AU654724B2 - Cloning and recombinant production of receptor(s) of the activin/TGF-beta superfamily - Google Patents

Description

OPI, DATE 30/12/92 AOJP DATE 11/02/93 APPLN. 10 19945/92 PCT NUMBER PCT/US92/03825 IIlllllllllll I1lllllII AU9219945 IN ITENAIIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 International Publication Number: WO 92/20793 C12N 15/12, C07: 15/00 C12Q 1/68, G01N 33/53 Al C12N 15/62, A61K 37/02 (43) International Publication Date: 26 November 1992 (26.11.92) C12P 21/08, A61K 39/395 (21) International Application Number: PCT/US92/03825 (74) Agent: REITER, Stephen, Pretty, Schroeder, Brueggemann Clark, 444 South Flower Street, Suite 2000, Los (22) International Filing Date: 8 May 1992 (08,05.92) Angeles, CA 90071 (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro 698,709 10 May 1991 (10.05.91) US pean patent), CA, CH (European patent), DE (Euro- 773,229 9 October 1991 (09.10.91) US pean patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent,. GR (European patent), IT (European patent), JP, LU (Euro- (71) Applicant: THE SALK INSTITUTE FOR BIOLOGICAL pean patent), MC (European patent), NL (European pa- STUDIES [US/US]; 10010 North Torrey Pines Road, La tent), SE (European patent).

Jolla, CA 92037 (US), (72) Inventors: MATHEWS, Lawrence, S. 3705 Coconino Published Court, San Diego, CA 92177 VALE, Wylie, W. With international search report.

1643 Valdez, La Jolla, CA 92037 Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

654724 (54)Title: CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTIVIN/TGF-P SUPER-

FAMILY

C-terminus N-terminus kinase domain Intracellular domain &econd hydrophobic domain Ligand-binding domain Transmembrane domain (57) Abstract In accordance with the present invention, there are provided novel receptor proteins characterized by having the following domains, reading from the N-terminal end of said protein: an extracellular, ligand-binding domain, a hydrophobic, trans-membrane domain, and an intracellular, receptor domain having serine kinase-like activity. The invention receptors optionally further comprise a second hydrophobic domain at the amino terminus thereof. The invention receptor proteins are further characterized by having sufficient binding affinity for at least one member of the activin/TGF-P superfamily of polypeptide growti factors such that concentrations of 10 nM of said polypeptide growth factor occupy 50 of the binding sites of said receptor protein. A presently preferred member of the invention superfamily of receptors binds specifically to activins, in preference to inhibins, transforming growth factor-, and other non-activin-like proteins, DNA sequences encoding such receptors, assays employing same, as well as antibodies derived therefrom, are also disclosed.

WO 92/20793 PCT/US92/03825 -1- CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTIVIN/TGF-3 SUPERFAMILY

ACKNOWLEDGEMENT

This invention was made with Government support under Grant Numbers HD 13527 and DK 26741, awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to receptor proteins, DNA sequences encuding same, and various uses therefor.

BACKGROUND OF THE INVENTION Activins are dimeric proteins which have the ability to stimulate the production of follicle stimulating hormone (FSH) by the pituitary gland. Activins share a common subunil: with inhibins, which inhibit FSH secretion.

Activins are members of a superfamily of polypeptide growth factors which includes the inhibins, the transforming growth factors-B (TGF-B), Mullerian duct inhibiting substance, the Drosophila decapentaplegic peptide, several bone Porphogenetic proteins, and the Vg-related peptides.

VO 92/20793 PcT/US92/03825 -2- As a result of their extensive anatomical distribution and multiple biological actions, members of this superfamily of polypeptide growth factors are believed to be involved in the regulation of numerous biological processes. Activin, for example, is involved in the proliferation of many tumor cell lines, the control of secretion and expression of the anterior pituitary hormones FSH, GH and ACTH), neuron survival, hypothalamic oxytocin secretion, erythropoiesis, placental and gonadal steroidogenesis, early embryonic development, and the like.

Other members of the activin/TGF-B superfamily of polypeptide growth factors are involved in the regulation of cell function and cell proliferation for numerous cell types, in adults and embryos. For example, cells which are subject to regulation by one or more members of the activin/TGF-B superfamily of polypeptide growth factors include mesenchymal cells, muscle cells, skeletal cells, immune cells, hematopoietic cells, steroidogenic cells, endothelial cells, liver cells, epithelial cells, and the like.

Chemical cross-linking studies with a number of cell types suggests that multiple binding sites receptors) exist on the surface of cells. However, little is known about the structure of these receptors, or about the second messenger signalling systems that they employ.

It would be desirable, therefore, if the nature of these poorly characterized receptor proteins could be more fully understood.

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, we have identified and characterized members of a new superfamily of receptor proteins which comprise three distinct domains: an extracellular, ligand-binding domain, a hydrophobic, at ta tIlt a..

trans-membrane domain, and an intracellular, receptor domain having serine kinase-like activity.

Also provided are DNAs encoding the above-described receptor proteins, and antibodies thereto, as well as bioassays, therapeutic compositions containing such proteins and/or antibodies, and applications thereof.

The DNAs of the invention are useful as probes for the identification of additional members of the invention superfamily of receptor proteins, and as coding sequences which can be used for the recombinant expression of the invention receptor proteins, or functional fragments thereof. The invention receptor proteins, and antibodies thereto, are useful for the diagnosis and therapeutic management of carcinogenesis, wound healing, disorders of the immune, reproductive, or central nervous systems, and the like.

According to the first embodiment of this invention there is provided a novel receptor protein characterised by having the following domains, reading from the Nterminal end of said protein: 1i an extracellular, ligand-binding domain, a hydrophobic, trans-membrane domain, and an intracellular, receptor domain having serine kinase-like activity, wherein said protein is further characterised by having sufficient binding affinity for at least one member of the activin/TGF-P superfamily of polypeptide growth factors such that concentrations of 10 nM of said polypeptide growth factor occupy 2 50% of the binding sites of said receptor protein.

According to a second embodiment of this invention there is provided a protein having an amino acid sequence substantially the same as set forth in Sequence ID No. 2, Sequence ID No. or Sequence ID No. 4.

According to a third embodiment of this invention there is provided a soluble, exiracellular, ligand-binding protein, characterised by: having a sufficient binding affinity for at least one member of the activin/TGF-0 superfamily of polypeptide growth, factors such that concentrations of 5 10 nM of said polypeptide growth factor occupy 50% of the binding sites on said receptor protein, and 30 having at least about 30% sequence identity with respect to: the sequence of amino acids 20-134 set forth in Sequence ID No. 2; the sequence of amino acids 20-134 set forth in Sequence ID No. 2, wherein the arginine residue at position number 39 is replaced by a lysine, and te isoleucine at residue number 92 is replaced by a valine; or the sequence of amino acids 21-132 set forth in Sequence ID No. 4.

According to a fourth embodiment of this invention there is provided a DNA encoding a protein according to any one of the preceding embodiments.

According to a fifth embodiment of this invention there is provided a DNA Sencoding a precursor-for of the protein of any one of first to third embodiments.

a N:\LIBRR100197:IAR/GSi. o 7 According to a sixth embodiment of this invention there is provided a method for the recombinant production of activin receptor protein(s), said method comprising expressing the DNA of the fourth embodiment in a suitable host cell.

According to an seventh embodiment of this invention there is provided a DNA fragment useful as a hybridisation probe, wherein said DNA fragment comprises at least a portion of the DNA according to the fourth embodiment, and wherein said DNA fragment is labelled with a readily detectable substituent.

According to a eighth embodiment of this invention there is provided a method to identify clones encoding receptors of the activin/TGF-3 superfamily, said method comprising: screening a genomic or cDNA library with a DNA fragment according to the seventh embodiment under low stringency hybridisation conditions, and identifying those clones which display a substantial degree of hybridisation to said DNA fragment.

According to a ninth embodiment of this invention there is provided a method for screening a collection of compounds to determine those compounds which bind to receptors of the activin/TGF-3 superfamily, said method comprising employing the receptor of the first embodiment in a competitive binding assay.

According to an tenth embodiment of this invention there is provided a bioassay for S: 20 evaluating whether compounds are agonists for receptor protein(s) according to the first embodiment, or functional modified forms of said receptor protein(s), said bioassay comprising: culturing cells containing: DNA which expresses said receptor protein(s) or functional modified forms of said receptor protein(s), and DNA encoding a hormone response element operatively linked to a reporter gene, wherein said culturing is carried out in the presence of at least one compound whose ability to induce transcription activation activity of said receptor protein is sought to be 30 determined; and thereafter monitoring said cells for expression of said reporter gene.

According to a eleventh embodiment of this invention there is provided a bioassay I t for evaluating whether compounds are antagonists for receptor protein(s) according to the first embodiment, or functional modified forms of said receptor protein(s), said bioassay comprising: culturing cells containing: DNA which expresses said receptor protein(s), or functional modified forms of said receptor protein(s), and [N:LIBRR00197:IARIGSA a o 7 DNA encoding a hormone response element operatively linked to a reporter gene; wherein said culturing is carried out in the presence of: increasing concentrations of at least one compound whose ability to inhibit transcription activation of said receptor protein(s) is sought to be determined, and a fixed concentration of at least one agonist for said receptor protein(s), or functional modified forms of said receptor protein(s); and thereafter monitoring in said cells the level of expression of the product of said reporter gene as a function of the concentration of said compound, thereby indicating the ability of o said compound to inhibit activation of transcription.

According to a twelfth embodiment of this invention there is provided an antibody generated against the protein of the third embodiment.

According to thirteenth embodiment of this invention there is provided a method for modulating the transcription trans-activation of activin receptor(s), said method comprising: contacting said receptor with an effective, modulating amount of the protein of the third embodiment or the antibody of the twelfth embodiment.

Brief Description of the Figures Figure 1 is a schematic diagram of receptors of the invention and the various domains thereof.

Figure 2 outlines the strategy used for expression cloning of a receptor of the activin/TGF-P receptor superfamily.

Figure 3 is a schematic of two mouse activin receptor clones. The top line of the figure is a restriction map, in kb, of mActR1 and mActR2, with numbering starting from bp 1 of mActR2. The dotted line in the figure represents 5' untranslated sequences *I present only in mActR1. The middle lines present a schematic representation of two activin receptor cDNA clones. Boxes represent coding sequences black is the signal peptide, white is the extracellular ligand-binding domain, grey is tN:\LIERRIO197:IAIGSA b of 7 WO 92/20793 PCT/US92/03825 -4the transmembrane, and the intracellular kinase domain is hatched. Amino acids are numbered beneath the schematics.

Figure 4 presents a comparison between activin receptor and daf-i [a C. elegans gene encoding a putative receptor protein kinase (with unknown ligand); see Georgi, et al., Cell 61: 635-645 (1990)]. Conserved residues between the activin receptor and daf-l are highlighted; conserved kinase domain residues are designated with an Figure 5A summarizes results of 12I activin A binding to COS cells transfected with pmActRl. Binding was competed with unlabeled activin A. For the runs reported herein, total binding was 4.6% of input cpm, non-specific binding was 0.9% of input cpm, and therefore the specific binding was 3.7% of input cpm. Data are shown as specific binding, normalized to 100%. The inset presents a Scatchard analysis of the data [Ann. NY Acad. Sci. 51: 660-672 (1979)].

Figure 5B summarizes results of 1 25 activin A binding to COS cells transfected with pmActR2. Binding was competed with unlabeled factors as indicated in the figure.

For the runs reported herein, total binding was 3.4% of input cpm, non-specific binding was 0.9% of input cpm, and therefore the specific binding was 2.5% of input cpm. Data are shown as specific binding, normalized to 100%.

Figure 6 is a phylogenetic tree, comparing the relationship of the activin receptor kinase domain to other protein kinases. To construct the tree, the catalytic domains of representative sequences were empirically aligned and evolutionary relatedness was calcU. ed using an algorithm designed by Fitch and Margoliash [Science 155: 279-284 (1967)], as implemented by Feng and Doolittle [J.

Mol. Evol. 25: 351-360 (1987)]. Known subfamilies of WO 92/20793 PCT/US92/03825 kinases are indicated in the figure. For those sequences that had similarity scores a relative sequence identity) of at least 4 standard deviations above the mean (in comparison with all other known kinase sequences), the percent identity with the activin receptor is indicated.

For further detail on kinase sequences, the reader is referred to Hanks and Quinn, Meth. Enzymol. 200: 38-62 (1991).

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, there is provided a novel superfamily of receptor protein(s) characterized by having the following domains, reading from the N-terminal end of said protein: an extracellular, ligand-binding domain, a hydrophobic, trans-..iembrane domain, and an intracellular domain having serine kinase-like activity.

The novel receptor protein(s) of the invention optionally further comprise a second hydrophobic domain at the amino terminus thereof.

