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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) Internationa] Patent Classification 5 
C12N 15/12, C07K 15/00 
C12Q 1/68, G01N 33/53 
C12N 15/62, A61K 37/02 
C12P 21/08, A61K 39/395 



Al 



(11) International Publication Number: 



WO 92/20793 



(43) International Publication Date: 26 November 1992 (26.1 1.92) 



(21) International Application Number: PCT/US92/03825 

(22) International Filing Date : 8 May 3 992 (08.05.92) 



(30) Priority data: 

698,709 
773,229 



10 May 1991 (10.05.91) US 
9 October 1991 (09.10.91) US 



(71) Applicant: THE SALK INSTITUTE FOR BIOLOGICAL 

STUDIES [US/US]; 10010 North Torrey Pines Road, La 
Jolla, CA 92037 (US). 

(72) Inventors: MATHEWS, Lawrence, S. ; 3705 Coconino 

Court, San Diego, CA 92177 (US). VALE, Wylie, W. ; 
1643 Valdez, La Jolla, CA 92037 (US). 



(74) Agent: REITER, Stephen, R.; Pretty, Schroeder, Bruegge- 
mann & Clark, 444 South Flower Street, Suite 2000, Los 
Angeles, CA 90071 (US). 



(81) Designated States: AT (European patent), AU, BE (Euro- 
pean patent), CA, CH (European patent), DE (Euro- 
pean patent), DK (European patent), ES (European pa- 
tent), FR (European patent), GB (European patent), GR 
(European patent), IT (European patent), JP, LU (Euro- 
pean patent), MC (European patent), NL (European pa- 
tent), SE (European patent). 



Published 

With international search report 
Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: CLONING AND RECOMBINANT PRODUCTION OF RECEPTOR(S) OF THE ACTTVIN/TGF-B SUPER- 
FAMILY H 



N-terminus 



C-terminus 



kinase 
domain 



Second 

hydrophobic 

domain 



Ligand-binding 
domain 



Trans- 
membrane 
domain 



Intracellular 
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-mem- 
brane 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 growth factors 
such that concentrations of < 10 nM of said polypeptide growth factor occupy > 50 % of the binding sites of said receptor pro- 
tein. A presently preferred member of the invention superfamily of receptors binds specifically to activins, in preference to inhi- 
bins, transforming growth factor-P, and other non-activin-like proteins. DNA sequences encoding such receptors, assays employ- 
ing same, as well as antibodies derived therefrom, are also disclosed. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify Slates party to ihe PCI" on the fiont pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


Fl 


I -in land 


Ml. 


Mali 


AIJ 


Australia 


FR 


hraiiu: 


MN 


Mongolia 


BB 


Barbados 


CA 


Uabon 


MR 


Mauritania 


BE 


Belgium 


GB 


United Kingdom 


MW 


Malawi 


BP 


Burkina husu 


GN 


Guinea 


NL 


Netherlands 


BG 


Bulgaria 


GR 


Greece 


NO 


Norway 


BJ 


Benin 


HU 


Hungary 


PL 


Poland 


BR 


Brazil 


IE 


Ireland 


RO 


Romania 


CA 


Canada 


IT 


Italy 


RU 


Russian Federation 


CF 


{central African Republic 


JP 


Japan 


SD 


Sudan 


CC 


Congo 


KP 


Democratic People** Republic 


SB 


Sweden 


CH 


Swit/eitamJ 




of Korea 


SH 


Senegal 


CI 


Cole d'lvuirc 


KK 


Republic or Korea 


su 


Soviet Union 


CM 


Cameroon 


LI 


Liechtenstein 


TD 


Chad 


CS 


(Vcciwtuvuiia 


LK 


Sri Lanla 


TC 


Togo 


DE 


(iurniany 


Ul 


Luxembourg 


US 


United States of America 


UK 


Denmark 


MC 


Mutuco 






£S 


Spain 


MC 


Madagascar 







WO 92/20793 



-1- 



PCT/US92/03825 



CLONING AND RECOMBINANT PRODUCTION 
OF RECEPTOR ( S ) OF THE ACTIVIN/TGF-fl SUPERFAMILY 



ACKNOWLEDGEMENT 

This invention was made with Government support 
5 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 

10 

The present invention relates to receptor 
proteins, DNA sequences encoding same, and various uses 
therefor . 

