 is a schematic diagram of the receptor of the invention and its various domains.

Figure 2 shows receptors of the activin/TGF-β receptor superfamily. An overview of the method used for expression cloning of .

FIG. 3 is a schematic diagram of two mouse activin receptor clones. The top of the figure The upper line is the restriction enzyme map (kb) of zActRl and 1ActR2, The numbering starts from bp 1 of mActR2. The dotted line in the figure indicates mAc The 5' untranslated sequence present only in tRl is shown. The middle line represents two types of activin. A schematic diagram of receptor cDNA clones is shown. Squares indicate coding sequences.

That is, black is the signal peptide, white is the extracellular ligand binding domain, and gray is the transmembrane. The normal domain, and the shaded area is the intracellular kinase domain. Amino acids are shown in the diagram above. Numbered below the formula.

Figure 4 shows activin receptor and daf-1 [putative receptor protein C. elegans (C,el) encoding a kinase (with unknown ligand) egans) Gene HGeorgi et al., Ce11, 61: 635-545 (1990) presents a comparison between ref. activin receptor and daf-1 Conserved residues between are highlighted; conserved kinase domain residues are indicated with an asterisk (*).

Figure 5A shows 125I activin A transfected with pmActRl. The results of binding to COS cells are summarized. Binding was competed with unlabeled activin A . In the experiments reported here, nonspecific binding accounted for 4.6% of the total binding capacity input cpm. 0.9% of input epm, and therefore specific binding was 347% of input cpm. Ta. Data are normalized to 100% and presented as % specific binding. The inserted graph Scatchard analysis of data [Ann, NY Acad , Sci, 51: 680-672 (1979)].

Figure 5B shows 125I activin A transfected with pmActR2. The results of binding to CO3 cells are summarized. As shown in the figure, binding occurs with unlabeled active It competed with Chibin A. In the experiments reported here, the total coupling is 3.4 of the input cps. %, nonspecific binding is 0.9% of input cp+m, and therefore specific binding is input cp+m It was 2.5% of m.

Data are normalized to 100% and presented as % specific binding.

Figure 6 shows how the activin receptor kinase domain interacts with other protein kinases. This is a phylogenetic tree that compares the relationships between To create this phylogenetic tree, a sample of representative sequences was Pitch and Ma Algorithm designed by rgol 1ash [5science, ■, 279 -284 (1967)], performed by Feng and Dooljttle. As [J, Mo1. Evol, ■: 351-360. (1987) ] and calculated. Known subfamilies of kinases are indicated in the figure. Average value of at least 4 standard deviations (compared to all other known kinase sequences). For sequences with similarity scores (i.e., relative sequence identity), activin Percent identity with receptor is shown.

For further details regarding kinase sequences, see Hanks and Quinn , Math, Enzymol, 200: 3g-62 (1991). About.

Mendou love 1 dish shop Gono According to the present invention, the protein has the following domains, read from the N-terminus. A novel superfamily of receptor proteins characterized by Provided with: extracellular glue Gant binding domain.

a hydrophobic transmembrane domain, and Intracellular domain with serine kinase-like activity.

The novel receptor protein of the present invention is further characterized by the amino terminal of the optional 1. Includes a second hydrophobic domain.

As used herein, the term "extracellular, ligand-binding domain" refers to It has a high affinity for (meaning the part of the receptor of the present invention that is located in the surface). As a result of the exposed to all components of the extravesicular medium (see Figure 1).

As used herein, the term "hydrophobic transmembrane domain" (i. refers to the part of the receptor that represents the receptor, and includes the extracellular and intracellular domains of the receptor. Acts as a "bridge" between the channels. Hydrophobicity of this domain (well, receptor - functions to fix the cell membrane to the cell membrane (see Figure 1).

As used herein, the term "intracellular domain with serine kinase-like activity" The term refers to the portion of the receptor of the invention that is located within the cytoplasm, and This part embodies the catalytic functional properties of all the receptors of the present invention (X (See Figure 1).

The optional second hydrophobic domain is located at the ami/terminus of the receptor of the invention and is the most The first receptor to be expressed is a 7-dimensional protein that promotes intracellular movement through the Golgi membrane. (See Figure 1).

Members of the superfamily of receptors of the invention further include polypeptides at least one member of the activin/TGF-β superfamily of growth factors sufficient binding affinity for the polypeptide, e.g. at a concentration of 10 nM or less. a binding site in which the growth factor occupies 50% or more of the binding sites of the receptor protein; It is characterized by having affinity for combination.

Binding affinity (can be expressed by association constant Ka, or dissociation constant Kd) refers to the strength of the interaction between a ligand and a receptor, and refers to the strength of the interaction between a ligand and a receptor. It can be expressed in terms of the density of the gantt required to occupy 1/2 (50%). There is a certain Receptors with high binding affinity for Gund have a low binding affinity (at least 50% requires little ligand to become active (thus, the Kd value is small) (a numerical value); conversely, a receptor has low binding affinity for a given ligand requires the presence of high levels of ligand to reach 50% binding (thus, Kd value is a large value).

If the receptor protein is “less than or equal to lonM (i.e. ≦ 10 nM) concentration of the polypeptide growth factor binds to its receptor protein. occupy ≧50% (i.e., greater than or equal to 1/2) of the site. The term ``having sufficient binding affinity for a ligand'' (i.e., a polypeptide) peptide growth factor) occupies at least 50% of the active site of said receptor. Requires concentrations of no more than about 10 nM for This means that only the highest concentration is required. Preferred receptors in the present invention are The Gand occupies at least 50% of the receptor's binding sites (i.e., all binding sites are binding affinity such that only a concentration in the range of about 100-5009 M is required for It has sex.

Members of the superfamily of receptors of the present invention are radiolabeled active Most of the cross-linked complexes obtained when Tivin is chemically cross-linked to cell extracts. can be classified into various subclasses based on the size [for example, Example 6 below, or Mathews and Vale, Ce1l, 65: 973-982 (1991)].

Type I activin/TGF-β receptors are approximately 65 kD cross-linked with activin. It is a receptor that forms a complex; Type II receptors have approximately 8 It is a receptor that forms a cross-linked complex of O-85kD; also type ■, type Activin receptors such as It is a receptor that forms a bridge complex.

Each member of a subclass shares such records with other members of the same subclass. High homology (e.g., >80% total amino acid homology; often >90% total amino acid homology); members of a certain subclass There is low homology between members of different subclasses and such receptors. (e.g., at least about 30% to 80% total amino acid homology; especially that The enzyme domains have approximately 40% to 90% amino acid homology). typical Generally related receptors have at least 50% total amino acid homology; The enzyme domains have at least about 60% amino acid homology. related receptors preferably have at least 60% total amino acid homology; Both are defined as receptors with approximately 70% amino acid homology. It will be done.

Based on the above criteria, the receptors described herein are Type H receptors. The type H receptor (i.e., mouse-derived receptor tipin receptor) is termed ActRU, while the subsequently identified type H The receptor for ActRI is not a homologue of Although it is clearly related due to homology, it is considered to be a variant of ActRII. ), ActRI[B, ActRIIC, etc. Ru.

Preferred members of the superfamily of receptors of the invention further include inhibitors of It has a greater binding affinity for activins than for activins. It is characterized by Such receptors are also often used in nidorans has substantially no binding affinity for forming growth factor-β, and have substantial binding affinity for non-activin-like proteins or compounds. Not yet.

Another member of the superfamily of receptors of the invention also A lower binding parent for inhibins than for inhibins or TGF-βs. It is characterized by having compatibility.

Another member of the superfamily of receptors of the invention also Higher binding to TGF-β than to inhibins or inhibins It is characterized by having affinity.

As used herein, the term "activin j" refers to activin A (two inohibins). Activin B (homodimer of β^ subunit) (two inhibin βB subunits) , activin AB (one inhibin β subunit, homodimer of and one inhibin βB subunit); The word “inhibin” refers to inhibin A (inhibin alpha subnininoto and inhibin Inhibin B (inhibin β^ subunit), inhibin B (inhibin , and the inhibin βB subunit); "Runforming growth factor beta or TGF-beta" is TGF-beta1 (two TG F-βl subunit homodimer), ‘FGF-β2 (Z TGF-β2 subunits) TGF-β3 (two TGF-β3 subdimers), TGF-β3 (two TGF-β3 subdimers), TGF-β4 (homodimer of two TGF-β4 subunits) , TGF-β5 (homodimer of two TGF-β5 subunits), TGF-β5 12 (one TGF-β1 subunit and one TGF-β2 subunit heterodimer), etc.

Transforming growth factor-β (TGF-β) is a polypeptide growth factor. It is a member of the activin/TGF-β superfamily. TGF-βs has a structure similar to that of activins, and has at least 20-30% amino acid content with them. They have amino acid sequence homology. TGF-βs and activins are Substantially similar distribution pattern of protein residues (or substitutions) throughout the chain I have it. Furthermore, both polypeptides are dimeric species in their active form. .

As used herein, the term "non-activin-like" protein refers to activin (as used herein) any protein that has no essential structural similarity to means.