As employed herein, the phrase "extracellular, ligand-binding domain" refers to that portion of receptors of the invention which has a high affinity for ligand, and which, when associated with a cell, resides primarily outside of the cell membrane. Because of its location, this domain is not exposed to the processing machinery present within the cell, but is exposed to all components of the extracellular medium. See Figure 1.

As employed herein, the phrase "hydrophobic, trans-membrane domain" refers to that portion of receptors of the invention which traverses the cell membrane, and serves as a "bridge" between the extracellular and WO 92/20793 PCT/US92/03825 -6intracellular domains of the receptor. The hydrophobic nature of this domain serves to anchor the receptor to the cell membrane. See Figure 1.

As employed herein, the phrase '~uairacellular domain having serine kinase-like activity" refers to that portion of receptors of the invention which resides within the cytoplasm, and which embodies the catalytic functionality characteristic of all receptors of the invention. See Fig 1.

The optional second hydrophobic domain, positioned at the amino terminus of receptors of the invention, comprises a secretion signal sequence which promotes the intracellular transport of the initially expressed receptor protein across the Golgi membrane. See Figure 1.

Members of the invention superfamily of receptors can be further characterized as having sufficient binding affinity for at least one member of the activin/TGF-8 superfamily of polypeptide growth factors such that concentrations of 5 10 nM of said polypeptide growth factor occupy 50% of the binding sites of said receptor protein.

Binding affinity (which can be expressed in terms of association constants, Ka, or dissociation constants, Kd) refers to the strength of interaction between ligand and receptor, and can be expressed in terms of the concentration of ligand necessary to occupy one-half of the binding sites of the receptor. A receptor having a high binding affinity for a given ligand will require the presence of very little ligand to become at least 50% bound (hence the Kd value will be a small number); conversely, receptor having a low binding affinity for a given ligand will require the presence of high levels of ligand to become 50% bound (hence the Kd value will be a large WO 92/20793 PCT/US92/03825 -7number).

Reference to receptor protein "having sufficient binding affinity such that concentrations of said polypeptide growth factor less than or equal to 10 nM 10 nM) occupy 50% greater than or equal to one-half) of the binding sites of said receptor protein" means that ligand polypeptide growth factor) concentration(s) of no greater than about 10 nM are required in order for the ligand to occupy at least 50% of the active sites of said receptor, with much lower ligand concentrations typically being required. Presently preferred receptors of the present invention have a binding affinity such that ligand concentration(s) in the range of only about 100 500 pM are required in order to occupy (or bind to) at least 50% of the receptor binding sites.

Members of the invention superfamily of receptors can be divided into various subclasses, based on the approximate size of the crosslinked complexes obtained when radiolabeled activin is chemically crosslinked to cell extracts [see, for example, Example VI below, or Mathews and Vale in Cell 65:973-982 (1991)]. Type I activin/TGF-f receptors are those which form a crosslinked complex of about 65 kD with activin; Type II receptors are those which form a crosslinked complex of about 80-85 kD with activin; while Type III, Type IV and the like receptors are those which form crosslinked complexes with activin having molecular weights greater than about 100 kD.

Each member of a given subclass is related to other members of the same subclass by the high degree of homology >80% overall amino acid homology; frequently having >90% overall amino acid homology) between such receptors; whereas members of a given subclass differ from members of a different subclass by the lower degree of homology at least about 30% up to 80% overall amino WO 92/20793 PCT/US92/03825 -8acid homology; with in the range of about 40% up to ami.no acid homology specifically in the kinase domains thereof) between such receptors. Typically, related receptors have at least 50% overall amino acid homology; with at least about 60% amino acid homology in the kinase domains thereof. Preferably, related receptors are defined as those which have at least 60% overall amino acid homology; with at least about 70% amino acid homology in the kinase domains thereof.

Based on the above criteria, the receptors described herein are designated Type II receptors, with the first discovered Type II receptor the mouse-derived activin receptor) being designated ActRII, while subsequently identified Type II receptors which are not homologs of ActRII (because while clearly related by size and some sequence homology, they differ sufficiently to be considered as variants of ActRII), are designated ActRIIB, ActRIIC, etc.

Presently preferred members of the invention superfamily of receptors are further characterized by having a greater binding affinity for activins than for inhibins. Such receptors are frequently also observed to have: substantially no binding affinity for transforming growth factors-B, and substantially no binding affinity for non-activin-like proteins or compounds.

Additional members of the invention superfamily of receptors are further characterized by having a greater binding affinity for inhibins than for activins or TGF-Bs.

Additional members of the invention superfamily of receptors are further characterized by having a greater binding affinity for TGF-Bs than for activins or inhibins.

WO 92/20793 PCT/US92/0382S -9- As employed herein, "activin" refers to activin A (a homodimer of two inhibin BA subunits), activin B (a homodimer of two inhibin BB subunits), activin AB (a heterodimer composed of one inhibin BA subunit and one inhibin BB subunit); "inhibin" refers to inhibin A (composed of the inhibin a subunit and an inhibin BA subunit), inhibin B (composed of the inhibin a subunit and an inhibin BB subunit); "transforming growth factor B or TGF-B" refers to TGF-B1 (a homodimer of two TGF-B1 subunits), TGF-B2 (a homodimer of two TGF-B2 subunits), TGF-83 (a homodimer of two TGF-B3 subunits), TGF-B4 (a homodimer of two TGF-84 subunits), TGF-B5 (a homodimer of two TGF-B5 subunits), TGF-B1.2 (a heterodimer of one TGF-B1 subunit and one TGF-B2 subunit), and the like.

Transforming growth factors-6 (TGF-s) are members of the activin/TGF-f superfamily of polypeptide growth factors. TGF-Bs are structurally related to activins, sharing at least 20-30% amino acid sequence homology therewith. TGF-Bs and activins have a substantially similar distribution pattern of cysteine residues (or substitution) throughout the peptide chain.

Furthermore, both polypeptides, in their active forms, are dimeric species.

As employed herein, the term "non-activin-like" proteins refers to any protein having essentially no structural similarity with activins (as defined broadly herein).

Preferred members of the invention superfamily of receptors comprise those having in the range of about 500 amino acids, and are further characterized by having the following designated sizes for each of the domains thereof, reading from the N-terminal end of said receptor: the extracellular, ligand-binding domain preferably will have in the range of about 114-118 WO 92/20793 PCT/US92/03825 amino acids, the hydrophobic, trans-membrane domain preferably will have in the range of about 23-28 amino acids, beginning at the carboxy terminus of the extracellular domain, and the intracellular domain having kinase-like :ictivity preferably will have in the range of about 345-360 amino acids, beginning at the carboxy terminus of the hydrophobic, trans-membrane domain.

Receptors of the invention optionally further comprise a second hydrophobic domain having in the range of about 16-30 amino acids at the extreme amino terminus thereof at the amino terminus of the extracellular, ligand-binding domain). This domain is a secretion signal sequence, which aids the transport of invention receptor(s) across the cell membrane. Exemplary secretion signal sequences include amino acids 1-19 of Sequence ID No. 1, amino acids 1-20 of Sequence ID No. 3, and the like. Such secretion signal sequences can be encoded by such nucleic acid sequences as nucleotides 71-127 of Sequence ID No. 1, nucleotides 468-527 of Sequence ID No. 3, and the like.

Members of the invention superfamily of receptors can be obtained from a variety of sources, such as, for example, pituitary cells, placental cells, hematopoietic cells, brain cells, gonadal cells, liver cells, bone cells, muscle cells, endothelial cells, epithelial cells, mesenchymal cells, kidney cells, and the like. Such cells can be derived from a variety of organisms, such as, for example, human, mouse, rat, ovine, bovine, porcine, frog, chicken, fish, mink, and the like.

Presently preferred amino acid sequences encoding receptor proteins of the invention include the sequence set forth in Sequence XD No. 2 (which represents a mouse activin receptor amilno acid sequence), a modified form of WO 92/20793 PCT/US92/03825 -11- Sequence ID No. 2 wherein the arginine at residue number 39 is replaced by a lysine, the isoleucine at residue number 92 is replaced by a valine, and the glutamic acid at residue number 288 is replaced by a glutamine (which modified form of Sequence ID No. 1 is referred to hereinafter as "Sequence ID No. and represents a human activin receptor amino acid sequence), and the sequence set forth as Sequence ID No. 4 (which represents a Xenopus activin receptor amino acid sequence), as well as functional, modified forms thereof. Those of skill in the art recognize that numerous residues of the above-described sequences can be substituted with other, chemically, sterically and/or electronically similar residues without substantially altering the biological activity of the reialting receptor species.

In accordance with another embodiment of the present invention, there is provided a soluble, extracellular, ligand-binding protein, further characterized by: having sufficient binding affinity for at least one member of the activin/TGF-B superfamily of polypeptide growth factors such that concentrations of 10 nM of said polypeptide growth factor occupy 50% of the binding sites on said receptor protein, and having at least about 30% sequence identity with respect to: th, sequence of amino acids 20-134 set forth in Sequence ID No. 2; the sequence of amino acids 20-134 set forth in Sequence ID No. 2, wherein the arginine residue at position number 39 is replaced by a lysine, and the isoleucine at residue number 92 is replaced by a valine; or the sequence of amino acids 21-132 set forth in Sequence ID No. 4.

WO 92/20793 PCT/US92/03825 -12- Presently preferred soluble, extracellular, ligand-binding proteins contemplated by the present invention can be further characterized by having at least about 50% sequence identity with respect to: the sequence of amino acids 20-134 set forth in Sequence ID No. 2; the sequence of amino acids 20-134 set forth in Sequence ID No. 2, wherein the arginine residue at position number 39 is replaced by a lysine, and the isoleucine at residue number 92 is replaced by a valine; or the sequence of amino acids 21-132 set forth in Sequence ID No. 4; with the presently most preferred soluble, extracellular, ligand-bjnding proteins having at least about 80% sequence identity with respect to the above-referenced fragmen.ts of Sequence ID Nos. 2 or 4 Members of the class of soluble, ligand-binding proteins contemplated by the present invention may be divided into various subclasses, as previously described, wherein members of one subclass may have a greater binding affinity for activins than for inhibins and/or TGF-Bs; or alternatively, members of another subclass may have a greater binding affinity for inhibins than for activins and/or TGF-Bs; or alternatively, members of yet another subclass may have a greater binding affinity for TGF-Bs than for activins and/or inhibins. It is, of course, understood by those of skill in the art, that members of more than one subclass may have a greater binding affinity for one member of the activin/TGF-B superfamily of polypeptide growth factors, relative to other members of the superfamily.

Presently preferred soluble, extracellular, licgand-binding proteins of the present invention are further characterized by: WO 92/20793 PCT/US92/03825 -13having a greater binding affinity for activins than for inhibins, having substantially no binding affinity for transforming growth factors-B, and having substantially no binding affinity for non-activin-like proteins.

Presently preferred soluble, extracellular, ligand-binding proteins of the present invention typically comprise in the range of about 114-118 amino acids.

Especially preferred soluble, extracellular, ligarnl-binding proteins of the invention are those having substantially the same amino acid sequence as that set forth as: residues 20-134 of Sequence ID No. 2; residues 20-134 of Sequence ID No. 2, wherein the arginine residue at position number 39 is replaced by a lysine, and the isoleucine at residue number 92 is replaced by a valine; or residues 21-132 of Sequence ID No. 4.

As employed herein, the term "substantially the same amino acid sequence" refers to amino acid sequences having at least about 80% identity with respect to the reference amino acid sequence, and will retain comparable functional and biological properties characteristic of the protein encoded by the reference amino acid. Preferably, proteins having "substantially the same amino acid sequence" will have at least about 90% amino acid identity with respect to the reference amino acid sequence; with greater than about 95% amino acid sequence identity being especially preferred.

The above-described soluble proteins can be employed for a variety of therapeutic uses, to block receptors of the invention from affecting processes which WO 92/20793 PCT/US92/03825 -14the receptors would otherwise mediate. The presence of the soluble proteins of the invention will compete with functional ligand for the receptor, preventing the formation of a functional receptor-l.Tand complex, thereby blocking the normal regulatory actioi of the complex.

In accordance with yet another embodiment of the present invention, there are provided antibodies generated against the above-described soluble proteins and receptor proteins. Such antibodies can be employed for diagnostic applications, therapeutic applications, and the like.

Preferably, for therapeutic applications, the antibodies employed will be monoclonal antibodies.

The above-described antibodies can be prepared employing standard techniques, as are well known to those of skill in the art, using the invention receptor proteins as antigens for antibody production.

In accordance with still another embodiment of the present invention, there are provided methods for modulating the transcription trans-activation of receptor(s) of the invention by contacting said receptor(s) with a modulating, effective amount of the above-described antibodies.