15 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 
20 common subunit with inhibins, which inhibit FSH secretion. 

Activins are members of a super family of 
polypeptide growth factors which includes the inhibins, the 
transforming growth factors-6 (TGF-6) , Mullerian duct 
25 inhibiting substance, the Drosophila decapentaplegic 
peptide, several bone morphogenetic proteins, and the 
Vg-related peptides. 



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As a result of their extensive anatomical 
distribution and multiple biological actions, members of 
this superf amily of polypeptide growth factors are believed 
to be involved in the regulation of numerous biological 
5 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 
(e.g., FSH, GH and ACTH) , neuron survival, hypothalamic 
oxytocin secretion, erythropoiesis, placental and gonadal 
10 steroidogenesis, early embryonic development, and the like. 

Other members of the activin/TGF-B superf amily of 
polypeptide growth factors are involved in the regulation 
of cell function and cell proliferation for numerous cell 

15 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, 

20 endothelial cells, liver cells, epithelial cells, and the 
like. 

Chemical cross-linking studies with a number of 
cell types suggests that multiple binding sites (i.e., 

25 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 

30 understood. 

BRIEF DESCRIPTION OF THE INVENTION 

In accordance with the present invention, we have 
35 identified and characterized members of a new superfamily 
of receptor proteins which comprise three distinct domains: 
an extracellular, ligand-binding domain, a hydrophobic, 



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

Also provided are DNAs encoding the above- 
5 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 
10 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 
15 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. 

20 BRIEF DESCRIPTION OF THE FIGURES 

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

25 Figure 2 outlines the strategy used for 

expression cloning of a receptor of the activin/TGF-/? 
receptor superfamily. 

Figure 3 is a schematic of two mouse activin 
30 receptor clones. The top line of the figure is a 
restriction map, in kb, of mActRl and mActR2, with 
numbering starting from bp 1 of mActR2. The dotted line in 
the figure represents 5 1 untranslated sequences present 
only in mActRl. The middle lines present a schematic 
35 representation of two activin receptor cDNA clones. Boxes 

represent coding sequences black is the signal peptide, 

white is the extracellular ligand-binding domain, gray is 



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the transmembrane, and the intracellular kinase domain is 
hatched. Amino acids are numbered beneath the schematics. 

Figure 4 presents a comparison between activin 
5 receptor and daf-1 [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-1 are highlighted; 
conserved kinase domain residues are designated with an 
10 "*". 

Figure 5A summarizes results of 125 I activin A 
binding to COS cells transfected with pmActRl. Binding was 
competed with unlabeled activin A. For the runs reported 

15 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: 

20 660-672 (1979)]. 

Figure 5B summarizes results of 125 I activin A 
binding to COS cells transfected with pmActR2 . Binding was 
competed with unlabeled factors as indicated in the figure. 
25 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%. 

30 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 calculated using 

35 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 



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kinases are indicated in the figure. For those sequences 
that had similarity scores (i.e., a relative sequence 
identity) of at least 4 standard deviations above the mean 
(in comparison with all other known kinase sequences) , the 
5 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) . 

10 DETAILED DESCRIPTION OF THE INVENTION 

In accordance with the present invention, there 
is provided a novel superf amily of receptor protein (s) 
characterized by having the following domains, reading from 
15 the N-terminal end of said protein: 

an extracellular, ligand-binding domain, 
a hydrophobic, trans -membrane domain, and 
an intracellular domain having serine kinase-like 
activity. 