Preferred members of the superfamily of receptors of the present invention range from about 500 and further each domain thereof has an amino acid of Characterized by having the following dimensions as read from the N-terminus: Extracellular The ligand binding domain preferably has amino acids in the range of about 114-118. Gain, A hydrophobic, transmembrane domain preferably extends from the carboxy terminus of the extracellular domain. starting from about 23-28 amino acids, and The intracellular domain with kinase-like activity is preferably the hydrophobic transmembrane domain. Starting from the carboquine terminus of the protein, it may have a range of about 345-360 amino acids. Ru.

The receptors of the invention further include their most advanced amine terminus (i.e. extracellular linkage). the amine terminus of the Gund-binding domain) with amino acids in the range of approximately 16-30. Optionally includes two hydrophobic domains. This domain is a secretion signal sequence. The sequence helps the receptors of the invention to translocate across cell membranes.

Examples of this secretion signal sequence include amino acids ↓-19 of SEQ ID NO: 1, amino acids ↓-19 of SEQ ID NO: 3, Includes amino acids 1-20, etc. These secretory signal sequences are SEQ ID NO: 1. nucleotides 71-127, nucleotides 468-527 of SEQ ID NO: 3, etc. encoded by a similar nucleic acid sequence.

Members of the receptor superfamily of the present invention may be of various origins, including: can be obtained from For example, pituitary cells, placental cells, hematopoietic cells, brain cells, reproductive cells, liver cells, bone cells, muscle cells, endothelial cells, epithelial cells, counting cells, kidney cells, etc. There is. Such cells can be found in various organisms such as humans, mice, rats, humans, etc. It can be obtained from azaleas, tongues, pigs, frogs, chickens, fish, mink, etc.

A preferred amino acid sequence encoding the protein of the receptor of the present invention is SEQ ID NO: Sequence shown in 2 (indicates the amino acid sequence of mouse activin receptor), SEQ ID NO. Modified version of No. 2, that is, arginine at residue number 39 is replaced with lysine, and residue number 39 is replaced with lysine. Isoleucine at 92 is replaced with valine, and residue number 288 nocultamic acid is substituted with glutamine (modified version of SEQ ID NO: 1 is hereinafter referred to as SEQ ID NO: 1). °”, which represents the amino acid sequence of human activin receptor), and the sequence Sequence shown as number 4 (African frog (Xenopus) activin receptor) (showing the amino acid sequence of Sebuter), and functionally modified forms thereof. Current The manufacturer has determined that a number of residues in the above sequence substantially affect the biological activity of the resulting receptor species. other chemically, sterically and/or electronically similar residues without chemically altering It is recognized that it can be substituted with a group.

According to another embodiment of the invention, a soluble cell further characterized by: External ligand binding proteins are provided: A member of the activin/TGF-β superfamily of polypeptide growth factors a concentration of ≦10 nM of said polypeptide growth factor for at least one member; occupies ≧50% of the binding sites of the receptor protein. have compatibility, and Have at least about 30% sequence identity to the following sequences: Sequence of amino acids 20-134 shown in SEQ ID NO: 2; arginine residue at position number 39 and isoleucine at residue number 92 is replaced with valine. The sequence of amino acids 20-134 shown in SEQ ID NO: 2; or the sequence shown in SEQ ID NO: 4 Sequence of amino acids 21-132.

Preferred soluble extracellular ligand binding proteins according to the invention further include the following: can be characterized by having at least about 50% sequence identity to the sequence : Sequence of amino acids 20-134 shown in SEQ ID NO: 2; Arginine residue at position number 39 group is replaced with lysine, and isoleucine at residue number 92 is replaced with valine. the sequence of amino acids 20-134 shown in SEQ ID NO: 2; or the sequence shown in SEQ ID NO: 4; a sequence of amino acids 21-132; most preferably a soluble extracellular ligand-binding amino acid sequence; The cucumber protein is at least They have approximately 80% sequence identity.

Members of the class of soluble ligand-binding mink chestnuts according to the invention are described above. It can be classified into various subclasses as described above, and here, one subclass ras's men/z+- than against inhibins and/or TGF-βs. May have greater binding affinity for activins; or other subclasses members are more inhibitory than activins and/or TGF-βs. may have a greater than 1 binding affinity for members of the class; Men/zH- of the May have greater binding affinity for GF-βs. one or more subclasses members of the polypeptide growth factor activin/TGF-β superfluid. A mink in the family (this versus the other mink in this superfamily) It is of course clear to those skilled in the art that it may have a greater binding affinity than for It is better understood from this.

Suitable soluble extracellular ligand-binding proteins of the invention are described further below. Characterized by: Has greater binding affinity for activins than for inhipins. to do, having substantially no binding affinity for transforming growth factor β; and Having substantially no binding affinity for non-activin-like proteins.

Suitable soluble extracellular ligand binding proteins of the invention typically have about 11 It includes amino acids ranging from 4-118.

Particularly preferred soluble extracellular ligand binding proteins of the invention have the sequences shown below: is a protein that has substantially the same amino acid sequence as: Residues 20-134 of SEQ ID NO: 2: The arginine residue at position number 39 is replaced with lysine, and the iso residue at residue number 92 is replaced with lysine. Residues 20-134 of SEQ ID NO: 2 in which leucine is replaced with valine; or Residues 21-132° of SEQ ID NO: 4 As used herein, the term "substantially the same amino acid sequence" refers to a reference amino acid sequence. have at least about 80% identity and are encoded by the reference amino acid. Amino acid sequences that can retain functional and biological characteristics comparable to proteins means column. Preferably, proteins having "substantially the same amino acid sequence" are may have at least about 90% amino acid identity to a reference amino acid sequence; preferably have greater than about 95% amino acid sequence identity.

The soluble proteins described above can be used for a variety of therapeutic applications, such as the receptors of the present invention. can be used to prevent other mediators from participating in otherwise mediated processes. of the present invention The presence of soluble protein may compete for functional ligand for the receptor; prevents the formation of a functional receptor-ligand complex, thereby Blocks normal regulatory action.

According to yet another embodiment of the present invention, the above-described soluble protein and receptor Antibodies raised against proteins are provided. Such antibodies have diagnostic uses, therapeutic It can be used for medical purposes. Monoclonal antibodies are preferably used for therapeutic applications. null antibodies can be used.

The above antibodies are produced using the receptor protein of the present invention as an antigen for antibody production. can be prepared using standard methods, as known to those skilled in the art.

In accordance with further embodiments of the present invention, said receptors may be administered a modulating effective amount of said receptors. The transcriptional transactivation of the receptor of the present invention is regulated by contacting the receptor with the body. method is provided.

The soluble proteins of the invention and the antibodies of the invention can be administered, for example, intraperitoneally, intramuscularly, intravenously. Can be administered to patients using standard methods such as intravenous or subcutaneous injection, implantation or transdermal administration . Furthermore, viral or retroviral vectors that feed the compositions of the present invention There are methods such as transfection/yong. Those skilled in the art will understand the administration method used. Therefore, the dosage form, treatment method, etc. can be easily determined.

According to yet another embodiment of the invention, the above soluble protein and receptor A DNA sequence encoding a protein is provided. This would be a NA sequence or The fragment is optionally with easily detectable substituents (e.g. hybridization). can be labeled (used as an oxidation probe).

The receptors described above contain a large number of DNA sequences, such as a contiguous sequence substantially the same as those described below. Can be encoded by a DNA sequence having the nucleotide sequence: Nucleotides 128-1609 of SEQ ID NO: 1 (mouse activin receptor code); Variants of nucleotides 128-1609 of SEQ ID NO: 1, which encode amino acids The codon for acid residue number 39 encodes lysine, and the encoded amino acid residue number Codon number 92 encodes valine, and encoded amino acid residue number 2 88 codons code for glutamine (encodes the human activin receptor) do); Nucleotides 52g-1997 of SEQ ID NO: 3 (, Xenopus activin or encode the same amino acid sequence but with fewer All variants of the above sequence utilize different codons for the amino acids.

As used herein, the term "substantially the same" means that the subject DNA is the same as the DNA fragment being compared. D having at least about 70% homology to the nucleotide sequence of the fragment means NA. "Substantially the same" DNA preferably means that the comparison DNA is may be at least about 80% homologous to a reference nucleotide sequence; particularly preferably about 90% There may be greater homology than homology.

Other DIJAs encoding acceptance of the present invention are substantially the same contiguous nuclei as those described below. DNA having a leotide sequence: nucleotides 71-1609 of SEQ ID NO: 1 (encodes the precursor type of mouse activin receptor); Variants of nucleotides 71-1609 of SEQ ID NO: 1, where the encoded amino acid The codon of residue number 39 of amino acid encodes lysine, and the residue of the amino acid that is hooded Codon number 92 encodes valine, and the encoded amino acid residue number 288 codons hood glutamine (precursor of human activin receptor) Hood your body shape): Nucleotides 468-1997 of SEQ ID NO: 3 (African frog activity) (encoding the precursor form of receptor): or encoding the same amino acid sequence However, all of the above sequences utilize different codons for some amino acids. Mutant.