The soluble proteins of the invention, and the antibodies of the invention, can be administered to a subject employing standard methods, such as, for example, by intraperitoneal, intramuscular, intravenous, or subcutaneous injection, implant or transdermal modes of administration, and the like. In addition, methods such as transfection with viral or retroviral vectors encoding the invention compositions. One of skill in the art can readily determine dose forms, treatment regiments, etc, depending on the mode of administration employed.

'WO 92/20793 PCT/US92/03825 In accordance with a further embodiment of the present invention, there are provided DNA sequences which encode the above-described soluble proteins and receptor proteins. Optionally, such DNA sequences, or fragments thereof, can be labeled with a readily detectable substituent (to be used, for example, as a hybridization probe).

The above-described receptor(s) can be encoded by numerous DNA sequences, a DNA sequence having a contiguous nucleotide sequence substantially the same as: nucleotides 128 1609 of Sequence ID No. 1 (which encodes a mouse activin receptor); variations of nucleotides 128 1609 of Sequence ID No. 1, wherein the codon for residue number 39 of the encoded amino acid codes for lysine, the codon for residue number 92 of the encoded amino acid codes for valine, and the codon for residue number 288 of the encoded amino acid encodes glutamine (which encodes a human activin receptor); nucleotides 528 1997 of Sequence ID No. 3 (which encodes a Xenopus activin receptor); or variations of any of the above sequences which encode the same amino acid sequences, but employ different codons for some of the amino acids.

As employed herein, the term "substantially the same as" refers to DNA having at least about 70% homology with respect to the nucleotide sequence of the DNA fragment with which subject DNA is being compared. Preferably, DNA "substantially the same as" a comparative DNA will be at least about 80% homologous to the comparative nucleotide sequence; with greater than about 90% homology being especially preferred.

Another DNA which encodes a receptor of the invention is one having a contiguous nucleotide sequence WO 92/20793 PCT/US92/03825 -16substantially the same as: nucleotides 71 1609 of Sequence ID No. 1 (which encodes a precursor-form of a mouse activin receptor); variations of nucleotides 71 1609 of Sequence ID No. 1, wherein the codon for residue number 39 of the encoded amino acid codes for lysine, the codon for residue number 92 of the encoded amino acid codes for valine, and the codon for residue number 288 of the encoded amino acid encodes glutamine (which encodes a precursor-form of a human activin receptor); nucleotides 468 1997 of Sequence ID No. 3 (which encodes a precursor form of a Xenopus activin receptor); or variations of any of the above sequences which encode the same amino acid sequences, but employ different codons for some of the amino acids.

Yet another DNA which encodes the above-described receptor is one having a contiguous nucleotide sequence substantially the same as set forth in Sequence ID No. 1, Sequence ID No. 1' or Sequence ID No. 3.

In accordance with a further embodiment of the present invention, the receptor-encoding cDNAs can be employed to probe library(ies) cDNA, genomic, and the liks) for additional sequences encoding novel receptors of the activin/TGF-, superfamily. Such screening is initially carried out under low-stringency conditions, which comprise a temperature of less than about 42 0 C, a formamide concentration of less than about 50%, and a moderate to low salt concentration. Presently preferred conditions for such screening comprise a temperature of about 370C, a formamide concentration of about 20%, and a salt concentration of about 5X standard saline citrate (SSC; 20X SSC contains 3M sodium chloride, 0.3M sodium citrate, pH Such conditions will allow the identification of sequences which have a substantial degree 'WO 92/20793 PCT/US92/03825 -17of similarity with the probe sequence, without requiring perfect homology for the identification of a stable hybrid.

The phrase "substantial similarity" refers to sequences which share at least 50% homology. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% homology with the probe, while discriminating against sequences which have a lower degree of homology with the probe.

In accordance with yet another embodiment of the present invention, there is provided a method for the recombinant production of :e-aceptor(s) of the invention by expressing the above-described DNA sequences in suitable host cells.

The use of a wide variety of recombinant organisms has been described for the production of peptides. One of skill in the art can readily determine suitable hosts (and expression conditions) for use in the recombinant production of the peptides of the present invention. Yeast hosts, bacterial hosts, mammalian hosts, and the like can be employed. Regulatory sequences capable of controlling the expresseion of invention peptides are well known for each of these host systems, as are growth conditions under which expression occurs.

In accordance with a further embodiment of the present invention, there is provided a binding assay emp.loying receptors of the invention, whereby a large number of compounds can be rapidly screened to determine which compounds, if any, are capable of binding to the receptors of the invention. Then, more detailed assays can be carried out with those compounds found to bind, to further determine whether such compounds act as agonists or antagonists of invention receptors.

WO 92/20793 PCT/US92/03825 -18- Another application of the binding assay of the invention is the assay of test samples biological fluids) for the presence or absence of members of the activin/TGF-B superfamily of polypeptide growth factors.

Thus, for example, serum from a patient displaying symptoms related to pathway(s) mediated by members of the activin/TGF-B superfamily of polypeptide growth factors can be assayed to determine if the observed symptoms are perhaps caused by over- or under-production of such polypeptide growth factor.

The binding assays contemplated by the present invention can be carried out in a variety of ways, as can readily be identified by one of skill in the art. For example, competitive binding assays can be employed, as well as radioimmunoassays, ELISA, ERMA, and the like.

In accordance with a still further embodiment of the present invention, there are provided bioassays for evaluating whether test compounds are capable of acting as agonists or antagonists of receptor(s) of the present invention.

The bioassays of the present invention involve evaluating whether test compounds are capable of acting as either agonists or antagonists for members of the invention superfamily of receptors, or functional modified forms of said receptor protein(s). The bioassay for evaluating whether test compounds are capable of acting as agonists comprises: culturing cells containing: DNA which expresses said receptor protein(s) or functional modified forms of said receptor protein(s), and DNA encoding a hormone response element operatively linked to a reporter gene; wherein said culturing is carried out in the WO 92/20793 PCT/US92/03825 -19presence of at least one compound whose ability to induce transcription activation activity of receptor protein is sought to be determined, and thereafter monitoring said cells for expression of the product of said reporter gene.

The bioassay for evaluating whether, test compounds are capable of acting as antagonists for receptor(s) of the invention, or functional modified forms of said receptor(s), comprises: culturing cells containing: DNA which expresses said receptor protein(s), or functional modified forms of said receptor protein(s), and DNA encoding a hormone response element operatively linked to a reporter gene wherein said culturing is carried out in the presence of: increasing concentrations of at least one compound whose ability to inhibit transcription activation of said receptor protein(s) is sought to be determined, and a fixed concentration of at least one agonist for said receptor protein(s), or functional modified forms of said receptor protein(s); and thereafter monitoring in said cells the level of expression of the product of said reporter gene as a function of the concentration of said compound, thereby indicating the ability of said compound to inhibit activation of transcription.

Host cells contemplated for use in the bioassay(s) of the present invention, include CV-1 cells, COS cells, and the like; reporter and expression plasmids employed typically also contain the origin of replication WO 92/20793 PCT/US92/03825 of SV-40; and the reporter and expression plasmids employed also typically contain a selectable marker.

The hormone response element employed in the bioassay(s) of the present invention can be selected from, for example, mouse mammary tumor virus long terminal repeat (MTV LTR), mammalian growth hormone promoter, and the reporter gene can be selected from chloramphenicol acetytransferase (CAT), luciferase, 1-galactosidase, and the like.

The cells can be monitored for the level of expression of the reporter gene in a variety of ways, such as, for example, by photometric means by colorimetry (with a colored reporter product such as B-galacto.idase), by fluorescence (with a reporter product such as luciferase), etc], by enzyme activity, and the like.

Compounds contemplated for screening in accordance with the invention bioassays include activin- or TGF-f?-like compounds, as well as compounds which bear no particular structural or biological relatedness to activin or TGF-f.

As employed herein, the phrase "activin- or TGF--like compounds" includes substances which have a substantial degree of homology (at least 20% homology) with the amino acid sequences of naturally occurring mammalian inhibin alpha and fA or fl, chains (either singly or in any combination) as well as alleles, fragments, homologs or derivatives thereof which have substantially the same qualitative biological activity as mammalian inhibin, activin, or TGF-. Examples of activin- or TGF-f-like compounds include activin A (a homodimer of two inhibin 8A subunits), activin B (a homodimer of two inhibin B 8 subunits), activin AB (a h-terodimer composed of one inhibin BA subunit and one inhibin B, subunit), inhibin A WO 92/20793 PCT/US92/03825 -21- (composed of the inhibin a subunit and an inhibin BA subunit), inhibin B (composed of the inhibin a subunit and an inhibin 8, subunit), TGF-B1 (a homodimer of two TGF-B1 subunits), TGF-B2 (a homodimer of two TGF-B2 subunits), TGF-B3 (a homodimer of two TGF-B3 subunits), TGF-84 (a homodimer of two TGP-B4 subunits), TGF-B5 (a homodimer of two TGF-B5 subunits), TGF--B1.2 (a heterodimer of one TGF-B1 subunit and one TGF-B2 subunit), and the like.

Examples of compounds which bear no particular structural or biological relatedness to activin or TGF-f, but which are contemplated for screening in accordance with the bioassays of the present invention, include any compound that is capable of either blocking the action of the invention receptor peptides, or promoting the action of the invention receptor peptides, such as, for example, alkaloids and other heterocyclic organic compounds, and the like.

The method employed for cloning the receptor(s) of the present invention involves expressing, in mammalian cells, a cDNA library of any cell type thought to respond to members of the activin/TGF-B superfamily of polypeptide growth factors pituitary cells, placental cells, fibroblast cells, and the like). Then, the ability of the resulting mammalian cells to bind a labeled receptor ligand a labeled member of the activin/TGF-B superfamily of polypeptide growth factors) is determined. Finally, the desired cDNA insert(s) are recovered, based on the ability of that ODNA, when expressed in maiamalian cells, to induce (or enhance) the binding of labeled receptor ligand to said cell.

In addition to the above-described applications of the receptor proteins and DNA sequences of the present invention, the receptor or receptor-encoding compositions of the invention can be used in a variety of ways. For WO 92/20793 PCT/US92/03325 -22example, since activin is involved in many biological processes, the activin receptor (or antibodies thereto) can be applied to the modulation of such biological processes.

For example, the stimulation of FSH release by activin can either be enhanced (for example, by supplying the subject with increased amounts of the activin receptor, relative to the amount of endogenous receptor, by transfecting the subject with a tissue specific activin-encoding construct), or depressed by administration to a subject of antibodies to the activin receptor, thereby preventing formation of activin-receptor complex, which would then act to stimulate the release of FSH). Thus, the compositions of the present invention can be applied to the control of fertility in humans, domesticated animals, and animals of commercial interest.

As another example, the effect of activin on mitosis of red and white blood cells can be modulated, for example, by administering to a subject (employing suitable means of administration) a modulating, effective amount of activin receptor (which would enhance the ability of activin present in the cell to modulate mitosis).

Alternatively, one could administer to a subject an antibody to the activin receptor (or a portion thereof), which would reduce the effect of activin by blocking the normal interaction between activin and activin receptor.

As additional examples of the wide utility of the invention compositions, receptors and/or antibodies of the invention can be used in such areas as the diagnosis and/or treatment of activin-dependent tumors, enhancing the survival of brain neurons, inducing abortion in livestock and other domesticated animals, inducing twinning in livestock and other domesticated animals, and so on.

As still further examples of the wide utility of the invention compositions, agonists identified for TGF-B WO 92/20793 PCT/US92/03825 -23specific receptors can be used to stimulate wound healing, to' suppress the growth of TGF-B-sensitive tumcrs, to -uppress immune response (and thereby prevent rejection of transplanted organs), and the like. Antagonists or the soluble, ligand-binding domain derived from ftF-B receptors can be used to block endogenous TGF-B, thereby promoting liver regeneration and stimulating some immune responses.

It can be readily seen, therefore, that the invention compositions have utility in a wide variety of diagnostic, clinical, veterinary and research applications.

The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAIPLES

Recombinant human (rh) activii A, rh activin B, and rh inhibin A were generously provided by Genentech, Inc. Porcine TGF-B1 was obtained from R+D Systems.

Double-stranded DNA was sequenced by the dideoxy chain termination method using the Sequenase reagents from US Biochemicals. Comparison of DNA sequences to databases was performed using the FASTA program [Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988)].