20 

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

25 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, 

30 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, 
35 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 



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intracellular domains of the receptor. The hydrophobic 
nature of this domain serves to anchor the receptor to the 
cell membrane. See Figure 1. 

5 As employed herein, the phrase "intracellular 

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 
10 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 
15 promotes the intracellular transport of the initially 
expressed receptor protein across the Golgi membrane. See 
Figure 1. 

Members of the invention superf amily of receptors 
20 can be further characterized as having sufficient binding 
affinity for at least one member of the activin/TGF-6 
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. 

25 

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 

30 concentration of ligand necessary to occupy one-half (50%) 
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, 

35 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 



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-7- 

number) . 

Reference to receptor protein "having sufficient 
binding affinity such that concentrations of said 
5 polypeptide growth factor less than or equal to 10 nM 
(i.e., < 10 nM) occupy > 50% (i.e., greater than or equal 
to one-half) of the binding sites of said receptor protein" 
means that ligand (i.e., polypeptide growth factor) 
concentration (s) of no greater than about 10 nM are 

10 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 

15 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 super family of receptors 
can be divided into various subclasses, based on the 

20 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-£ 
receptors are those which form a crosslinked complex of 

25 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. 

30 

Each member of a given subclass is related to 
other members of the same subclass by the high degree of 
homology (e.g., >80% overall amino acid homology; 
frequently having >90% overall amino acid homology) between 
35 such receptors; whereas members of a given subclass differ 
from members of a different subclass by the lower degree of 
homology (e.g., at least about 30% up to 80% overall amino 



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-8- 

acid homology; with in the range of about 40% up to 90% 
amino acid homology specifically in the kinase domains 
thereof) between such receptors* Typically, related 
receptors have at least 50% overall amino acid homology; 
5 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. 

10 

Based on the above criteria, the receptors 
described herein are designated Type II receptors, with the 
first discovered Type II receptor (i.e., the mouse-derived 
activin receptor) being designated ActRII, while 
15 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. 

20 

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 
25 have: 

substantially no binding affinity for transforming 
growth factors-B, and 

substantially no binding affinity for non-activin-like 
proteins or compounds. 

30 

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. 

35 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. 



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-9- 

As employed herein, "activin" refers to activin 
A (a homodimer of two inhibin B A subunits) , activin B (a 
homodimer of two inhibin B B subunits) , activin AB (a 
heterodimer composed of one inhibin B A subunit and one 
5 inhibin B B subunit) ; "inhibin" refers to inhibin A (composed 
of the inhibin a subunit and an inhibin B A subunit) , inhibin 
B (composed of the inhibin a subunit and an inhibin fi B 
subunit) ; "transforming growth factor B or TGF-B" refers to 
TGF-B1 (a homodimer of two TGF-B1 subunits) , TGF-B2 (a 
10 homodimer of two TGF-B2 subunits) , TGF-B3 (a homodimer of 
two TGF-B 3 subunits), TGF-B4 (a homodimer of two TGF-B4 
subunits) , TGF-B5 (a homodimer of two TGF-B5 subunits) , 
TGF-B1.2 (a heterodimer of one TGF-B 1 subunit and one 
TGF-B2 subunit), and the like. 

15 

Transforming growth factors -/? (TGF-jfe) are 
members of the activin/ TGF-/? superf amily of polypeptide 
growth factors, TGF-Bs are structurally related to 
activins, sharing at least 20-30% amino acid sequence 
20 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. 

25 

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

30 

Preferred members of the invention superf amily 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, 
35 reading from the N-terminal end of said receptor: 

the extracellular, ligand-binding domain 
preferably will have in the range of about 114-118 



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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 
5 domain, and 

the intracellular domain having kinase-like 
activity preferably will have in the range of about 
345-3 60 amino acids, beginning at the carboxy terminus 
of the hydrophobic, trans-membrane domain. 