Still other DNAs encoding the above receptors include SEQ ID NO: 1, SEQ ID NO: 1' or SEQ ID NO: 1'. or having substantially the same contiguous nucleotide sequence as shown in SEQ ID NO: 3. It is DNA.

According to yet another embodiment of the invention, cDNAs encoding receptors are Another sequence encoding a novel receptor of the Tibin/TGF-β superfamily probe a library (e.g., cDNA, genomic, etc.) for a sequence It can be used for.

Such screening is initially performed under conditions of low stringency. These conditions include a temperature below about 42°C and a formamide concentration below about 50%. and moderate to low concentrations of salt. Conditions suitable for such screening is a temperature of about 37°C, a formamide concentration of about 20%, and a concentration of about 5x standard citric acid. Acid solution (SSC + 20X SSC is 3M sodium chloride, 0.3M citric acid solution) Conditions include a salt concentration of 7.0, containing sodium chloride acid pH 7.0. This way conditions that require complete homology to identify stable hybrids. Sequences that exhibit substantial similarity to the probe sequence can be identified without having to do so. "fruit The term "qualitative similarity" means sequences with at least 50% homology. Ru. Preferably, the hybridization conditions provide at least 70% homology with the probe. sequences with lower homology to the probe can be identified and distinguished from sequences with lower homology to the probe. may be selected.

According to yet another embodiment of the invention, the above DNA sequences are expressed in a suitable host cell. Provided are methods for recombinant production of the receptors of the present invention.

The use of a wide variety of recombinant organisms to produce peptides has been disclosed.

Those skilled in the art will know the appropriate hosts (and Expression conditions) can be easily determined. Can be used with yeast hosts, bacterial hosts, mammalian hosts, etc. Wear. The control sequences capable of controlling the expression of the peptides of the present invention include growth conditions under which expression occurs. are known for each of these host systems.

Other embodiments of the invention provide binding assays using the receptors of the invention. Served. This allows large numbers of compounds to be screened quickly and, if one can then determine which compounds are capable of binding to the receptors of the invention. Then, More detailed assays were performed on compounds found to bind and Such compounds may be agonists or antagonists of the receptors of the invention. It can be determined whether a compound acts as a compound.

Other uses of the binding assays of the invention include adding polypeptides to a test sample (e.g., biological fluid). There are members of the activin/TGF-β superfamily of growth factors. This is an assay to test whether or not the Thus, for example, polypeptide growth factors Activin 7' TGF-β superfamily member Serum from individuals exhibiting symptoms related to the route(s) mediated by the , probably caused by overproduction or underproduction of the polypeptide growth factors mentioned above. can be assayed to determine whether

Binding assays according to the invention can be performed in a variety of ways that can be readily identified by those skilled in the art. Ru. For example, competitive binding as well as radioimmunoassay, ELISA, ERMA, etc. Assays can be used.

According to yet another embodiment of the invention, the test compound is an amplifier of the receptors of the invention. Biochemistry to evaluate whether it can act as an agonist and as an agonist. 7 nosei will be provided.

The bioassay of the present invention is characterized in that the test compound is a superfamily of the receptors of the present invention. A functionally modified form of the receptor protein, or a member of the S. This includes evaluating whether it can act as an antagonist or antagonist. Contains. Bioassays to assess whether a test compound can act as an agonist The method includes the following steps: (a) obtaining the receptor protein or the receptor protein; DNA expressing a functionally modified form of the receptor protein and the reporter gene a cell containing DNA encoding a hormone response element operably linked to the offspring; A step of culturing the cells; here, the culturing involves culturing the transcriptional activation activity of the receptor protein. carried out in the presence of at least one compound whose ability to induce (b) monitoring the cell for expression of the product of the reporter gene. .

The test compound binds to a receptor of the invention, or a functionally modified form of said receptor. Bioassays to evaluate whether or not it can act as an antagonist for Includes the following steps: (a) the receptor protein, or the functional DNA expressing the modified version, and a host operably linked to the reporter gene. culturing cells containing DNA encoding a Lumon response element: Culture to measure the ability to inhibit transcriptional activation of said receptor protein. increasing the concentration of at least one compound being targeted; and protein, or a functionally modified form of said receptor protein. carried out with a fixed concentration of at least one agonist; and then (b) determining the expression level of the product of said reporter gene as a function of the concentration of said compound; and monitoring within said cell, thereby inhibiting transcriptional activation of said compound. demonstrate the ability to

Host cells used in the bioassay of the present invention include CV-1 cells, CO3 The reporter and expression plasmid used also typically contains from the 5V-40 origin of replication; and the reporter and expression protein used Lasmids also typically contain a selectable marker.

The hormone response element used in the present invention can be used, for example, in mouse mammary cancer tumors. virus long terminal repeat (MTV LTR), mammalian growth hormone promoter The reporter gene may be selected from chloramphenicyl acetylate Select from Ranfuerase (CAT), Luciferase, β-galactosidase, etc. You can choose.

Cells can be measured using e.g. charge assays [e.g. colorimetric methods (such as β-galactonase (due to colored reporter products), fluorescence (due to reporter products such as luciferase) The expression of the reporter gene can be controlled in various ways, such as by enzyme activity (by product), or by enzyme activity. Expression levels can be monitored.

Compounds screened by the bioassay of the present invention are actipin-like or or TGF-β-like compounds, and activin or TGF-β and certain structural or include compounds that have no biological relevance.

As used herein, the term "activin-like or TFG-β-like compound" α and β chains or βB chains (single or all) of naturally occurring mammalian inhipin amino acid sequences (including combinations), as well as alleles, fragments, and mammalian Has substantially the same qualitative biological activity as activin, activin or TFG-β have substantial homology (at least 20% homology) with homologs or derivatives of It includes substances that Examples of activin-like or TGF-β-like compounds include activin-like or TGF-β-like compounds. Bin A (homodimer of two inbin βq subunits), activin B (two homodimer of inhibin βB subunit), actipin AB (one inhibitor Consisting of a bin β-ro subunit and one inhibin βB subunit inhibin A (inhibin α subunit and inhibin βQ subunit), inhibin A (inhibin α subunit and inhibin βQ subunit), inhibin B (composed of inhibin α subunit and and inhibin βB subunits), TGF-β1 (two TGF - homodimer of β1 subunit), TGF-β2 (two TGF-β2 subunits), TGF-β3 (homodimer of two TGF-β3 subunits), monodimer), TGF-β4 (homodimer of two TGF-β4 subunits), TGF-β5 (a homodimer of two TGF-β5 subunits), TCP-β1 ．． 2 (one TGF-β1 subunit and one TGF-β2 subunit heterodimer), etc.

No specific structural or biological relatedness to activin or TGF-β. However, examples of compounds used for screening by the bioassay of the present invention are , such as alkaloids and other heterocyclic organic compounds. or the effect of the receptor peptide of the present invention. It includes all compounds that can promote

The method used to clone the receptors of the invention involves polypeptide growth. In response to the activin/TGF-β-T4 family of factors, All possible cell types (e.g., pituitary cells, placental cells, fibroblasts, etc.) etc.), expressed in mammalian cells. Next The resulting mammalian cells are then treated with a labeled receptor ligand (i.e., a polypeptide). Labeled activin/TGF-β superfamily of peptide growth factors Measures the ability to bind to the molecule. Finally, insert the desired cDNA insert into , when expressed in mammalian cells, said cells of the labeled receptor ligand. The cDNA is recovered based on its ability to induce (or promote) binding to.

In addition to the above-described uses of the receptor proteins and DNA sequences of the present invention, the present invention The receptor or composition encoding the receptor can be used in a variety of ways.

For example, activin is involved in many biological processes, so activin Activin receptors (or antibodies against activin receptors) are Can be used for process regulation. For example, stimulation of FSFI release by activin (e.g., if a patient receives an increased amount of activin receptors relative to the amount of in vivo receptors) For example, by supplying a patient with a protein that encodes a tissue-specific actipin. can be increased (by transfecting with a construct) or suppressed (e.g. For example, a patient may be given an antibody against the activin receptor, thereby stimulating FSH release. (inhibits the formation of the activin receptor complex, which acts to stimulate the production of activin receptors).

Accordingly, the compositions of the present invention are suitable for the reproductive management of humans, livestock, and commercially important animals. It can be used for.

As another example, the effects of activin on leukocyte and red blood cell mitosis are e.g. For example, activin receptor (activin present in cells that regulates mitosis) administering to the patient (using an appropriate means of administration) a modulating effective dose (which will enhance the patient's It can be adjusted by giving Alternatively, activin receptor ( Antibodies against activin and activin (or a portion thereof) can be administered to the patient; Reduces the effects of activin by blocking the normal interaction between receptors It is possible.