EXAMPLE I Construction and Subdivision of cDNA Library Polyadenylated RNA was prepared from AtT20 cells using the Fast Track reagents from InVitrogen. cDNA was commercially synthesized and ligate into the plasmid vector pcDNA1 using non-palindromic BstXI linkers, yielding a library of approximately 5x10 6 primary recombinants. The unamplified cDNA library was plated at 1000 clones per 100 mm plate, then scraped off the plates, frozen in glycerol WO 92/20793 PCT/US92/03825 -24and stored at Activin suppresses adrenocorticotrophic hormone (ACTH) secretion by both primary anterior pituitary cell cultures [Vale et al., Nature 321: 776-779 (1986)] and mouse corticotropic cells. Because AtT20 cells possess activin receptors indistinguishable from those on other cell types (based on binding affinity measuremen':s with activin these cells were chosen to be the source of cDNA for transfection. A cDNA library of approximately 5x10 independent clones from AtT20 cells was constructed in the mammalian expression vector, pcDNAl, and screened using an expression cloning approach [Gearing et al., EMBO J. 8, 3667-3676 (1989)) based on the ability to detect activin binding to single transfected cells. The library was divided into pools of 1000 clones, DNA was prepared from each pool of clones and transiently transfected into COS '-ells, and the cells screened for the capacity to bind iodinated activin A. Binding was assessed by performing th? transfections and binding reactions directly on chambered microscope slides, then dipping the slides in photographic emulsion and analyzing them under a microscope. Cells which had been transfectL4 with an activin receptor cDNA, and consequently bound radioactive activin, were covered with silver grains. DNA from pools of clones were analyzed either singly or in groups of three. Of 300 pools (approximately 300,000 clones) assayed in this manner, one group of three generated two positive cells when transfected into COS cells. The positive pool was identified by transfecting and analyzing DNA from each pool of 1000 singly, and then was further fractionated until a single clone (pmActRl) was purified which generated >104 positive cells after transfection (see Table 1).

'WO 92/20793 PCT/US92/03825 Table 1 Purification of the activin receptor clone from the AtT20 library Pool Clones/pool Positive cells/slide 62,63,64 3x1000 2 64 1000 1-3 64-51 400 4-10 64-51-R10;64-51-C13 20 25-40 pmActRl 1 >104 The total number of transfected cells capable of binding activin A in a field of 2x10 5 COS cells was counted for pools of clones at each stage of the purification process.

pmActR1 contained a 1.7 kb insert, coding for a protein of 342 amino acids (Figure however, it was incomplete on the 3' end, thus the last 17 amino acids were encoded by vector sequences. In order to obtain the entire sequence, the AtT20 library was rescreened by hybridization with the 1.6 kb SacI-PstI fragment (Figure Screening 6x10 5 colonies yielded one additional positive clone (pmActR2) which had a 2.6 lb insert and contained the entire coding sequence for the mouse activin receptor (Figare The nucleic acid sequence and the deduced amino ril sequence of the insert in pmActR2 are set forth 'n RFec ID No. 1.

EXAMPLE II COS Cell Transfection Aliquots of the frozen pcols of clones were grown overnight in 3 ml cultures of terrific broth, and mini-prep DNA prepared from 1.5 ml using the alkaline lysis method rManiatis et al. Molecular Cloning (Cold Spring Harbor Laboratory (1982)]. 1/10 of the DNA from a mini-prep WO 92/20793 PCT/US92/03825 -26- Ml of 100 Ml) was used for each transection.

2x10 5 COS cells were plated on chambered microscope slides (1 chamber Nunc) that had been coated rith 20 gg/ml poly-D-lysine and allowed to attach for at least 3 hours. Cells were subjected to DEAE-Dextran mediated transfection as follows. 1.5 ml of serum-free Dulbecco's Modified Eagle's medium (DME) containing 100 mM chloroquine was added to the cells. DNA was precipitated in 200 ml DME/chloroquine containing 500 mg/ml DEAE- Dextran, then added to the cells. The cells were incubated at 370 for 4 hours, then the media was removed and the cells were treated with 10% DMSO in HEPES buffered saline for 2 minutes. Fresh media was added and the cells assayed 3 days later. For transfections with the purified clone, 2.5x106 cells were transfected in 100 mm dishes with 5 ig purified DNA. The total transfection volume was 10 ml, and the DNA was precipitated in 400 pl.

EXAMPLE III iindinc Assay Cells were washed 2x with HEPES buffered saline (HDB) containing 0.1% BSA, then incubated for 90 minutes at 220 in 0.5 ml HDB, 0.1% BSA containing 7x10 5 cpm 125I activin A (approximately 7 ng, 500 pM). The cells were then washed 3X with cold HDB, fixed for 15 minutes at 220 in glutaraldehyde/HDB and washed 2X with HDB. The chambers were then peeled off the slides, and the slides dehydrated in 95% ethanol, dried under vacuum, dipped in NTB2 photographic emulsion (Kodak) and exposed in the dark at for 3 days. Following development of the emulsion, the slides were dehydrated in 95% ethanol, stained with eosin and coverslipped with DPX mountiant (Electron Microscopy Sciences). The slides were analyzed under darkfield illumination using a Leitz microscope.

WO 92/20793 PCT/US92/03825 -27- EXAMPLE IV Subdivision of Positive Pool Of 300 pools screened (each pool containing about 1000 cDNAs), one positive pool which produced two positive cells, was identified. Bacteria from the frozen stock of this positive pool were replated at approximately 400 clones per plate, replica plates were made, and DNA was prepared from each subpool and analyzed employing the binding assay described above. Several positive subpools were found, which generated from 4-10 positive cells per slide. The bacteria from the replica plate of one positive subpool were picked onto a grid, and DNA prepared from pools of clones representing all the rows and all the columns, as described by Wong [Science 228:810- 815 (1985)]. The identification of one positive row and one positive column unambiguously identified a single clone, which when transfected yielded >10 4 positive cells/2x105 cells.

EXAMPLE V Radloreceptor Assay 105 COS cells transfected with either pmActRl or pmActR2, or 106 untransfected COS cells, were plated in 6 well dishes and allowed to grow overnight. The cells were washed 2X with HDB, 0.1% BSA, and incubated at 220 for minutes in 0.5 ml HDB, 0.1% BSA containing 100,000 cpm (approximately 1 ng, 75 pM) 125I activin A (5 gg activin A was iodinated by chloramine T oxidation to a specific activity of 50-90 pCi/gg; iodinated activin A was purified on a 0.7x20 cm G-25 column) and varying amounts of unlabeled competitor hormone. Following binding, the cells were washed 3X with cold HDB, solubilized in 0.5 ml 0.5 N NaOH, removed from the dish and radioactivity was measured in a gamma counter. Data presented in Figure 5 are expressed as specific binding, where 100% specific WO 92/20793 PCT/US92/03825 -28binding is the difference between binding in the absence of competitor and binding in the presence of a 100 fold molar excess of unlabeled activin A. Binding parameters were determined using the program LIGAND [Munson P.J. and Rodbard, Anal. Biochem. 107:220-259 (1980)].

EXAMPLE VI Chemical Cross-linking 2x106 COS cells, or 5x106 AtT20 cells, were washed 2x with HDB, scraped off the dish, incubated for 90 minutes at 220 under constant rotation in 0.5 ml HDB containing 7x105 cpm (approximately 500 pM) 15I activin A with or without 500 ng (37 nM) unlabeled activin A. Cells were diluted with 1 ml HDB, pelleted by centrifugation and resuspended in 0.5 ml HDB. Disuccinimidyl suberate (DSS; freshly dissolved in DMSO) was added to 500 MM, and the cells incubated at 0° for 30 minutes. The cross-linking was terminated by addition of 1 ml 50 mM Tris-HCl pH 100 mM NaCI, then the cells were pelleted by centrifugation, resuspended in 100 Al 50 mM Tris-HCl pH 1% Triton X-100 and incubated at 00 for 60 minutes.

The samples were centrifuged 5 minutes at 13,000xg, and the Triton-soluble supernatants analyzed by SDS-PAGE using polyacrylamide gels. The gels were dried and subjected to autoradiography for 4-14 days.

EXAMPLE VII RNA Blot Analysis Total RNA was purified from tissue culture cells and tissues using LiCl precipitation. 20 Ag total RNA was run on 1.2% agarose, 2.2M formaldehyde gels, blotted onto nylon membranes (Hybond NEN), and hybridized with a 0.6 kb KpnI fragment (see Figure 3} which had been labeled with 32P by random priming using reagents from US Biochemicals.

Hybridization was rprformed at 420 in 50% formamide, and WO 92/20793 PCT/US92/03825 -29the filters were washed at 650 in 0.2X SSC.

EXAMPLE VIII Sequence Analysis Full length mouse activin receptor clone encodes a protein of 513 amino acids, with a 5' untranslated region of 70 bp and a 3' untranslated region of 951 bp. pmActR2 does not contain a poly A tail, although it does have a potential pol;\denylylation site at bp 2251. The insert in clone pmActRl had an additional 551 bp of 5' untranslated sequence, was identical in the overlapping range, and stopped at the 3' end at base 1132 of pmActR2. The first methionine codon (ATG), at bp 71, in pmActR2 is in a favorable context for translation initiation [Kozak, M., Nucl. Acids Res. 15:8125-8148 (1987)], and is preceded by an in-frame stop codon. pmActRl contains 3 additional ATGs in the 5' untranslated region; however, none of these is in an appropriate context for initiation, and all are followed by in-frame stop codons. While this unusually long leader sequence may have functional significance, it is clearly not necessary for proper expression, because pmActR2, which lacks most of that sequence, can be functionally expressed in COS cells (see below).

Hydropathy analysis using the method of Kyte and Doolittle Mol. Biol. 157:105-132 (1982)] revealed two hydrophobic regions: a 10 amino acid stretch at the amino terminus assumed to be a single peptide, and a single putative 26 residue membrane-spanning region between amino acids 119-142 (see Figure 1 and Sequence ID No. The signal peptide contains the conserved h- and c- domains common to signal sequences; the site of cleavage of the signal peptide, before Ala is predicted based on rules described by von Heijne [Biochim. Biophys. Act. 947:307-333 (1988)?, As is common for the cytoplasmic side of membrane-spanning domains, the predicted transmembrane WO 92/20793 PCr/US92/03825 region is closely followed by two basic amino acids. The mature mouse activin receptor is thus predicted to be a 494 amino acid type I membrane protein of Mr 54 kDa, with a 116 amino acid N-terminal extracellular ligand binding domain, and a 346 amino acid intracellular signalling domain.

Comparision of the activin receptor sequence to the sequence databases revealed structural similarity in the intracellular domain to a number of receptor and nonreceptor kinases. Analysis of the sequences of all kinases has led to the identification of a 300 amino acid kinase domain characterized by 12 subdomains containing a number of highly conserved amino acids [Hanks, S.K. and Quinn, Meth. Enzymol. 200:38-62 (1991) and Hanks et al., Science 241:42-52 (1988)]; the activin receptor sequence has all of these conserved subdomains in the proper order (Figure A conserved Gly in subdomain I is replaced by Ala 18 0 in the activin receptor, but this residue has also been observed in other kinases. Based upon structural relatedness, therefore, this receptor is expected to be a functional protein kinase.

The sequences in two of these subdomains (VIB and VIII) can be used to predict tyrosine vs. serine/threonine substrate specificity [Hanks et al., (1988) supra]. The sequence of the mouse activin receptor in both of these subdomains is characteristic of serine kinases.

0 %0J Table 2 Kiriase Domain Predictive Sequences serine kinase consensus activin receptor tyrosine kinase consensus

VIB

DLKPEN

DIKSKN

DLAARN

SEO ID NO. VIII SEQ ID NO.

5 G(T/S)XX(Y/F)X 6 7 GTRRYM 8 9 XP(I/V) WO 92/20793 PCT/US92/03825 -32- Therefore, the activin receptor is expected to have serine/threonine specificity. Furthermore, the activin receptor does not have a tyrosine residue in the standard autophosphorylation region between subdomains VII and VIII, indicating that it is not a standard tyrosine kinase. The receptor could potentially autophosphorylate at Ser or Thr One interesting additional possibility is that the activin receptor kinase may have specificity for serine, threonine and tyrosine residues. Several kinases with these properties have recently been described [see, for example, Howell et al., Mol. Cell. Biol. 11:568- 572 (1991), Stern et al., Mol. Cell. Biol. 11:987-1001 (1991) and Featherstond, C. and Russell, Nature 349:808-811 (1991)].

Phylogenetic analysis of the activin receptor compared to 161 other kinase sequences revealed that the activin receptor and the C.elegans protein, daf-l [Georgi et al., Cell 61:635-645 (1990)] may constitute a separate subfamily of kinases (see Figure daf-l is a putative transmembrane receptor involved in the developmental arrest of a non-feeding larval state and shares 32% identity with the activin receptor (see Figure Like the activin receptor, daf-l is predicted to be a transmembrane serine/threonine-specific kinase; furthermore, both daf and the activin receptor have short, conserved inserts in the kinase domain sequence between subdomains VIA-VIB and X-XI that are not present in any other kinase (underlined in Figure 4B). This additional similarity lends credence to their belonging to a unique subfamily of kinases. The activin receptor is quite distantly related (18% amino acid sequence identity) to the only other known transmembrane serine/threonine protein kinase, encloded by the ZmPK gene of maize [Walker, J.C. and Zhang, Nature 345:743-746 (1990)].