10 

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 (i.e., at the amino terminus of the extracellular, 

15 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 

20 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 
25 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 
30 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 
35 receptor proteins of the invention include the sequence set 
forth in Sequence ID No. 2 (which represents a mouse 
activin receptor amino acid sequence) , a modified form of 



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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 
5 modified form of Sequence ID No. 1 is referred to 
hereinafter as "Sequence ID No. 1 ,M , 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 

10 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 

15 resulting receptor species. 

In accordance with another embodiment of the 
present invention, there is provided a soluble, 
extracellular, ligand-binding protein, further 
20 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 
25 on said receptor protein, and 

having at least about 30% sequence identity with 
respect to: 

the sequence of amino acids 20-134 set forth in 
Sequence ID No. 2; 
30 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 

35 the sequence of amino acids 21-132 set forth in 

Sequence ID No. 4. 



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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: 
5 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 
10 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, 
15 ligand-binding proteins having at least about 80% sequence 
identity with respect to the above-referenced fragments of 
Sequence ID Nos. 2 or 4 . 

Members of the class of soluble, ligand-binding 
20 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 
25 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 
30 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. 

35 Presently preferred soluble, extracellular, 

ligand-binding proteins of the present invention are 
further characterized by: 



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having a greater binding affinity for activins than 
for inhibins, 

having substantially no binding affinity for 
transforming growth factors-B, and 
5 having substantially no binding affinity for 

non-activin-like proteins. 

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

Especially preferred soluble, extracellular, 
ligand-binding proteins of the invention are those having 
substantially the same amino acid sequence as that set 
15 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 
20 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 

25 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 

30 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. 

35 The above-described soluble proteins can be 

employed for a variety of therapeutic uses, e.g., to block 
receptors of the invention from affecting processes which 



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the 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-ligand complex, thereby 
5 blocking the normal regulatory action 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 
10 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. 

15 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. 

20 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 

25 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, 

30 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 

35 readily determine dose forms, treatment regiments, etc, 
depending on the mode of administration employed. 



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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 
5 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 
10 numerous DNA sequences, e.g., 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 
15 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 
20 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 
25 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 
30 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. 

35 

Another DNA which encodes a receptor of the 
invention is one having a contiguous nucleotide sequence 



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substantially 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 
5 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 
10 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 
15 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 
20 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 

25 employed to probe library (ies) (e.g., cDNA, genomic, and 
the like) 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 °C, a 

30 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 37 °C, a formamide concentration of about 20%, and a 
salt concentration of about 5X standard saline citrate 

35 (SSC; 2 OX SSC contains 3M sodium chloride, 0.3M sodium 
citrate, pH 7.0). Such conditions will allow the 
identification of sequences which have a substantial degree 



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of 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, 
5 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. 

10 In accordance with yet another embodiment of the 

present invention, there is provided a method for the 
recombinant production of receptor (s) of the invention by 
expressing the above-described DNA sequences in suitable 
host cells. 

15 

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 

20 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 

25 conditions under which expression occurs. 

In accordance with a further embodiment of the 
present invention, there is provided a binding assay 
employing receptors of the invention, whereby a large 

30 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 

35 antagonists of invention receptors. 



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Another application of the binding assay of the 
invention is the assay of test samples (e.g., biological 
fluids) for the presence or absence of members of the 
activin/TGF-B superfamily of polypeptide growth factors. 
5 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 
10 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 
15 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 
20 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 
25 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 
30 comprises: 

(a) culturing cells containing: 

DNA which expresses said receptor 
protein (s) or functional modified forms of 
said receptor protein (s), and 
35 DNA encoding a hormone response element 

operatively linked to a reporter gene; 
wherein said culturing is carried out in the 



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presence of at least one compound whose ability 
to induce transcription activation activity of 
receptor protein is sought to be determined , and 
thereafter 

5 (b) 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 
10 receptor (s) of the invention, or functional modified forms 
of said receptor (s) , comprises: 

(a) culturing cells containing: 

DNA which expresses said receptor 
protein (s), or functional modified forms of 
15 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; 

20 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 

25 agonist for said receptor protein (s) , or 

functional modified forms of said receptor 
protein (s) ; and thereafter 

(b) monitoring in said cells the level of expression 
of the product of said reporter gene as a 

30 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 
35 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 



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of SV-40; and the reporter and expression plasmids employed 
also typically contain a selectable marker. 