As another example of the wide range of uses of the compositions of the invention, the receptors and/or or antibodies for the diagnosis and/or treatment of actipin-dependent tumors, brain neuron Promoting survival, inducing miscarriages and twin births in livestock and domesticated animals, etc. It can be used in the field of

As yet another example of the wide range of uses of the compositions of the invention, TGF-β specific receptor Agonists identified against TGF-β stimulate wound healing and stimulate TGF-β sensitive tumors. growth inhibition, suppressing the immune response (and thereby preventing rejection of the transplanted organ) It can be used for things like stopping. Antagonist or TGF-β receptor derived The resoluble ligand-binding domain of Promote liver regeneration, and how many.

can be used to stimulate an immune response.

Therefore, the compositions of the present invention have a wide range of diagnostic, clinical, veterinary and research applications. It is easy to understand that

The present invention will be explained in detail by the following examples, but the present invention is not limited to these examples. Not done.

Emperor L Recombinant human (rh) activin ASrh activin B1 and rh inhibin A was generously provided by Genenteeh, Inc. Pig TGF- β1 is! ! +D Obtained from Systems.

Double-stranded DNA was sequenced using 5 strands obtained from US Biochemicals. Performed by dideoxy chain terminus method using equenase reagent. It was. Comparison of DNA sequences with databases was performed using the FASTA program [Pear son and Lipman, Proc, Natl, Acad, Sciυ It was carried out using SA its: 2444-2448 (1988>).

fruit 11shi 2 cDNA library and 1 Polyadenylated RNA was obtained from FastTr obtained from lnVitrogen. Prepared from AtT2O cells using ack reagent. cDNA is commercially synthesized , the plasmid vector p cDNA1, and a library of approximately 5×IG6 initial recombinants was obtained. non-increase The wide cDNA library was plated with 1000 clones per plate in Loomm. then scraped from the plate at 1, frozen in glycerin, and incubated at -70 °C. Saved with.

Activin is present in primary cultures of anterior pituitary cells [Vale et al., Nature 3 21: 776-779 (1986) Co and AtT2O mouse corticotro It suppresses the secretion of adrenocorticotropic hormone (ACT) by both human and human cells. A tT2O cells have activin receptors that are indistinguishable from those of other cell types. receptor (based on binding affinity measurements with activin A). ), these cells were selected as the source of cDNA for transfection. Ta. c DNA library of approximately 5xio6 independent clones derived from AtT2O cells construct into the mammalian expression vector pcDNA1 and perform a single transfection. Expression clone units based on the ability to detect activin binding to cells [Gearing et al., EMBOJ, 83667-3676 (1989 )] was used for screening.

Divide the library into two pools of 1000 clones, each pool of clones. DNA was prepared from the sample and transiently transfected into CO3 cells. The cells were screened for their ability to bind iodinated activin A. connectivity For evaluation, transfection and binding reactions are performed using a microscope equipped with a chamber. performed directly on a slide, then immersing the slide in a photographic emulsion, and This was done by analyzing them under a microscope. With activin receptor cDNA transfected and thus supplemented with radioactive activin. cells are silver particles It was covered with DNA from pools of clones, singly or in groups of three It was analyzed in 300 pools (approximately 300,000 clones) were assayed in this way. Among the three groups, one group was transfected into COS cells. When tested, two positive cells were generated. DNA from each pool of 1000 The positive pool (364) was isolated by transfecting and analyzing alone After identification and further fractionation and transfection, >101 To purify a single clone (p+oActR1) that produces positive cells (see Table 1) .

(Margin below) Table”- Activin receptor clone 11 from AtT20 library 62, 63.64 3X tooo 264-51-R10, 64-5l-C1 32025-40p25-40p>10' can bind 125 Activin A within a field of 2×105 CO3 cells, Calculate the total number of transfected cells and clones for each step of the purification process. The pool was counted.

pmActRl is a 1.7k protein that encodes a 342 amino acid protein. Contains the inserted fragment of b (Fig. 3); was cut and the 3° end was incomplete. Therefore, the last 17 amino acids were encoded by the vector sequence. complete arrangement To obtain the ALT20 library, the 1.6 kb 5acl-Pstl rescreened by hybridization with the fragment (Fig. 3 ).

2.6kb insertion flag by screening 6x10 columns containing the entire coding sequence of the mouse activin receptor. In addition, another positive clone (pmActR2) was obtained (Fig. 3). pIIIAc The nucleic acid sequence and deduced amino acid sequence of the inserted fragment in tR2 are It is shown in number 1.

(Margin below) Fruit "1a" engineering Transfection of COS A portion of the frozen pool of clones was added to Terrific Broth. oth) overnight in 3 ml of medium, and then grown using the alkaline lysis method [Man iatis et al., Molecular Cloning (Cold Spring gHarbor Laboratory (19g2)], 1.5a+1 Mini-prep DNA was produced from. From mini prenobu 1/10 of the DNA (10 Ml out of 100 Ml) was added to each transfection. It was used for

Microscope slide with 20 μg/+1 poly-lysine coated chamber. 2x105 CO3 cells were plated on a cell plate (1 chamber, 1func). and allowed to adhere for at least 3 hours. Cells were treated with DEA as described below. Transfected via E-dextran. 1.5 ml of 100 mM Serum-free Dulbecco's Modified Eagle's Medium (DME) containing chloroquine was added. was added to the cells. 2 containing 500 mg/ml DEAE-dextran Precipitate the DNA in 00 ml of DME/chloroquine and then add to the cells. Ta.

Cells were incubated at 37°C for 4 hours, then the medium was removed and 10% D Cells were treated with HEPE Sii buffered saline containing MSO for 2 minutes. new Media was added and cells were assayed 3 days later. Testing with purified clones Transfection was carried out in 2.5xl with 5μg of purified DNA in 100 μg of dinocytium. G' cells were transfected. Total transfection volume is 10 ml The DNA was then precipitated to 400 ttl.

Real J1 Gai”- In the hindrance, - in the work Cells were incubated twice with HEPES-buffered saline (HDB) containing 0.1% BSA. Wash, then add 0.5 ml of I(DB, 7 Bottle A (approximately 7 ng, 500 pM) in 0.1% BSA at 22 degrees. Incubated for 90 minutes. Cells were then washed three times with cold HDB and 2.5% glucose. Fix in taraldehyde/HDB for 15 min at 22°C and twice in HDB. Washed. Next, strip the chamber from this slide and evaporate the slide to 95%. Dehydrated with alcohol, dried under reduced pressure, and immersed in NTB2 photographic emulsion (manufactured by Kodak). and exposed for 3 days at 4°C in the dark. After developing the emulsion, the slides were 95% etched. Dehydrated with tanol, stained with eosin, mounted with a cover glass, and DPX m auntiant (Electron Microscopy 5science (manufactured by S company). This slide glass was examined in the dark using a Leitz microscope. Analyzed under field illumination.

Fruit] U town soil of the pool We screened 300 pools (each pool containing approximately 1000 cDNAs). One positive pool ($54) that produced two positive cells was identified. It was. Bacteria from a frozen stock of this positive pool ($64) were collected at approximately Re-brate with 400 clones, make replica plates, and DNA was prepared from the pool and analyzed using the binding assay described above.

Several positive subpools were found, these were 4-10 positives per slide. generated cells. Grip the bacteria from the replica plate in one positive subpool. [5science 228:810-81] 5. (1985)], DNA was extracted from a pool of clones representing all columns and rows. Manufactured. One positive column and one positive row identified and clearly a single clone was confirmed, and when the clone was transfected, 2 x 105 cells were obtained. More than 104 positive cells were obtained per sample.

Sangketsu Goi receptor assay 105 transfected with either pmActRl or pmActR2. of CO3 cells or 106 untransfected CO3 cells were cultured in 6 mice. Breeded into El's dish and multiplied overnight. )IDB, 0. The cells were washed twice with 1% BSA and 0.5 ml HDB, 100. OO Ocpm (1 ng, 75 cps) +251 activin A (5 μg activin Bottle A was iodinated by chloramine T oxidation to give a specific activity of 50-90 μCi/μg. The iodinated activin A was purified using a 0.7 x 20 cm G-25 column. 0.1% BSA (approximately 1 ng, 75 pM) containing , incubated for 90 min at 22°C in different amounts of unlabeled competitive hormone. Ta. After binding, cells were washed three times with cold HDB and 0.5 ml of 0.5 N NaOH. Solubilize with Established. The data shown in Figure 5 is expressed as % specific binding; 100% specific binding is Binding in the absence of compound and the presence of a 100-fold molar excess of unlabeled activin A This is the difference from the bond below. The binding parameters are the program LIGAND [Mun son, P. J., and Rodbard, D.