WO 92/20793 PCT/US92/03825 -33- The extracellular domain of the activin receptor did not show similarity to any other sequences in the databases. This ligand binding domain is relatively small in comparison to those found in other growth factor receptors, but like those receptors this domain has a high cysteine content. The pattern of these Cys residues, however, is not like either an immunoglobulin fold or the cysteine rich repeats of the EGF receptor. There are also two potential sites of N-linked glycosylation in the extracellular domain, as well as a number of potential phosphorylation sites for protein kinase C and casein kinase II in the intracellular domain.

EXAMPLE IX Binding Properties of the Cloned Activin Receptor To verify that the cloned receptor is activin specific, competition binding experiments were performed on COS cells transiently transiected with either pmActRl or pmActR2. Cells transfected with either construct bound activin A with a single high affinity component (Kd 180 pM; Figure indicating that a functional (structurally complete) intracellular kinase domain is not required for ligand binding. This binding affinity is consistent with that measured on other activin-responsive cell types [see, for example, Campen, C.A. and Vale, W., Biochem. Biophys. Res. Comm. 157:844-849 (1988); Hino et al., J. Biol. Chem. 264:10309-10314 (1989); Sugino et al., J. Biol. Chem. 263: 15249-15252 (1988); and Kondo et al., Biochem. Biophys. Res. Comm, 161:1267-1272 (1989)].

Untransfected COS cells do not bind activin A. The transfected cultures as a whole expressed approximately 26,000 receptors per cell; however, because only 15% of the cells express the transfected gene (as measured by quantitating transfected cells as a fraction of all cells following dipping in emulsion), each transfected cell expressed an average of 175,000 receptors per cell. The WO 92/20793 PCT/US92/03825 -34level of expression per cell varies considerably, though, based on the number of accumulated silver grains. This value is comparable to the expression o'f other transfected cell surface proteins in COS cells.

Binding of iodinated activin A to COS cells transiently transfected with pmActR2 could be competed by activin B with slightly reduced potency-compared to activin A; by inhibin A with approximately 10-fold lower potency; and could not be competed by TGF-B1 (Figure 5B). This affinity and specificity of binding match those observed following binding of activin A to a number of other activin-responsive cell types. Although activin B appears to bind the transfected receptor with lower affinity than activin A, the activin B preparation used in these experiments may have suffered a reduction in potency, based on a comparison of bioactivity with activin A, since the recombinant synthesis of the activin B employed herein had been carried out some time ago [recombinant synthesis of activin B is described by Mason et al., in Mol. Endocrinol.

3: 1352-1358 (1989)). It is likely that this cDNA encodes a receptor for multiple forms of activin.

The size of the cloned activin receptor was analyzed by affinity cross-linking 125I activin A to COS cells transfected with pmActR2 using the bifunctional chemical cross-linker, disuccinimidyl suberate (DSS). A major cross-linked band of 84 kDa was observed in transfected, but not in untransfected cells. Subtracting the molecular weight of activin, this represents a protein of 56 kDa, which corresponds well to the molecular weight predicted from the nucleic acid sequence data. Crosslinking 125 activin A to AtT20 cells yields a major band of kDa, with minor bands of approximately 78 and 84 kDa.

The size of the largest band matches that generated by the cloned receptor. The smaller bands could be either separate proteins, different phosphorylated forms of the WO 92/20793 PCT/US92/03825 same protein, or degradation products of the full length clone; the sequences DKKRR at amino acid 35 and KKKR at amino acid 416 could be potential sites of proteolysis.

Alternatively, these bands could come from alternatively spliced products of the same gene.

The 84 and 65 kDa cross-linked bands have also been observed in other activin-responsive cell types [Hino, supra; Centtella et al., Mol. Cell. Biol. 11:250-258 (1991)], and interpreted to represent the signalling receptor, although complexes of other sizes have also been seen as well. The size of the activin receptor is very similar to a putative TGF-B receptor, to the limited extent it has been characterized by chemical cross-linking [see Massague et al., Ann. N.Y. Acad. Sci. 593: 59-72 (1990)].

EXAMPLE X Expression of Activin Receptor mRNA The distribution of activin receptor mRNA was analyzed by Northern blot. Two mRNA species, of 6.0 and kb, were observed in AtT20 cells as weUl as a number of mouse tissues, including brain, testis, pancreas, liver and kidney. The total combined size of the inserts from pmActRl and pmActR2 is 3.1 kb, which corresponds to the size of the smaller transcript. Neither the extent of similarity between the two mRNAs, nor the significance of having two transcripts is clear. The genes for several other hormone receptors have been shown to be alternatively spliced to generate both a cell surface receptor and a soluble binding protein, and it is possible that the activin receptor is processed in a similar manner.

Interestingly, the relative abundance of the two transcripts varies depending on the source. While cells have approximately equal levels of both mRNAs, most tissues had much greater levels of the 6.0 kb transcript, WO 92/20793 PCT/US92/03825 with little or no expression of the 3.0 kb transcript.

Testis, on the other hand, had a greater amount of the kb band. Expression of activin receptor mRNA in brain, liver and testis is in accord with described biological actions of activin in those tissues [Mine et al., Endocrinol. 125:586-591 (1989); Vale et al., Peptide Growth Factors and Their Receptors, Handbook of Experimental Pharmacology, M.A. Sporn and A.B. Roberts, ed., Springer-Verlag (1990), in press].

EXAMPLE XI Identification of a Human Activin Receptor A human testis library (purchased from Clontech; catalog no. HL1010b) was probed with the full length mouse activin receptor gene (see Sequence ID No. 1) under the following conditions: Hybridization stringency: formamide, 6X SSC at 4P C; Wash stringency: 2X SSC, 0.1% SDS at 42°C.

A sequence which is highly homologous with the mouse activin receptor was identified (Sequence ID No. Due to the high degree of homology between this receptor and the mouse activin receptor, this receptor is designated as the human form of the activin receptor from the same subclass as the mouse receptor described above.

EXAMPLE XII Identification of a Xenopus Activin Receptor A Xenopus stage 17 embryo cDNA library (prepared as described by Kintner and Melton in Development 99: 311- 325 (1987) was probed with the full length mouse activin receptor gene (see Sequence ID No. 1) under the following conditions: WO 92/20793 PCT/US92/03825 -37- Hybridization stringency: formaride, 6X SSC at 42 0

C;

Wash stringency: 2X SSC, 0.1% SD at 420C.

A sequence having a substantial degree of homology with respect to the mouse activin receptor was identified (Sequence ID No. The degree of overall amino acid homology (relative to the mouse acitvin receptor) is only about 69% (with 7T% homology in the intracellular domain and 58% homology in the extracellular domain). Due to the moderate degree of homology between this receptor and the mouse activin receptor, this receptor is designated as an activin receptor from a different subclass than the mouse receptor described above.

EXAMPLE XIII Functional Assays of ActRs in Xenopus embryos To determine whether xActRIIB can transmit a signal in response to activin, xActRIIB RNA was synthesized in vitro and injected into Xenopus embryos at two different concentrations. Injected embryos were allowed to develop to stage 9, at which ti m= animal caps were dissected and treated overnight with different concentrations of activin.

The xActRIIB cDNA was cloned into rp64T [see Krieg and Melton in Metho's in Enzymology, Abelson and Simon, Eds.

(Academic Press, New York, 1987), vol. 155, p. 397] and transcribed in vitro to generate a capped, synthetic xActRIIB RNA (see Melton et al., in Nucleic Acids Res.

12:7035 (1984) and Kintner in Neuron 1:545 (1988)j.

Embryos at the two- to four-cell stage were injected with about 20 nl of RNA at conc.ntrations of 0.02 ng/nl, or 0.1 ng/nl, spread between four quadrants of the animal pole.

At stage 9, animal caps were removed from RNA-injected embryos and incubated in 0.5x modified mammalian Ringer's (KMR), 0.1% bovine serum albumin (BSA) with different WO 92/20793 WO 9220793PC1'/US92/038i5 -38concentrations of purified, porcine activin A (six caps per incubation). After 20 hours in culture, total RNA was preFared.

The response of the caps to activin was assessed by quantifying muscle-specific actin RNA with a ribonuclease protection assay as yp'er B18ckwell and Weintraub, Science ,L50:1104 (1990). E-mbryos injected with 0.4 and 2.0 ng of xActR"IB R.NA were approximately 10- and~ 100-fold more sensitive, respectively, to activin than control embryos. The low amount of muscle actin found in animal caps in the absence of added activin A is proba vI a consequence of contanination of the animal cap with a small amount of marginal zone tissue, The amount of muscle actin decreased with increasi~ig concentration of activ:Lt in the embryos injected with 2 ng of xActRIIB RNA, This is coiia$stent with the observation that isolated an'iial -ap el nfrl exposed to different conceritrations of activin only form muscle calls in rest Onse to a narrow4 range of activin concentrations (see Blackxann and 1(adesch in Genes and Development A:1057 (1990)). The present re~ults indlicate that th-n conoentratzion of ligand and the amount of receptor are both importaaut in determining the signal trar~nitted.

Thus, the rango of activin concentrations that lead to muscle differentiation is lower in animal cap cells from injected embryos, which are expressing more receptor than nor~ al, than from ninjected embryos.

EX ~PLt XIV Analysis of ki~,YiaeactivZity of mActTRII A fragment of cDNA corresponding to the entire 353 intracellular domain of mActRII (amino acids 143-494) was subcloned into the vector pGEX(-2T (see Smith and Johnson in Gene 67:31-40 (1988)3, creating a fusdon protein between 'WO 92/20793 PCT/US92/03825 -39glutathione S-transferase (GST) and the putative kinase domain of the receptor. This plasmid was introduced into bacteria and the expressed fusion protein was purified using glutathione affinity chromatography as described by Smith and Johnson. Approximately 100-200 ng of fusion protein, or of purified GST, were incubated with 25 gCi 32] ATPin a buffer containing 50 mM Tris, 1G .aM Mgrl 2 for minutes at 37 0 C. The products were analyzed by SDS-PAGE and autoradiography. The fusion protein, but not the GST alone, became phosphorylated, indicating that the kinase domain of the fusion protein was functional. Phosphoamino acid analysis, performed according to Cooper at al. [Meth.

Enzym. 99:387 (1983)], indicated that the predominant amino acid residue that became phosphorylated was threonine.

While the invention has been described in detal with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

WO 92/20793 PCr/US92/03825 SUMMARY OF SEQUENCES Sequence ID No. 1 is the nucleic acid sequence (and the deduced amino acid sequence) of a cDNA encoding a mouse-derived activin receptor of the present invention.

Sequence ID No. 1' is a nucleic acid sequence encoding a human-derived activin receptor of the present invention. Sequence ID No. 1' is substantially the same as Sequence ID No. 1, except that the codon for amino acid residue number 39 encodes lysine nucleotides 185-187 are AAA or AAG) the codon for amino acid residue 92 encodes valine nucleotides 344-346 are GTN, wherein N is A, C, G or and the codon for amino acid residue number 288 encodes glutamine nucleotides 932-934 are CAA or CAG).

Sequence ID No. 2 is the deduced 'mino acid sequence of a mouse-derived activin receptor of the present invention.

Sequence ID No. 2' is an 'no acid sequence for a human-derived activin receptor of present invention.

Sequence ID No. 2' is substantially i mie as Sequence ID No. 2, except that amino acid residu ,er 39 is lysine, amino acid residue 92 is valine, amino acid residue number 288 is glutamine.

Sequence ID No. 3 is the nucleic acid sequence (and the deduced amino acid sequence) of a cDNA encoding a Xenopus-derived activin receptor of the present invention.

Sequence ID No. 4 is the deduced amino acid sequence of a Xenopus-derived activin receptor of the present invention.

WOo 92/20793 PCT/US92/03825 -41- Sequence ID No. 5 is the amino acid sequence of the VIB subdomain of the serine kinase consensus sequence.

Sequence ID No. 6 is the amino acid sequence of the VIII subdomain of the serine kinase consensus sequence.

Sequence ID No. 7 is the amino acid sequence of the VIB subdomain of the invention activin receptor.

Sequence ID No. 8 is the amino acid sequence of the VIII subdomain of the invention activin receptor.

Sequence ID No. 9 is the amino acid sequence of the VIB subdomain of the tyrosine kinase consensus sequence.

Sequence ID No. 10 is the amino acid sequence of the VIII subdomain of the tyrosine kinase consensus sequence.

WO 92/20793 PCT/US92/03825 -42- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Mathews, Ph.D., Lawrence S.

Vale, Ph.D., Wylie W.