Anal, Biochem, 107:220-259 (1980)] It was determined using

L standing five engineering Others 2x106 COS cells or 5xlO6 AtT20 cells in )IDB. Washed twice and peeled off from Dinongyu, 7xlO5cpm (approx. 500 pM) 500 ng (3 7 nM) with or without unlabeled activin A. Incubate for 90 minutes at 22 degrees with rotation. Transfer the cells to 1 ml of 1 (diluted in DB). The cells were diluted, pelleted by centrifugation, and resuspended in 0.5 ml HDB. vinegar Disuccinimidyl perate (DSS; immediately dissolved in DMSO) was added. 500 μM and cells were incubated at 0 degrees for 30 minutes. crosslinking reaction , 1 ml of 50 mM Tris-HCl pH 75, supplemented with 100 IIM NaCl. The cells were then pelleted by centrifugation and incubated at 100 μm (50 mM). Resuspend in Lys-HCl pH 7,5,1% Triton X-100 and Incubate for 60 minutes at 13.o”c. Centrifuge the sample for 5 minutes at 13. and the Triton-soluble supernatant was run on an 8.5% polyacrylamide gel. Analyzed by 5DS-PAGE. Dry the gel and autoradiate for 4-14 days. I applied it to the graph.

Bloodline Yuuko ■E Mushi Nitarioka Total RNI was purified from cultured cells and tissues using the LiCl precipitation method.

20 μg of total RNA was added to 1.2% agar 1-ose, 2.2M formaldehyde. The gel was run on a nylon membrane (Hybond.

(manufactured by NEN), and then 0.6kb with 32p mark by random brining using reagents from ls. was hybridized with the Kpnl fragment (see Figure 3). hybrid Dization was carried out in 50% formaldehyde at 42°C and the filter Washed in 0.2X SSC at 65°C.

Five supporting pillars [Gokyujo The full-length mouse activin receptor clone contains a 70 bp 5° untranslated region. encodes a 513 amino acid protein with a 951 bp 3° untranslated region do. pmActR2 does not contain a polyA tail, but there is a latent polyA tail at bp 2251. It definitely has a unique polyadenylation site. Insertion within clone pIIIActR1 The fragment contains an additional 551 bp of 5' untranslated sequences, which are identical within the overlap. are the same, and the 3° end ends at base position 1132 of pmActR2.

The first methionine codon (ATG) at bp 71 in pmActR2 is the translational opener. It is in a favorable condition at the beginning [Kozak, M, Nucl, Ac1ds R es, 15:8125-8148 (1987)], and one inflation It is located immediately after the stop codon. pmActRl has 3 in the 5゛ untranslated region contains two different ATGs; however, none of these are suitable for initiation. None, and all of these are followed by an in-frame stop codon. child Even if the unusually long 5' leader sequence of It is clearly not necessary for this purpose. Because pIII, which lacks most of its sequence, This is because ActR2 can be functionally expressed in CO3 cells (see below). .

Kyte and Doolittle [J, Mo1. Biol, 157. 105 -132. In hydrophilic analysis using the method of 19821, two hydrophobic regions were diamified. a stretch of 10 amino acids, presumed to be a single peptide, at the A single membrane spanning region, putatively consisting of 26 residues in the acid 119-142 region, was found. (see FIG. 1 and SEQ ID NO: 2). This signal peptide is co-located with the signal sequence. Contains the same conserved n-1h- and C-domains; The cleavage site before Ala' of Tido is determined by the rule described by von Heljne [Bi ochim, Biophys, Act, 947:307-333. (1 98g)]. On the cytoplasmic side of the membrane spanning domain As is often the case, the predicted transmembrane region has two basic amino acids immediately adjacent to it. It follows. Therefore, the mature mouse activin receptor has a 116 amino acid N Terminal extracellular ligand binding domain and 346 amino acid intracellular signaling domain A 494-amino acid type I membrane protein with Mr. 54 kDa and is expected.

By comparing the activin receptor sequence with sequence databases, many receptors have been identified. found structural similarities with the intracellular domains of receptor and non-receptor kinases. . Analysis of the sequences of all kinases reveals that they contain many highly conserved amino acids. The 300 amino acid kinase domain is characterized by 12 subdomains. Confirmed [Hanks, S., and. u4nn, A, M, (7), M eth, Enzymol, 200:38-62 (1991), and Han ks et al., 5science 241:42-52 (1988)]; The receptor sequence has all of these conserved subdomains in the correct order. (Figure 4). sub domain! The conserved Gly in the activin receptor In the tar, this residue is substituted with A l a l 9 G, but this residue is It is also observed in ze. Therefore, based on structural relevance, this receptor is functionally It is predicted to be a sexual protein kinase.

The sequences within two of these subdomains (vlB and ■) are tyrosine versus serine halide. It can be used to predict leonine substrate specificity [Hanks et al., supra (1988)].

The sequence of the mouse activin receptor contains selinkinase in these subtomains. Therefore, the activin receptor has serine-haleonine specificity. It is predicted that the Furthermore, the activin receptor has subdomains ■ and ■. It has no tyrosine residue in the canonical autophosphorylated region between the canonical tyronone residues This shows that it is not a naze. This receptor is 5er333 or 7 It is potentially possible to autophosphorylate at hr337. Another one that's interesting The possibility that activin receptor kinase This means that it can have specificity for syn residues. I want to have these characteristics Several kinases have been recently described [eg Howell et al., Mo1. C. e11. Biol.

11+568-572 (1991); 5tern et al., Mo1. Ce11. B iol, ll:987-1001 (1991); and Feathersto nd, C., and Ru5sell, P., Nature 349:808 -811 (1991)].

Phylogenetic analysis comparing activin receptor with 161 other kinase sequences 7cutin receptor and C. elegans protein daf-1 [G eorgi et al., Ce1l 61:635-645 (1990)]. (See Figure 6). daf-1 is , a putative transmembrane receptor involved in the arrest of differentiation in the larval state; It shares 32% homology with activin receptor (see Figure 6). Aku Similar to the tibin receptor, daf-1 is a serine/threonine-specific transmembrane kinase. Moreover, both daf and 7 cutin receptors are predicted to be Subdomains VIA-VIB and X-XI ( There is a short conserved sequence within the kinase domain between (underlined in Figure 4B) Contains an insert. Another similarity is that these are unique subfamilies of kinases. It confirms that it belongs to Millie. Actipin receptors are found in corn The only other known enzyme encoded by the ZIIIPK gene of Transmembrane serine/threonine protein kinase [Walker, J., C., O. and Zhang, R., Nature 345 Near 43-746 (199 0)] has a very low relationship (18% amino acid sequence identity).

The extracellular domain of the activin receptor does not match any other sequence in the database. They showed no similarity. This ligand-binding domain is a receptor for other growth factors. Although relatively small compared to domains found in these receptors, 7 stain content is high. However, the pattern of these Cys residues is The folding structure of Robulin or the continuous repetition of EGF receptor It does not resemble any of the other parts. Also, N-linked glycosylation in the extracellular domain two potential sites, and protein kinase C and casei in the intracellular domain. There are many potential phosphorylation sites for Kinase H.

Real] 1self”- Mucloning of the cloned activin receptor/Sebuter To confirm activin specificity, pmActRl or p+a Competitive binding experiments were performed in CO3 cells transiently transfected with ActR2. Ta. Cells transfected with either construct produced a single high-affinity d = 180 pM; Figure 5), function The (structurally intact) intracellular kinase domain is not required for Gant binding. It was shown that This binding affinity has been measured with other activin-responsive cell types. consistent with the defined binding affinity [e.g. Campen, C, A, et al. and Vale, W., Biochem, Biophys, Res, Co1n , 15 Uni 844-849 (1988); Hino et al., J. Biol, Ch. ew, 264:10309-10314 (1989); Sugino et al., J , Biol, Chet 263:15249-15252 (1988) ; and Kondo et al., Biochem, Biophys, Res, Comm , Month u: 12S7-1272 (1989)].Untreated CO3 cells were Does not bind to cutin A.

In total, this transfected culture had approximately 26,000 receptors per cell. However, only 15% of the cells expressed the transfected gene. (transfected as a fraction in whole cells after immersion in emulsion) (measured by cell quantification), each transfected cell had an average of 1 75,000 receptors were expressed. Expression levels per cell were accumulated Although the value is based on the number of silver particles, it varies considerably. This value is This value is comparable to that of other transfected cell surface proteins.

Iodination on CO3 cells transiently transfected with pmActR2 Activin A binds to activin B, which has a slightly lower ability than activin A. Inhibin A is about 10 times lower in its ability to compete; As a result, TGF-β1 could not compete. The affinity of this bond specificity and specificity between activin A and many other activin-responsive cell types. Consistent with what was observed after binding. Activin B was added to the transfected receptor. The receptor appears to bind with lower affinity than activin A, so The activin Bg product used in these experiments was compared in biological activity to activin A. There is a possibility that the ability will decrease. This is because the activin used herein This is because the recombinant synthesis of activin B was carried out a long time ago [recombinant synthesis of activin B]. The synthesis was carried out by Mason et al. in Mo1. Endocrinol, 暉135 2-1358 (1989)]. This cDNA exists in multiple forms. 7 appears to encode a receptor for cutin.

The size of the cloned activin receptor was determined using a difunctional chemical crosslinker. Transfection with pmActR2 using nosuccinimidyl phosphate (DSS) Affinity cross-linking of 1251 activin A to the infected CO3 cells was performed and analyzed. Ta. 84 kDa cross-linked major band in transfected cells but not in non-transfected cells. Acti Subtracting the molecular weight of the bottle (and showing that it is a 56 kDa protein, which is The molecular weight was in good agreement with the predicted molecular weight from the nucleic acid sequence data. 125I activin A When cross-linked to yf:AtT2O cells, a major band of 65 kDa and a Minor bands of 78 kDa and 84 kDa were obtained. this biggest band The size of the band corresponds to that occurring in the cloned receptor.

The two small bands are different proteins, i.e. different phosphorylations of the same protein. form, or a degradation product of the full-length clone; at amino acid position 35 The DKKRR sequence and the KKKR sequence at amino acid position 416 have a high proteolytic potential. It can be an existing site.

Alternatively, these bands may result from differently spliced products of the same gene. It's possible in the future.

The cross-linked bands of 84 kDa and 65 kDa are similar to those of other activin-responsive cells. It has also been observed in species [(Hino, supra; Centrella et al., Mol.

Ce11. Biol, 11:250-258 (1991)], and Sig. Although interpreted to represent a nulling receptor, other sized complexes are recognized as well. I can't stand it. The size of the actipin receptor is similar to that of the putative FGF-β receptor. are very similar and are characterized to some extent by chemical cross-linking reactions [Massag ue et al., Ann, N.Y., Acad, Sci., Issue: 59-72. (19 90)].

Real J1 Egami”- activin receptor-zRNA The distribution of activin receptor mRNA was analyzed using Northern Blonometry. 6.0k b and 3. Two mRNA species of Okb include brain, jujube, pancreas, liver and kidney Observed in numerous tissues of mice and AtT2O cells. pmActRl and p The total size of the mActR2-derived insert fragments is 3.1 kb, which is small. corresponds to the size of the transcript. The degree of similarity between these two mRNAs also The meaning of having two transcripts is also not clear. some other hormone receptors -genes are spliced differently to allow soluble binding to cell surface receptors. The activin receptor has been shown to produce both proteins. It is possible that they are processed in a similar manner.

Interestingly, the relative abundance of these two transcripts varies depending on their origin. A tT2O cells have approximately equal levels of both mRNAs, but most tissues have much greater levels of the 6.0 kb transcript; 3. Okb transcription The body exhibits little or no expression. On the other hand, Suimaru has a large amount of 3. Okb van has a code. Activin receptor mRNA expression in the brain, liver, and Suimaru , consistent with the described biological effects of activin in these tissues [Min et al., Endocrinol, 125+586-591 (1989); Vale et al., Peptide Growth Factors and The ir Receptors, Handbook of Experimenta l Pharmacology, M, A, 5porn and A, B, Ro berts (ed.), Springer-Verlag (1990), in press).

Four U Human activin receptor - No. 5 Human lightning library (purchased from Clontech; catalog number: HLl olob) under the following conditions: the full-length mouse activin receptor gene (sequence (see number 1).

Hybridization stringency: 20% formamide, 6X S SC, 42°C; Sequence highly homologous to mouse activin receptor was identified Sequence number 1°). Homology between this receptor and mouse activin receptor This receptor is a subclass of the same mouse receptor as its own mouse. This is called the human form of the activin receptor.

African frog stage 17 embryo cDNA library (Kintner et al. and Melton, Development 99+ 311-325 (1 (prepared as described in 987)) was grown as a full-length mouse active under the following conditions. The probe was probed with the gene for the protein receptor (see SEQ ID NO: 1).

Hybridization stringency 20% formamide, 6X Stringency of SSC, 42°C: 2X SSC, 0.1% SDS, 42℃ against mouse activin receptor Sequences with a significant degree of homology were to be identified (SEQ ID NO: 3). of total amino acids The degree of homology (to mouse activin receptor) is approximately 69% (intracellular domain). The ins have a degree of homology of 77% and the extracellular domain only has a degree of homology of 58%). . The degree of homology between this receptor and the mouse activin receptor is moderate. Therefore, we put this receptor in a subclass different from the mouse receptor mentioned above. It is called activin receptor.

Kojaku 1ju 1'd Assay of ActR in African frog 1 x AetRI IB is active To examine whether xActRII transduces/signal in response to cutin. B. RNA was synthesized in vitro and injected into Africa at two different concentrations. injected into embryos.

The injected embryos were allowed to develop to stage 9, at which point the animal cap was removed and ,different! Treated with one dose of activin overnight.

This xActRIIB cDNA was cloned into rp64T [Kreig and Melton, Methods in Enzymology, Abe lson and Simonm collection, (Academic Press, New York, 1987), vol, 155. See p. 397], Then, it is transcribed in vitro to generate synthetic xActR11B RNA with a cap structure. [Melton et al., Nucleic Ac1ds Res, 12 Near 0 35, (1984) and Kintner, Neuron 1:545 (1 98g)].

0.02 ng/nl or 0.1 ng/nl concentration for embryos at cell stage 2-4. Approximately 20 nl of RNA at a time was injected into the embryos throughout all four quadrants of the animal ball.

At step 9, the animal cap is removed from the RNA-injected embryo and placed in a 0.5X modified mammalian lid. gel solution (MMR), purified bovine serum albumin (BSA) at different concentrations, pig of activin A (6 kanoka per incubation). . After culturing for 20 hours, total RNA was prepared.

The response of the cap to 7 cutin was determined by [Blackwell and 1 Vein Traub, 5science 250:1104 (1990)] Accordingly, muscle-specific actin RNA was quantified in a bonuclease protection assay. It was evaluated. 0.4ng and 2. Inject Ong's xActRIIB RNA. The embryos were approximately 10 and 100 times more sensitive to activin than control embryos, respectively. It was a feeling. Small amounts found in animal caps not injected with activin A Muscle actin is probably caused by contamination by animal caps that have small amounts of marginal layer tissue. This is the cause.

Increased activin concentration in embryos injected with 2 ng of xActRIIB RNA The amount of muscle actin decreased. This applies uniformly to different concentrations of activin. Isolated animal cap cells respond to a narrow concentration range of activin in muscle tissue. The observation that only cells [B1aek++ann and Kadesch, Genes and Development 5:105) (1990)] matches. This result indicates that both the concentration of ligand and the amount of receptor are transmitted. shown to be important for determining the signal. Therefore, muscle differentiation A range of activin concentrations that result in more expressed receptors than normal lower in animal cap cells from injected embryos than in non-injected embryos.

L-shiki five-story construction mActRII kinase cD corresponding to the entire intracellular domain of mActRll (amino acids 143-494) The fragment of NA was subcloned into the vector pGEX-2T [Sm 1th and Johnson, Gene 67:31-40 (198g). ], putative glutathione S-) transferase (GST) and receptor A fusion protein with a kinase domain was created. Introduce this plasmid into bacteria. The resulting fusion protein was published by Sm1th and Johnson. It was purified using glutathione affinity chromatography. about 10 0-200 nH of fusion protein or purified GST in 50mM Tris, 1 with 25 μCi of [γ-32P]ATP in a buffer containing 0 mM MgCb. Then, the cells were incubated at 37°C for 30 minutes. The product was analyzed by 5DS-PAGE and auto Analyzed by radiography. This fusion protein, rather than GST alone, oxidized, indicating that the kinase domain of the fusion protein was functional. C According to the method of Ooper et al. [Meth, Enzym, 99:387 (1983)] Therefore, the analysis of phosphoamino acids performed mainly focused on amino acid residues that are phosphorylated. The group was shown to be threonine.

Although the invention has been described in detail with reference to certain preferred embodiments thereof, modifications thereof It is understood that modifications and variations are within the spirit and scope of the invention as described and claimed. I want to be understood.

[Sword o1 toe SEQ ID NO: 1 is a cDN encoding the mouse-derived activin receptor of the present invention. The nucleic acid sequence (and its deduced amino acid sequence) of A.

SEQ ID NO: 1° is a nucleic acid sequence encoding the human-derived activin receptor of the present invention. It is a column. SEQ ID NO: 1° shows that the codon for amino acid residue 39 immediately hoods lysine. nucleotides 185-187 are AAA or AAG), amino acid residues 92 codons are valine hoods, i.e. nucleotides 344-346 are GTN , where N is A%CSG, or T), and amino acid residue 288 codon encodes glutamine (i.e., nucleotides 932-934 are CA A or CAG).

SEQ ID NO: 2 is the deduced amino acid sequence of the mouse-derived actipin receptor of the present invention. It is.

SEQ ID NO: 2° is the amino acid sequence of the human-derived activin receptor of the present invention. Ru. In SEQ ID NO: 2', amino acid residue 39 is lysine and amino acid residue 92 is lysine. phosphorus, and amino acid residue 288 is glutamine. It is essentially the same as SEQ ID NO:2.

SEQ ID NO: 3 encodes the activin receptor from African frog of the present invention. This is the nucleic acid sequence (and its deduced amino acid sequence) of the cDNA that encodes the protein.

SEQ ID NO: 4 is a putative activin receptor derived from African frog of the present invention. It is an amino acid sequence.