(ii) TITLE OF INVENTION: CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTIVIN/TGF-BETA SUPERFAMILY (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: PRETTY, SCHROEDER, BRUEGGEMANN CLARK STREET: 444 South Flower Street, Suite 2000 CITY: Los Angeles STATE: California COUNTRY: United States ZIP: 90071-2921 COMPUTER READABLE FORM MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE: 08-MAY-1992

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Reiter, Mr., Stephen E.

REGISTRATION NUMBER: 31192 REFERENCE/DOCKET NUMBER: P31 9309/FP31 9291 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (619) 546-4737 TELEFAX: (619) 546-9392 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2563 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 71..1609 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTCCGAGGAA GACCCAGGGA ACTGGATATC TAGCGAGAAC TTCCTACGGC TTCTCCGGCG CCTCGGGAAA ATG GGA GCT GCT GCA AAG TTG GCG TTC GCC GTC TTT CTT 109 Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu 1 5 I WO 92/20793 WO 9220793PCY/US92/03825 -43- ATC TCT TGC TOT TCA GOT GOT ATA CTT GGC AGA TCA GAA ACT Ile

TOT

Cye

GGT

S.L

GCT

Ala

TGT

Cys

AAA

Lys

TOT

Cys 110

ACT

Thr

TAT

Tyr

TTT

Phe

OCT

Pro

AAG

Lys 190

GTC

Val

CCA

Pro

CTA

Leu

AAA

Lye

CAT

His 270 Ser is

CTT

Leu

OTT

Val

ACC

Thr

TGG

Trp,

AAA

Lye

AAT

Asn

TCA

Ser

TCC

Ser

TG

Trp

ACT

Thr 175

CA

Pro

TG

Trp

ATA

Ile

OCT

Pro

AGA

Arg 255 Oye

TTC

Phe

GAA

Oiu

TOO

Trp

CTG

Leu

GAC

Asp

GMA

Glu

AAT

Asn

TTG

Leu

GTO

Val 160

CAA

GlE.

TTG

Leu

AAA

Lye

CAG

Gin

GGA

Oly 240

GG

Gly Ser

TTT

Phe

OCT

Pro

MAG

Lye

OAT

Asp

AGO

Ser

AAG

Lye

COT

Pro

GTA

Val 145

TAO

Tyr

GAO

Asp

CAG

Gin 000 Ala

GAO

Asp 225

ATG

Met

ACC

Thr Ser

AAT

Aen

TOO

Cys

AAT

Asn

GAT

Asp

OCT

Pro

TTO

Phe

GTT

Val 130

OCA

Pro

AGA

Arg

CCA

Pro

OTG

Leu

CAG

Gin 210

AMA

Lys

MAG

Lye

AGT

Ser Gly

GOT

Ala 35

TAT

Tyr

ATT

Ile

ATO

Ile

GMA

Giu

TOT

Ser 115

ACA

Thr

CTA

Leu

CAT

His

GGA

Oly

TTA

Leu 195

TTG

Leu

CAG

Gin

CAT

His

GTG

Val Ala 20

MAT

Asn

GOT

Gly

TOT

Ser

MOC

Aen

OTG

V a 1 100

TAT

Tyr

CG

Pro

ATG

Met

CAC

His

OCA

Pro 180

GMA

Olu

CTC

Leu

TOO

Ser

GAG

Glu

GAT

Asp 260 Ile

TG

Trp

GAT

Asp

GGT

Giy

TOO

Cys 85

TAO

Tyr

TTT

Phe

MAG

Lys

TTA

Leu

MAG

Lye 165

COO

Pro

GTO

Vai

MAT

Asn

TOO

Trp

MAC

Aer 245

GTG

Val Leu

GMA

Giu

AMA

Lye

TOO

Ser 70

TAT

Tyr

TTT

Phe

CG

Pro

OCA

Pro

ATT

Ile 150

ATO

Met

CCA

Pro

AMA

Lys

GMA

Giu

CAG

Gin 230

ATA

Ile

GAO

Asp Gly

AGA

Arg

OAT

Asp 55

ATT

Ile

GAO

Asp

TOT

Cys

GAG

Glu

CCC

Pro 135

OCA

Ala

CO

Ala

COT

Pro

OCA

Ala

TAT

Tyr 215

MAT

Asn

CTA

Leu

CTO

Leu Arg

GAO

Asp 40

AMA

Lye

GMA

Oiu

AGG

Arg

TOO

Cys

ATG

Met 120

TAT

Tyr

OGA

Gly

TAO

Tyr

TOO

Ser

AGO

Arg 200

OTG

Val

GMA

Giu

CAG

Gin

TOO

Trp Ser

AGA

Arg 000 Arg

ATA

Ile

ACT

Thr

TOT

Cys 105

GMA

Giu

TAO

Tyr

ATT

Ile

COT

Pro

OCA

Pro 185

GGA

Gly

OCT

Ala

TAT

Tyr

TTO

Phe

OTA

Leu 265 Glu

ACC

Thr

CGA

Arg

GTG

Vai

OAT

Asp

GAG

Giu

OTO

Val

MOC

Aen

GTO

Val

OCT

Pro 170

TTA

Leu

AGA

Arg

OTC

Val

GAA

Giu

ATX

Ile 250

ATC

Ile Thr

MOC

Asn

CAT

His

MAG

Lye

TOT

Cys

GC

Gly

ACA

Thr

ATT

Ile

ATT

Ile 155

GTA

Vai

CTA

Leu

TTT

Phe

A

Lye

OTO

Val 235

OCT

Gly

ACA

Thr

CAG

Gin

CAG

O in

TOT

Cys

CAA

Gin

ATA

Ile

MAT

Asn

CAG

Gin

CTG

Leu 140

TOT

Cys

OTT

Leu 000 Oly

GT

Giy

ATA

le 220

TAT

Tyr

GOA

Ala

OCA

Ala

GAO

Oiu

ACT

Thr

TTT

Phe

GT

Giy

GMA

Giu

ATO

Met 000 Pro 125

CTG

Leu

OCA

Ala

OTT

Val

TTG

Leu

TOT

Cys 205

TTT

Phe

AGT

Ser

GAG

Glu

TTT

Phe 157 205 253 301 349 397 445 493 541 589 637 685 733 781 829 877 925 GAM MG GGC TCA CTG TCA GAO TTT OTT MAG OCT MAT OTO OTO TOT Oiu Lye Giy Ser Leu Ser Asp Phe Leu Ala Asn Val Val Ser WO 92/20793 WO 9220793PCr/US92/038i5 -44-

TGG

Trp

TAT

Tyr

ATC

Ile

CTG

Leu

GGC

G ly 350

ATO

Met

TTT

Phe

GCT

Ala

CCA

Pro

GAA

Giu 430

CAG

Gin

TGG

Trp

AGA

Arg

ATT

Ile

AAT

Asn

TTA

Leu

TCT

Ser

ACA

Thr 335

AAG

Lys

GCT

Ala

CTG

Leu

TCT

Ser

TTT

Phe 415

GTT

Val

AAA

Lys

GAT

Asp

ATT

Ile

GTA

Val GAA CTT TGT CAT ATT GCA GAA ACC ATG GCT AGA OGA TTG GCA Giu

CAT

His

CAC

His 320

GCT

Ala

TCT

Ser

CCA

Pro

AGG

Arg

CGT

Arg 400

GAG

Giu

OTT

Val

CAT

His

CAT

His

ACT

Thr 480

ACA

Thr Leu

GRG

Olu 305

AGO

Arg

TGC

Cys

GCA

Ala

GAG

Giu

ATA

Ile 385

TOC

CYs

GAA

Oiu

GTG

Val

GCA

Ala

GAT

Asp 465

CAG

Gin

GTG

Val Cys His 290 OAT ATA Asp Ile GAC ATO Asp Ile ATT GCT Ile Ala OCT GAC Gly Asp 355 OTG TTG Val Leu 370 OAT ATO Asp Met ACT OCT Thr Ala OAA ATT Giu Ile CAT AAA His Lys 435 OGA ATO Gly Met 450 OCA GAA Ala Giu ATG CAA Met Gin GTC ACA Ile Ala CCT GGC Pro Gly AAA AGT Lys Ser 325 GAC TTT Asp Phe 340 ACC CAT Thr His GAG GOT Glu Gly TAC 0CC Tyr Ala OCA OAT Ala Asp 405 GOC CAG Gly Gin 420 AAA AAG Lys Lys OCA ATO Ala Met GCC AGO Ala Arg AGA CTA Arg Leu 485 ATO GTG Oiu Thr 295 TTA AAA Leu Lye 310 AAA AAT Lys Aen 000 TTG Oly Leu 000 CAG Gly Gin OCT ATA Ala Ile 375 ATO OGA Met Gly 390 OGA CCC Oly Pro CAT CCA His Pro AGO CCT Arg Pro CTC TOT Leu Cys 455 TTA TCA Leu Ser 470 ACA AAT Thr Asn ACA AAT Met Ala Arg Oly Leu Ala 300

OAT

Asp

OTO

Val 0CC Aia

OTT

Val 360

AAC

Asn

TTA

Leu

OTA

Val

TCT

Ser

OTT

Val 440

GAA

Oiu

OCT

Ala

ATC

Ile

OTT

GC

Gly

CTG

Leu

TTA

Leu 345

GT

Gly

TTC

Phe

OTC

Val

OAT

Asp

CTT

Leu 425

TTA

Lou

ACO

Thr

OGA

Gly

ATT

Ile

GAC

Asp 505

CAC

His

TTG

Lou 330

AAG

Lys

ACC

Thr

CAA

Gin

CTA

Leu

GAG

Glu 410

OAA

O lu

AGA

Arg

ATA

Ile

TOT

Cys

ACT

Thr 490

TTT

AAG CCT Lye Pro 315 AAA AAC Lye Aen TTC GAG Phe Oiu COO AGO Arg Arg AGO GAC Arg Asp 380 TOO OAA Trp Oiu 395 TAC ATO Tyr Met GAT ATO Asp Met OAT TAT Asp Tyr OAA OAA Olu Glu 460 GTA GOT Val Gly 475 ACA GAG Thr Glu CCT CCC

OCA

Ala

AAT

Asn

OCT

Ala

TAT

Tyr 365

OCA

Ala

TTO

Leu

TTA

Leu

CAG

Gin

TOO

Trp 445

TOT

Cys

OAA

Glu

GAC

Asp

AAA

Lys 973 1021 1069 1117 1165 1213 1261 1309 1357 1405 1453 1501 1549 1597 1649 1709 1769 1829 1889 Vai Thr Met Val Thr Aen Val 500 Phe Pro Pro 495 GAA TCT AGT CTA Giu Ser Ser Lou TOATGGTOOC ACCGTCTGTA CACACTGAGG ACTGGGACTC

TGAACTGGAG

GATGCCTCCA.

ATCTGGOAAA

CTGCAAACTG

CTOCTAAGCT AAGGAAAOTG CTTAGTTGAT TTTCTGTOTG-AAATOAGTAG GGACATOTAC OCAAGCAGCC CCTTGTOOAA AGCATGGATO TOOGAGATOG CTTACTOCAT CGTCTOCAGC ACAGATATGA AGAGOAOTCT AAGOAAAAG TAAAOAACTT CTGAAAATGT ACTCGAAGAA TGTOOCCCTC TCCAAATCAA WO 92/20793 WO 9220793PCT/LJS92/03825

GGATCTTTTG

TGTCAGAAGA

TCATTTCAGA

CTATTGTAAT

ATAGTCATCA

CAAGGTATAC

GAATAAATCA

AAAAATGGTA

GCTTTTCTTC

AATATGGTGT

TCAAAGACAG

CTTTACAAAT

GACCTGGCTA

CACTAATTCC

TTTTAAAAAG

GCCAACATGA

AAGTGGGGTA

CTCAGTTCCA

GTCCATGTTT

AGCTATGCTT

TACTGGCTTG

CACCCTACCC

CACTTTGAAA

AGCC

ATCAAGTATT

TTAAATGAAC

GGTAACTTTT

CACAGCTTGT

CAGTAAAGAG

CGGTTGTTAA

TATAACAAGG

AGTGCCAATA

TAATTTAGGG

CCCATACTTA

ACCCTAAATT

TGCAAAACTG

TACTGCTATT

TATTGCATTT

GAATGTGTAG

GCTTCCAAGC

ATTATAAAAT

TTAATTACAA

GTAAGTGGCT

AAAACAAGTG

TATCAAGGTC

ACAAGCCAGT

ACATCAGATT

TTTTTTAAAT

GCTGTTGTTT

TGTGCTGCTG

ATTACTTTAA

TGAAAACACT

ATTCACTGTG

ATTTGTAAAG

CTGTCTTTGA

CCAAAATATT

AGAAGAAAAG

TCTTAATGTC

GAAAAACTTT

CTATAAATGP,

TTCTGTGTAC

CCTCCCTCAA

AACAGAATTT

TTATTTAAGA

CAGTGTTTTA

AATGGAAAAG

CTTTTCCATT

CTAAAACACG

1949 2009 2069 2129 2189 2249 2309 2369 2429 2489 2549 2563 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 513 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Gly Ala Ala Ala TYPE: protein DESCRIPTION: SEQ ID Lys Leu Ala Phe Ala NO: 2.