SEQ ID NO: 5 is the amino acid of the VIB subdomain of the serine kinase consensus sequence. This is a noic acid sequence.

SEQ ID NO: 6 is the '/II+ subdomain of the serine kinase consensus sequence. It is an amino acid sequence.

SEQ ID NO: 7 is the amino acid sequence of the VIB subdomain of the activin receptor of the present invention. It is an array.

SEQ ID NO: 8 is Vll! of the activin receptor of the present invention. subdomain amino It is an acid sequence.

SEQ ID NO: 9 is the accession of the VIB subdomain of the tyrosine kinase consensus sequence. It is a amino acid sequence.

SEQ ID NO: IO is the VII+ subdomain of the tyrosine kinase consensus sequence This is the amino acid sequence of

Sequence list (1) General information (i) Applicants: Manoyous, BH, D1. Lawrence varnish, pale , BH, D, l Wiley D'Abriny.

(ii) Name of the invention Niactivin/TGF-β superfamily receptor Cloning and recombinant production of (iii) Number of arrays: 10 (iv) Correspondence address: (A) Address: Pretty, Schroeder, Prusogeman and Clark (B) Address: South Flower Street 444 Suite 2000゜ (C) City: Los Angeles (D) State: California (E) Country: United States of America (F) Postal code + 90071-2971 (V) Computer readout format :(A) Media type: Fronoby disc (B) Computer: IBM PC compatible (C) Operation system: PC-D OS/MS-DOS(D) software: Patentln Re1ease #1. OS version $1.25 (vi> Data of this application: (A) Application number: US (B) Application date: May 8, 1992 (C) Classification: (viii) Agent/office information: (A) Name: Niraita-1 Mr.-1, Stephen Yi.

(B) Registration number: 31192 (C) Inquiry/record number: P319309/FP319291 (ix) Telephone line information: (A) Episode 11: (619) 546-4737 (B) Fax: (619) 546-9392 (2) Information on sequence number = 1: (1) Array characteristics: (A) Length: 2563 base pairs (B) type: Nucleic acid (C) Number of strands/single strand (D) Topology: linear (ii) Type of molecule: cDNA (ix) Features: (A) Symbol representing characteristics: CD5 (B) Location: 71. ．． 1609 (xi) Sequence: Sequence number: 1: CT mouth CG entered cc entered AG 8 to CCAGGG 8 80τCGATATC’J%GC GAC, input λCT? CC-DingACGGCTfuCτCCCGC■@60 ? CAA 3 people CC'T'CC entering ＾acτ entering AGCk entering ＾C-,G (?rA ) Ding GAT -- τCfu CτC, TG input TGAG”FλG near 09 (Mu'bow margin) (2) Information on sequence number 2: (i) Array wait? r1= <A) Long E: 51s amino acid (B) type diamino acid (D) Toboronow bilinear (Type of molecule: protein (χyu) Sequence/Sequence number 2: Mat Gly Ala Ala Lym Lau Ala Pha Ala　νJLI　Pha　lllu　A111　Bar　O7m Yu S 10 15 Ser　5＠r　Gly　81&　2l＊　Leu　C1y　enter rg　Sir　 Glu Thx Gin Glu Cys Lau PhePh・8sn A R input sn Trp Glu Arg 8sp Arg Thr Asn G engineering n Thr Gly Val GluPro Cym Tyr Gly Asp Lys Asp Lys Arg Enter rq His Cys Phs Enter 1a Thr　τrpso　ss　b.

Lye A11n D1a 5er Gly Bar: Yuka Glu Xla νNaoyu Lys Gin Gly Cyll Trp 2■■ Asp Asp Engineering L* Asn cyts Tyr Asp Enter xq Thr Amp Cym Xla lu Lys Lye As■ Sar　Pro　Glu　￥ Crystal Tyr　Ph5e　Cys　Cym　Cym　 Gikl　Gly　kjIn　Mat　Cys　11nG繧t Yu05 Uni0 Pro Val Thr P2. LyII Pro Pro Tyr Tyr A11ll Xla L, au Leu Tyr Sur@Lmu Yu 30 135 140 VA engineering Pro Lau H life t Leu Xla Ala Gly engineering 1m Val 11m Cys Ala Pha Trp val Yu45 A50 1 55 160 ? yr Arg HLx Hlt Lyw Mat AIJL Tyr Pro Pro Val Lmu Val Pro Thr Gi■ Yu 65 Near 0 175 Person-p Pro Gly Pro Pro Pro Bar Pro Lau Lau Gly Lau Lye Pro L*uyu80 185 1 90 Gin Lau Lau Glu Van Lys Ala enter rg Gly Arg Pha Gly Cys Val Trp Lys Yu95 2o Ro 2 05 Asp Lys Gin Sar Trp Gin Asn Glu Tyr C1u Val Tyr Sir Lau Pro GlyM@t Lym H Lm Glu Asn rle Lau Glr+ Pha XLa to Ay Artificial a (Lu Ly-human rgG engineering y?hr 5@r Val A-p Val xsp Lmu Trp Lsu XLea Dinghr AL breath Phe HL m　GLu　Ly■ Gly　5@r　Lau　Sar　8-p　Ph　Lau　Lys　Ala　 ^-n Van Val 9r Ding+7 Asn GluL*u Cys Hi s Xla Ala Glu Thr Met 71a Arg Gly La u Ala Tyr Lau HLmGlu Asp Xh Kai Pro C1y 　Lau　Lys　入sp　aly　）lis　Lys　Pro　A1　锜工 i M! ;sr HP Person z9 Amp: le Lye Bar Lye sn Val Lau Lau　Ly-Asn　8an　Lau　Thr　1a325　33o　33 5 Cys xlII entering 1a Asp Pha GLy Lau entering IJL La u Lym Pha Glu 8 la Gly Lys 5≠■ 340 ko45 350 People n G17 Amp Thr HLs Gly G engineering Val Gly Thr Enter r9 Person rg Tyr M・℃ Enter 1a Pr mouth Glu Val L ・u Glu C1y Enter 1a Engineering 1e Asn Phe Gin ^x9 8 sp 8 la Pha Leu ArgCys Thr λ1 & 1a s p cly Pro Vil Enter Hp C1u Tyr Met Lsu Pr o Phe Glu405 4 engineering 0 4 u 5 Glu　C1u　ffle　Gly　Gyun　J(ij　Bar　La u Glv Amp Met G engineering n Glu Val V≠P Val HLs Lys Lye Lys Arg Pro Val Lau Enter r9 people sp Tyr Trp Gin Lyg HLs4ko5 440 44 5 Ala (:ly Mat Ala Met Lmu Cys Glu Thr :le Glu Glu Cym Ding rp enter sp HL■ Human sea P 8la Glu entering 1a entering rg Lsu 5ar Ala C1y Cys Val Gly Glu 8rg Eng 1e ThrC Yun Mat G ll'l krq LalJ Thr Ajn h Yukai zle Thr Th r Glu Amp Engineering @VJII@τhr Val V 1 Thr Mat Val hr Asn Val enter sp l’he Pro Pro Lye Glu 5@r 5e■ (2) News about sequence number 3: (1> Characteristics of array: (A) Long mini 2335 base pairs (B) type: Nucleic acid (Number of Cun i Sada 2 - Unchained (D) Topo: G: Straight chain (11) Molecule type: cDNA (Origin of vi + N Nao wo Concubine: (B) Ric-7 people + X, ACTll (1x) Features: (A) Symbol representing characteristics: CD5 (B) Location: 46g, 1997 (xl) Sequence: SEQ ID NO: 3゜ GTCAAGαuλga; C’TGCAG 2335 (2) Information on SEQ ID NO: 4 (1) Array characteristics: (A) Length: 510 amino acids (B) type amino acid (D) Toborono: Straight triangular shape (11) Molecule type: protein (Xl) Sequence, SEQ ID NO: 4 set Gly A1a 5ar Val Ala L@u Thr Phe Leu Lau Lau Ala Thr Phe 5 Yu O2s Enter r9 Person 1a Gly Sar Gly HLts Asp Glu Val C1u Thr Arg Glu Cyts Engineering 1e T 凾■ ? 1Gin Cys Glu Lys Glu :la Phe Thr Th r　Pro　Gly　Met　Lys　His　GluAsn　Lau　L@u Glu Pha X1a Ala enter La Glu Lys Arg Gly 5er A++n Leu Gl■ Ribs Glu L@u Trp Lau Ila Thr Ala Pha H Lm xsp LYll Gly Sac Lau Thr8sp Tyr L au Lyg Gly Asn Leu Val Sar Trp Asn G lu　Lau　Cys　HLs　:1eτhr　Glu　Thr　Met　Al a Arg Gly Leu Enter La Tyr Leu His Glu As p Val F mouth Arg Cys Lys Gly GLu Gly HLm Lys Pro Ala Xis Xl Todoroki HLm Arg Asp Pha 305 3yu 0 315 320 Lys Sur Lys Asn Val Lau Lau Enter rg Asn 8sp Lau Thr Ala Engineering 14 Lau Ala325 330 = 35 ”y=Enter 1a Mat Gly Lau VaL L@u Trp Glu E* Val Sac ＾rgCy-Thr 1 piece Aim Amp Gly Pro VaL sp GLu Tyr Lau Lau Pro Ph・ Glu Glu Glu l1a405 4yu0 415 Gly Gin His Pro Ssr Lau Glu Asp Lau (:ln　Glu　Val　Val　Val　14is　Lㇾ■ LyII Ee enter rg E’ro Vayo Pha Lys ＾lip H Lm:rp Lau Lye HLm Pr mouth Gly kau Person 1a Gin LJu Cys VaL Dinghr rL@Glu clu CY@Trp 8sp His input-p input 1mu Glu artificial field Arg La u 5・r Enter λa GLY Cys Val Glu Glu Enter r9 Engineering 1 mSa! ″ ni Gin engineering @ k nitrogen■ 465 470 475 41i10 LY-5@r Val Asn Gly Thr Thr Sar 8ap C ys Lau Val Sir 工’L@Val dinghrSar Val ding hr Asn Val input sp Leu Pro Pro Lys Glu S ur 5er engineering 1esoo SO5510 (Shito Chikura, b) Sequence number: 5 DLKPEN Sequence number 6 G(T/5)XX(Y/F)X Sequence number, 7 IKSKN Sequence number, 8 TRRYM Sequence number, 9 L.A.A.R.N. Sequence number: 10 XP (1/V) (K/R) W (T/M) FIGURE 2 FIGtlu 3 nM Fushichi Nephew F engineering GUλ35 ψ FICllRE 6 Country@Investigation Report , -PCT/11592103825 International Survey Report ・-1-m-・・- Continuation of front page (51) Int, C1,5 identification symbol Internal office reference number A61K 39/395 ADU D 9284-4CC12N 15/10 C12P 21102 ZNA C8214-4B21108 8214-4B C12Q 1/68 A 7823-4B // A61K 37102 AAB 8314-4CGOIN 33153 D 8310-2J (81) Designated country EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), AU, CA, J.P. I

Claims (31)

[Claims] 1. A novel receptor protein, read from the N-terminus of the protein. The following domains are: extracellular ligand-binding region; a hydrophobic transmembrane region, and an intracellular receptor region with serine kinase-like activity; A protein characterized by having 2. 2. The method according to claim 1, further comprising a second hydrophobic domain at its amino terminus. protein on the plate. 3. The protein further comprises a polypeptide growth factor activin/TGF- against at least one member of the β superfamily at a concentration of 10 nM or less. The peptide growth factor occupies 50% or more of the binding sites of the receptor protein. according to claim 1, characterized in that it has a binding affinity sufficient to protein on the plate. 4. The protein further includes: greater binding affinity for activin than for inhibin to have, Has substantial binding affinity for transforming growth factor-β that there is no, and Virtually no binding affinity for non-activin-like proteins , 4. Protein according to claim 3, characterized by: 5. Substantially the same as that described in SEQ ID NO: 2, SEQ ID NO: 2', or SEQ ID NO: 4 The protein according to claim 1, having the amino acid sequence. 6. In addition, the activin/TGF-β superfamily of polypeptide growth factors of the polypeptide growth factor at a concentration of 10 nM or less for at least one of the occupies 50% or more of the binding sites on the receptor protein. and The sequence of amino acids 20-134 shown in SEQ ID NO: 2; the arginine residue at position 39 is A sequence in which lysine is substituted, and isoleucine at position 92 is substituted with valine. the sequence of amino acids 20-134 set forth in number 2; or at least about 30 for the sequence of amino acids 21-132 set forth in SEQ ID NO: 4. A soluble, extracellular, ligand characterized by having % sequence identity binding protein. 7. Furthermore, there is a greater binding affinity for activin than for inhibin. binding affinity for transforming growth factor-β. have virtually no identity; and Virtually no binding affinity for non-activin-like proteins , 7. Protein according to claim 6, characterized by: 8. 6. The protein has amino acids in the range of about 114-118. Proteins described in. 9. A DNA encoding the mature protein according to claim 1. 10. A DNA encoding the mature protein according to claim 3. 11. DHA encoding a precursor-form of the protein of claim 1. 12. DNA encoding the protein according to claim 2. 13. A DNA encoding the soluble protein according to claim 6. 14. A DNA encoding the soluble protein according to claim 8. 15. A DNA encoding a precursor form of the protein of claim 6. 16. Nucleotides at positions 128-1609 of SEQ ID NO: 1; encoded amino acids The codon of residue number 39 encodes lysine, and the residue number of the encoded amino acid is 92 codons code for valine and encoded amino acid residue number 28 Nucleus at positions 128-1609 of SEQ ID NO: 1, where codon 8 encodes glutamine Mutants of leotide; Nucleotides at positions 528-1997 of SEQ ID NO: 3; or the same amino acids as the above sequence encodes an acid sequence but contains different codons for some amino acids , variants of all the above sequences, 10. The DNA of claim 9, having substantially the same contiguous nucleotide sequence. 17. Nucleotides at positions 71-1609 of SEQ ID NO: 1; encoded amino acid The codon for residue number 39 encodes lysine, and the encoded amino acid residue number 9 Codon 2 encodes valine and encoded amino acid residue number 288 The nucleo at positions 71-1609 of SEQ ID NO: 1, in which the codon encodes glutamine Chido mutant; Nucleotides at positions 468-1997 of SEQ ID NO: 3; or the same amino acids as the above sequence encodes an acid sequence but contains different codons for some amino acids , variants of all the above sequences, 10. The DNA of claim 9, having substantially the same contiguous nucleotide sequence. 18. Substantially the same as that described in SEQ ID NO: 1, SEQ ID NO: 1' or SEQ ID NO: 3 The DNA according to claim 9, having the same contiguous nucleotide sequences. 19. Nucleotides at positions 71-127 of SEQ ID NO: 1 or 468 of SEQ ID NO: 3 14. Having substantially the same flanking sequence as the nucleotide at position -527. DNA. 20. A single method comprising expressing the DNA of claim 9 in a suitable host cell. or methods for recombinant production of multiple activin receptors. 21. expressing the DNA of claim 13 in a suitable host cell. A method for recombinant production of one or more soluble activin receptors. 22. A DNA fragment useful as a hybridization probe. hand, the DNA fragment comprises at least a portion of the DNA according to claim 9; and the DNA fragment is labeled with an easily detectable substituent, DNA fragment. 23. The easily detectable substituent may be a radiolabeled molecule, a fluorescent molecule, an enzyme, etc. 23. A DNA fragment according to claim 22, selected from , or a ligand. 24. It encodes a receptor of the activin/TGF-β superfamily. 1. A method for identifying clones with low stringency hybridization. claim 2, wherein the genomic library or cDNA library is screening with the DNA fragment described in 2; and Clones showing a high degree of hybridization to the DNA fragment the process of identifying How to include. 25. binds to the receptors of the activin/TGF-β superfamily A method of screening sets of these compounds to identify compounds. and the step of using the receptor according to claim 1 in a competitive binding assay. , Method. 26. The compound comprises one or more receptor proteins according to claim 1. is an agonist of the receptor protein(s). A bioassay for evaluating whether a functionally modified form is a functionally modified form, comprising: (a) a single the number or plurality of said receptor proteins or the number or plurality of said receptor proteins; DNA expressing a functionally modified form of the protein protein, and Encodes a hormone response element operably linked to a reporter gene DNA to a step of culturing cells containing Here, the culture has the same ability to induce transcriptional activation activity of the receptor protein. carried out in the presence of at least one compound believed to have been After the, (b) monitoring the cell for expression of the reporter gene; Bioassays encompassing. 27. The compound comprises one or more receptor proteins according to claim 1. is an agonist or functions of the receptor protein(s) A bioassay for evaluating whether a modified form of a single or a plurality of said receptor proteins or one or more said receptors. - DNA expressing a functionally modified form of the protein, and Encodes a hormone response element operably linked to a reporter gene DNA to a step of culturing cells containing Here, the culture: The ability to inhibit the transcriptional activity of one or more of the receptor proteins has been identified. in the presence of increased concentrations of at least one compound believed to be number or plurality of said receptor proteins, or one or more said receptor proteins; a constant concentration of at least one agonist of a functionally modified form of a target protein; carried out in the presence of And then, (b) determining the expression level of the product of said reporter gene as a function of the concentration of said compound; Intracellularly, monitoring shows the ability of the compound to inhibit activation of transcription. A bioassay comprising a process. 28. Regulating transcriptional transactivation of one or more activin receptors 7. A method of: controlling said receptor in an effective modulating amount of the protein of claim 6. a step of bringing it into contact with the material; How to include. 29. 7. An antibody raised against the protein of claim 6. 30. 30. The antibody of claim 29, wherein said antibody is a monoclonal antibody. 31. 30. Contacting said receptor with an amount of said antibody of claim 29 that provides effective modulation. transcriptional receptor of one or more activin receptors, including the step of exposing one or more activin receptors to How to modulate ence activation.