Val Phe Ser Ser Gly Ala Phe Aen Ala Asn Pro Cys Tyr Gly so Ile Leu Gly Arg Trp Giu Arg Asp 40 Glu Thr Gln Leu Ile Ser Cys Glu Cys Leu Phe Thr Gly Val Giu Phe Ala Thr Trp Arg Thr Asn Gin Asp Lys Lys Arg Arg His Cys Lys Gin Lys Asn Ile Ser Gly Ile Glu Ile Val Gly Cys Trp Asp Asp Ile Asn Tyr Asp Arg Thr Cys Ile Glu Lys Lys Asp Ser Pro Gliu Lys Phe Ser 115 Pro Val Thr Val 100 Tyr Tyr Phe Cys Cys Giu Gly Asn Phe Pro Glu Pro Lye Pro Met Leu Ile IS0 Pro 135 Glu Val Thr Gin Tyr Asn Ile Leu 140 Ile Val Ile Cys 155 Met Cys Azn Giu 110 Pro Thr Ser Asn 125 Leu Tyr Ser Leu Ala Phe Trp Val 160 Val 145 Pro Leu Ala Gly WO 92/20793 WO 9220793Pcr/US92/ 03825 -46- Tyr Asp Gin Ala Asp 225 Met Thr Gly Leu Giu 305 Arg Cys Ala Glu Ile 385 Cys Glu Val Ala Asp Gin Val Leu Arg His His Pro Giy Pro 180 Leu Leu Giu 195 Gin Leu Leu 210 Lys Gin Ser Lys His Giu Ser Val Asp 260 Ser Leu Ser 275 Cys His Ile 290 Asp Ile Pro Asp Ile Lys Ile Ala Asp 340 Giy Asp Thr 355 Val Leu Giu 370 Asp Met Tyr Thr Aia Ala Glu Ile Gly 420 His Lys Lys 435 Gly Met Ala 450 Ala Glu Ala Met Gin Arg Val Thr Met 500 Lys 165 Pro Vai Asn Trp Asn 245 Val Asp Ala Gly Ser 325 Phe His Gly Al a Asp 405 Gin Lys Met Arg Leu 485 Val Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gin Pro Lys Giu Gin 230 Ile Asp Phe C lu Leu 310 Lys Gly Giy Ala Met 390 Gly His Arg Leu Leu 470 Thr Thr Pro Ala Tyr 215 Asn Leu Leu Leu Thr 295 Lys Asn Leu Gin Ile 375 Giy Pro Pro Pro

CYR

455 Ser Asn Asn 170 Leu Arg Val Giu Ile 250 Ile Asn Arg His Leu 330 Lys Thr Gin Leu Giu 410 Giu Arg Ile Cys Thr 490 Phe Leu Phe Lys Val 235 Gly Thr Val Gly Lys 315 Lys Phe Arg Arg Trp 395 Tyr Asp Asp Giu Val 475 Thr Pro Leu Cys 205 Phe Ser Giu Phe Ser 285 Ala Aia Asn Ala Tyr 365 Ala Leu Leu Gin Trp 445 Cys C iu Asp Lys 175 Lys Pro Leu 190 Val Trp Lys Pro Ile Gin Leu Pro Giy 240 Lys Arg Gly 255 His Glu Lys 270 Trp Asn Giu Tyr Leu His Ile Ser His 320 Leu Thr Ala 335 Giy Lys Ser 350 Met Ala Pro Phe Leu Arg Ala Ser Arg 400 Pro Phe Giu 415 Glu Val Vai 430 Gin Lys His Trp Asp His Arg Ile Thr 480 Ile Val Thr 495 Glu Ser*Ser 510 I WO 92/20793 WO 9220793PCI'/US92/03825 -47- INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2335 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (vii) IMMEDIATE SOURCE: CLONE: XACTR (ix) FEATURE: NAME/KEY: CDS LOCATION: 468.. 1997 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCGCCCACAC AGTGCAGTGA ATAATAGCCG GTGCGGCCCC TGTGTATCTG TCACATTGAA GTTTGGGCTC CTGTGAGTCT TGTGAAGCTG CTGCTGCAGA AGGTGGAGTC GTTGCATGAG TTTGATCTGC CTCTGCTCCC CATTCACACT CTCATTTCAT ACTCGCCTTT AACCCTTTCC CTGGCGGAGC CCACGCGTCT CGCTGAGCGA CCAGAGCGCG ACATTGTTGC GGCGGGGGAT AATCGGAGCT GCTGGGGGGG AACTGATACA ACGTTGCGAC CGAATGGGAT TTTATCTGTG TCGGTGAGAG AAGCGGATCC

TCCCCTCTTT

GAGCCTCCCC

GGTGGGGGGA

TCCCACGGAT

TTCATCCCTC

TGGGCGACAT

TGTAAAGCJAA

CCCTGGCAGT

CTGTGTCTCA

GTCGCTGCTG

CCACATTACA

CTGCCGCGGC

TGTTGCGAAT

TTAACTCGGC

120 180 240 300 360 420 476 CAGGAGC XTG GGG GCG :Aet Gly Ala 1 TCT GTA GCG CTG ACT TTT Ser Val Ala Leu Thr Phe GGA CAC GAT GAR Gly His Asp Glu

CTA

Leu

GAG

Glu

ACC

Thr CTT CTT CTT GCA Leu Leu Leu Ala ACA AGA GAG TGC Thr Arg Glu Cys MAC CAM AGT GGG Asn Gln Ser Gly CAC TGT TAC GCG His Cys Tyr Ala

ACT

Thr

ATC

I le TTC CGC GCA GGC Phe Arg Ala Gly TAT TAC MAT Tyr Tyr Asn TOG GMA CTG Trp Glu Leu GTG GMA AGC Val Glu Ser TGC GMA Cys Glu so GGG GMA MG Gly Glu Lys CGA CTC Arg Leu TCT TOG AGO MAC MAT Ser Trp Arg Asn Aen TOG CTG GAT GAC TTC Trp Leu Asp Asp Phe s0 620 668 716 764 TCO GGC TTC ATA Ser Gly Phe Ile MAC TOT TAT GAO Asn Cys ~yr Asp GAG CTG GTG Glu Leu Val AGA CAG GMA Arg Gln Olu AAA GGA TGC Lys Gly Cys TOT ATT GCC MAG Cys Ile Ala Lys GMA AAC CCC CMA Glu Asn Pro Gln WO 92/20793 WO 9220793Pcr/US92/03825 -48-

GTC

Val.

100

CAT

His

TCC

Ser

CCC

Pro

TCT

Ser 180

CGA

Arg

GTC

Val

GAG

Giu

GAG

Giu

TGG

Trp 260

AAA

Lys

ATG

Met

GGT

G ly

AAT

Asn

CTG

Leu 340

CAG

Gin

TTT

Phe

TTG

Leu rTTA

ATG

Met

TAC

Tyr 165

CCT

Pro 000 Gly

OCA

Ala

AAA

Lys

TTC

Phe 245

CTC

Leu

GGG

Gly

GCT

Ala

GMA

0 iu

GTA

Val 325

GCC

Ala

GTT

Val

TOC

Cys

GM%

Giu

AAC

Aen 135

ATT

Ile

CAT

His

GTC

Val

TTC

Phe

AAA

Lys 215

ATC

Ile

GCC

Aia

ACT

Thr

TTG

Leu

GGG

Giy 295

CAC

His

CTA

Leu

CGA

Arg

ACC

Thr

TGC

Cys

GTC

Val 120

ATT

Ile

CTC

Leu

GTA

Val.

000 Gly

GGT

Gly 200

ATC

Ile

TTC

Phe

OCT

Ala

GCA

Ala

GTG

Val.

280

CTG

Leu

MA

Lys

AGA

Arg

TTT

Phe

AGO

Arg 360

TGC

Cys 105

GMA

Giu

CTG

Leu

CTG

Leu

GAG

0 lu

CTG

Leu IB5

TGC

Cys

TTC

Phe

ACC

Thr

GAG

Glu

TTT

Phe 265

AGC

Ser 0CC Ala

CCT

Pro

MAC

Asn

GAG

Giu 345

AGO

Arg GAG GGA Giu Gly ACA TTT Thr Phe ATC TAT Ile Tyr GCG TTC Ala Phe 155 ATC AAT Ile Asn 170 AAG CCG Lys Pro GTC TG Val Trp CCC GTG Pro Val ACO COO Thr Pro 235 MAG AGG Lys Arg 250 CAT GAT His Asp TGG MAT Trp, Asn TAC TTA Tyr Leu OCA ATC Ala Ile 315 GAC CTG Asp Leu 330 CCT GGA Pro Gly TAT RTG Tyr Met

MOC

Asn

OAT

Asp

TOO

Ser 140

TG

Trp

GAG

Giu

CTG

Leu

AMA

Lys

CAG

Gin 220

GGC

Giy

GGA

Gly

RAG

Lys

GMA

Giu

CAT

His 300

GOT

Ala

ACT

Thr

AMA

Lys

GCT

Ala TAO TGO Tyr Cys 110 COG MAG Pro Lye 125 CTG OTT Leu Leu ATO TAC Met Tyr GAO CCC Asp Pro CAG TTG Gin Leu 190 OCT CGT Ala Arg 205 OAT MAG Asp Lys ATO AMA Met Lye AGO MAC Ser Asn GOT TOT Gly Ser 270 CTG TOT Leu Cys 285 GMA OAT Giu Asp CAC AGA His Arg 000 ATA Ala Ile COT CCG Pro Pro 350 CCT GAG Pro Giu 365

MOC

Asn

CCC

Pro

OCA

Pro

COT

Arg

GT

Oly 175

CTG

Leu

CTG

Leu

CAG

Gin

CAT

His

CTG

Leu 255

OTO

Leu

CAC

His

OTO

Val

OAT

Asp

TTA

Leu 335

OGA

Oly

GTT

Val MAG AMA TTT Lye Lye Phe CAG CO TCA Gin Pro Ser 130 ATT OTT GOT Ile Vai Gly 145 CAT OGA MAG His Arg Lye 160 CTG CCC COT Leu Pro Pro GAG ATA MAG Giu Ile Lye OTO MAT GMA Leu Asn Giu 210 TCG TOO CAG Ser Trp Gin 225 GMA MC CTA Oiu Aen Leu 240 GAO ATG GAG Giu Met Giu ACO GAO TAO Thr Asp Tyr ATA ACA GMA Ile Thr Olu 290 CCC COO TOT Pro Arg Cys 305 TTT AMA AGT Phe Lye Ser 320 OCA GAO TTC Ala Asp Phe OAT ACA CAC Asp Thr His OTA GAG OGA Leu Oiu Oly 370

ACT

Thr 115 0CC Ala

CTT

Leu

COT

Pro

CCA

Pro 000 Ala 195

TAT

Tyr

TOT

Cys

TTO

Leu

OTG

Leu

CTO

Leu 275

ACA

Thr

A

~Lys

MAG

Lys 000 Gly 000 Gly 355 GCh Ala 812 860 908 956 2004 1052 1100 1148 1196 1244 1292 1340 1388 1436 1484 1532 1580 'WO 92/20793 'WO 9220793PCT/US92/03825 -49- ATT AAC TTT Ile Asn Phe GGA CTG GTA Gly Leu Val 390 CGA GAT TCC TTT Arg Asp Ser Phe AGG ATA GAT ATG Arg Ile Asp Met CTC, TGG GAA ATA Leu Trp Giu Ile CGA TOT Arg Cys CCA GTA Pro Val 403 CCT TCC Pro Ser GAT GAG TAT CTO Asp Giu Tyr Leu CTA GAO OAT CTO Lou Oiu Asp Lei, 425 TTC AAA GAO CAC Phe Lys Asp His CCA TTC GAA GAA Pro Phe Giu Giu ACA OCA Thr Ala 400 GAG ATT Oiu Ile 415 CAC AAG His Lys TAT 0CC ATG Tyr Ala Met 385 OCA OAT 000 Al 1a Asp Gly 000 CAA CAT Gly Gin His 420

COT

GAA GTT GTC Oiu Val Vai CTO AAA CAC Lou Lys His 445

GTT

Val 430

OCT

Pro AAG ATA Lys Ile

OTA

Val.

TG

Trp GOT CTG GCC Giy Leu Ala TOO OTO ACC ATT Cys Vai Thr Ile 455 TOG GCA GO TOO Ser Ala Gly Cys 470 !.aO 000 ACT ACC Asn Gl]y Thr Thr 440- GAA GAA TOO Giu Oiu Cys GTA GAG GAG Val Glu Glu

TG

Trp CAA OTO Gin Lou 450 OAT GAT 000 GAA His Asp Ala Oiu GCA COO OTT Ala Arg Leu 465 A.AA TCA OTO Lys Ser Val 1628 1676 1724 1772 1820 1868 1916 1961 2017 2077 2137 2197 2257 2317 2335 TCLU CAA ATO Ser Gin Ile TOG GAO Ser Asp 485 AAT GTO Asn Val

TOO

Cys 490

AAA

Lys OTA TOO ATT Val Ser Ile

OTT

Val 495 ACA TOT OTO ACC Thr Ser VF~l Thr GAO TTG COO Asp Leu Pro GAG TOO AOT Glu Ser Ser TGAOGTTTOT TTGGTCTTTC

CAOAOTOAOT

TTTAATATTT

AAAGTATTAA

AGOTGOCGAO

CTAGAGAATG

GTCAAGCAAA

OACTTTTAAA

TTTTTOTTGG

AAAAAAAAAA

TTATGAATGC

TTCTOOTGGT

TGCTGCAG

AAAAAAAOTC ACGAATGCAG ?LTTTTAOTTG TCGGATCA CAAAAOAAAA AAGCAAAAAC CAATAOGTGC AGGOAACTTCA TTCCTTTATO TCAGAAGAGG

CTGCTATTT

ATTTAOOAGO

AGAOATOTCA

GAACCTCAAC

ACCCATAGGA

ATOTTOAOTT

ACGTCATTCG

GCAAGCATTC

AA=CTCATTT

AAACACCTAA

INFORMATION FOR SEQ ID NO:4: SEQUEN4CE CHARACTERISTICS: LENGTH: 510 amino acids TYPE: amino acid TOPOLOGY: linear (ii) 14OLECULE IiPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NOz4: Met Gly Ala Ser Val. Ala Lou Thr Phe Leu LP,4 Leu Leu Ala Thr Phe 1 5 10 Arg Ala Gly Ser Gly His Asp Glu Val Glu Thr Arg Glu Cys Ile Tyr 25 WO 92/20793 WO 9220793PCT/US92/03825 Tyr Ser Arg Asp Asn Lys Pro Val 145 Arg Pro Ile Asn Trp 225 Ass Met Asp Thr Arg 305 Lys Asp Thr Gin Asn Ala Cys Gin s0 Aen Aen Asp Phe Pro Gin Pbe Thr 115 Ser Ala 130 Gly Len Lye Pro Pro Pro Lye Ala 195 Giu Tyr 210 Gin Cys Leu Leu Giu Leu Tyr Leu 275 Giu Thr 290 Cys Lye Ser Lye Phe Gly His Gly 355 Gly Ala 370 Asn Gly Ser Ass Val 100 His Ser Ser Pro Ser 180 Arg Val Giu Giu Trp 260 Lye Met Giy Ass Leu 340 Gin Ile Trp Giu Giu Lye Gly Phe 70 Cys Tyr Phe Phe Leu Pro Val Leu Met Ala 150 Tyr Gly 165 Pro Leu Gly Arg Ala Val Lye Giu 230 Phe Ile 245 Leu le Gly Aen Ala Arg Gin Gly 310 Val Leu 325 Ala Val Val Gly Ass Phe Len Asp 55 Ile Asp Cys Gin Asn 135 Ile His Val Phe Lys 215 Ile Ala Thr Leu Gly 295 His Leu Arg Thr Gin 375 Gin 40 Lys Giu Arg Cys Val 120 Ile Leu Val Gly Gly 200 Ile Phe Ala Ala Val 280 Len Lys Arg Phe Arg 360 Arg Lys Arg Leu Gi1n Cys 105 Gin Leu Leu Giu Len 185 Cys Phe Thr Gin Phe 265 Ser Ala Pro Ass Gin 345 Arg Asp Thr L'eu Val Gin 90 Gin Thr Ile Ala Ile 170 Lys Val Pro Thr Lye 250 His Trp Tyr Ala Asp 330 Pro Tyr Ser Ass His Lys 75 Cys Gly Phe Tyr Phe 155 Asn Pro Trp Val, Pro 235 1\rg Asp Aen Leu Ile 315 Leu Gly Met Phe Gin Ser Cy8 Tyr Lye Gly Ile Ala Ass Tyr Asp Pro 125 Ser Leu 140 Trp Met Giu Asp Len Gin Lye Ala 205 Gin Asp 220 Giy Met Gly Ser Lye Gly Gin Leu 285 His Gin 300 Ala His Thr Ala Lye Pro Ala Pro 365 Len Arg 380 Gly Ala Cys Lye Cys 110 Lys Len Tyr Pro Len 190 Arg Lye Lye Asn Ser 270 Cys Asp Arg Ile Pro 350 Gin Ile Val Ser Trp Gin Ass Pro Pro Arg Gly 175 Leu Len Gin His Leu 255 Len His Val Asp Len 335 Gly Val Asp Gin Trp Leu Gin Lys Gin Ile His 160 Leu Gin Len Ser Gin 240 Gin Thr Ile Pro Phe 320 Ala Asp Len Met IWO 92/20793 WO 92/2793 !CTUS92/03825 Al a Asp Gin Ile Gin 450 Arg Ser Val Val 390 Asp Leu Phe Thr Gly 470 Thr Asp -51- Ile Val 395 Leu Pro 410 Gin Giu Trp Leu Cys Trp Giu Arg 475 Cys Leu 490 Lys Giu Cys Giu Val 430 Pro Asp Gin Ile Ile 510 SEQ ID NO.:

DLKPEN

SEQ ID ItO 6 G S )XX X SEQ ID N-Q. 7

DIKSKN

SEQ ID No.: 8

GTIPRYH

SEQ ID NO.: 9

DLAARN

SEQ ID No.:

Claims (26)

1. A novel receptor protein characterised by having the following domains, reading from the N-terminal end of said protein: an extracellular, ind-binding domain, S a hydrophobic, trans-membrane domain, and an intracellular, receptor domain having serine kinase-like activity, wherein said protein is further characterised by having sufficient binding affinity for at least one member of the activin/TGF-0 superfamily of polypeptide growth factors such that concentrations of 10 nM of said polypeptide growth factor occupy 50% of the binding sites of said receptor protein.

2. A protein according to claim 1, further comprising a second hydrophobic domain at the amino terminus thereof.

3. A protein having an amino acid sequence substant !ly the same as set forth in Sequence ID No. 2, Sequence ID No. or Sequence ID No. 4. 1i 4. A soluble, extracellular, ligand-binding protein, characterised by: having a sufficient binding affinity for at least one member of the activin/TGF-P superfamily of polypeptide growth factors such that concentrations of 10 nM of said polypeptide growth factor occupy 50% of the binding sites on said receptor protein, and having at least about 30% sequ 'nce identity with respect to: the sequence of amino acids 20-134 set forth in Sequence ID No. 2; the sequence of amino acids 20-134 set forth in Sequence ID No. 2. wherein the arginine residue at position number 39 is replaced by a lysine, and the isoleucine at residue number 92 is replaced by a valine; or the sequence of amino acids 21-132 set forth in Sequence ID No. 4.

5. A protein according to any one of claims 1 to 4, further characterised by: having a greater binding affinity for aetiviis tLan for inhibins, S. having substantially no binding affinity for transforming growth factors-p, and having substantiy no binding affinity for non-activin-like proteins.

6. A protein according to claim 4 or claim 5, wherein said protein comprises in 30 the range of about 114-118 amino acids.

7. A DNA encoding a protein according to any one of claims 1 to 6.

8. A DNA encoding a precursor-form of the protein according to any one of claims 1 to 6.

9. A DNA according to claim 7 having a contiguous nucleotide sequence substantially the same as set forth in Sequence ID No. 1, Sequence ID No. 1' or Sequence ID No. 3. A DNA according to claim 7 having a contiguous nucleotide sequence sulstantially the same as: K tucleotides 71-1609 of Sequence ID No. 1; r IN\LIORRIO0197MIARIGSA 52 of 7 variations of nucleotides 71-1609 of Sequence ID No. 1, wherein the codon for residue number 39 of the encoded amino acid codes for lysine, the codon for residue number 92 of the encoded amino acid codes for valine, and the codon for residue number 288 of the encoded amino acid encodes glutamine; nucleotides 468-1997 of Sequence ID No. 3; or variations of any of the above sequences whichl encode the same amino acid sequences, but employ different codons for some of the amino acids.

11. A DNA according to claim 10, wherein the contiguous nucleotide sequence comprises nucleotides 128-1609 of Sequence ID No. 1, and nucleotides 528-1997 of o1 Sequence ID No. 3.

12. A DNA according to clair a contiguous nucleotide sequence substantially the same as nucleotides 71-127 ,saaence ID No. 1, or nucleotides 468- 527 of Sequence ID No. 3.

13. A method for the recombinant production of activin receptor protein(s), said method comprising expressing the 5)NA of claim 7 in a suitable host cell.

14. A method according to claim 13, wherein the a-tive receptor is a soluble activin receptor(s). A DNA fragment useful as a hybridisation probe, wherein said DNA fragment S 20 comprises at least a portion of the DNA according to claim 7, and wherein said DNA fragment is labelled with a readily detectable substituent.

16. A DNA fragment according to claim 15 wherein said readily detectable substituent is selected from a radiolabelled molecule, a fluorescent molecule, an enzyme, or a ligand.

17. A method to identify clones encoding receptors of the activin/TGF-p superfamily, said method comprising: .,screening a genomic or cDNA library with a DNA fragment according to claim under low stringency hybridisation conditions, and identifying those clones which display a substantial degree of hybridisation to said S 30 DNA fragment.

18. A method for screening a collection of compounds to determine those compounds which bind to receptors of the activin/TGF-p superfamily, said method 4 comprising employing the receptor of claim 1 in a competitive binding assay,

19. A bioassay for evaluating whether compounds are agonists for receptor protein(s) according to claim 1, or functional modified forms of said receptor protein(s), said bioassay comprising: culturing cells containing: fDNA which expresses said receptor protein(s) or functional modified forms of said receptor protein(s), and '(N:LIaBRnI0097:IAR/ISA 53 o 7 54 DNA encoding a hormone response element operatively linked to a reporter gene, wherein said culturing is carried out in the presence of at least one compound whose ability to induce transcription activation activity of said receptor protein is sought to be determined; and thereafter monitoring said cells for expression of said reporter gene. A bioassay for evaluating whther compounds are antagonists for receptor protein(s) according to claim 1, or functional modified forms of said receptor protein(s), said bioassay comprising: culturing cells containing: DNA which expresses said receptor protein(s), or functional modified forms of said receptor protein(s), and DNA encoding a hormone response element operatively linked to a reporter gene; wherein said culturing is carried out in the presence of: increasing concentrations of at least one compound whose ability to inhibit transcription activation of said receptor protein(s) is sought to be determined, and a fixed concentration of at least one agonist for said receptor protein(s), or functional modified forms of said receptor protein(s); and thereafter monitoring in said cells the level of expression of the produ't of said reporter gene as a function of the concentration of said compound, thereby indicating the ability of said compound to inhibit activation of transcription.

21. A method for modulating the transcription trans-activation of activin receptor(s), said method comprising: contacting said receptor with an effective, modulating amount of the protein of claim 4.

22. An antibody generated against the protein of claim 4. .23. An antibody according to claim 22, wherein said antibody is a monoclonal *antibody. 30 24. A method according to claim 21, wherein the said modulation is carried out with the antibody of claim 22 or claim 23.

25. A novel receptor protein, substantially as hereinbefore described with reference to any one nf the Examples.

26. A soluble, extracellular, ligand-binding protein, substantially as hereinbefore described with reference to any one of the Examples.

27. A DNA encoding a protein, substantially as hereinbefore described with roeference to any one of the Examples.

28. A DNA encoding a precursor-form of the protein, substantially as hereinbefore described with reference to any one of the Examples. (NALIORRIa0197:IARIGSA 54 of 7

29. A method to identify clones encoding receptors of the activin/TGF-p superfamily, substantially as hereinbefore described with reference to any one of the Examples. A method for screening a collection of compounds to determine those compounds which bind to receptors of the activin/TGF-p superfamily, substantially as hereinbefore described with reference to any one of the Examples.

31. A bioassay for evaluating whether compounds are agonists for receptor protein, substantially as hereinbefore described with reference to any one of the Examples.

32. A bioassay for evaluating whether compounds are antagonists for receptor proteins, substantially as hereinbefore described with reference to any one of the Examples.

33. A method for modulating the transcription trans-activation of activin receptors, substantially as hereinbefore described with reference to any one of the Examples. Dated 7 September, 1994 The Salk Institute for Biological Studies Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON S I 0* e e INALIRR1001 97:IARIGSA 55 o17