The hormone response element employed in the 
5 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 
acetytransf erase (CAT), lucif erase, B-galactosidase, and 
10 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 [e.g., by color imetry 
15 (with a colored reporter product such as B-galactosidase) , 
by fluorescence (with a reporter product such as 
lucif erase) , etc] , by enzyme activity, and the like. 

Compounds contemplated for screening in 
20 accordance with the invention bioassays include activin- or 
TGF-/?-like compounds, as well as compounds which bear no 
particular structural or biological relatedness to activin 
or TGF-0. 

25 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 fi k or fi B chains (either singly or in any 

30 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-/?-like 
compounds include activin A (a homodimer of two inhibin B A 

35 subunits) , activin B (a homodimer of two inhibin B e 
subunits) , activin AB (a heterodimer composed of one 
inhibin B A subunit and one inhibin B B subunit) , inhibin A 



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( composed of the inhibin a subunit and an inhibin B A 
subunit) , inhibin B (composed of the inhibin a subunit and 
an inhibin B B subunit) , TGF-B1 (a homodimer of two TGF-B1 
subunits) , TGF-B2 (a homodimer of two TGF-B2 subunit s) , 
5 TGF-B3 (a homodimer of two TGF-B3 subunits) , TGF-B4 (a 
homodimer of two TGF-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. 

10 Examples of compounds which bear no particular 

structural or biological relatedness to activin or TGF-/?, 
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 

15 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. 

20 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 (e.g., pituitary cells, placental cells, 

25 fibroblast cells, and the like) . Then, the ability of the 
resulting mammalian cells to bind a labeled receptor ligand 
(i.e., 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 

30 of that cDNA, when expressed in mammalian cells, to induce 
(or enhance) the binding of labeled receptor ligand to said 
cell. 

In addition to the above-described applications 
35 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 



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example, 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 
5 either be enhanced (for example, by supplying the subject 
with increased amounts of the activin receptor, relative to 
the amount of endogenous receptor, e.g., by transfecting 
the subject with a tissue specific activin-encoding 
construct), or depressed (e.g., by administration to a 

10 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 

15 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 

20 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) , 

25 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 
30 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. 

35 

As still further examples of the wide utility of 
the invention compositions, agonists identified for TGF-6 



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specific receptors can be used to stimulate wound healing, 
to suppress the growth of TGF-B-sensitive tumors, to 
suppress immune response (and thereby prevent rejection of 
transplanted organs) , and the like. Antagonists or the 
5 soluble, ligand-binding domain derived from TGF-6 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 
10 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. 



15 



EXAMPLES 



Eecombinant human (rh) activin A, rh activin B, 
and rh inhibin A were generously provided by Genentech, 
20 Inc. Porcine TGF-B 1 was obtained from R+D Systems. 

Double-stranded DNA was sequenced by the dideoxy 
chain termination method using the Seguenase reagents from 
US Biochemicals. Comparison of DNA seguences to databases 
25 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 

30 

Polyadenylated RNA was prepared from AtT20 cells 
using the Fast Track reagents from InVitrogen. cDNA was 
commercially synthesized and ligated into the plasmid 
vector pcDNAl using non-palindromic BstXI linkers, yielding 
35 a library of approximately 5xl0 6 primary recombinants. The 
unamplified cDNA library was plated at 1000 clones per 100 
mm plate, then scraped off the plates, frozen in glycerol 



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and stored at -70°. 

Activin suppresses adrenocorticotropic hormone 
(ACTH) secretion by both primary anterior pituitary cell 
5 cultures [Vale et al., Nature 321: 776-779 (1986)] and 
AtT20 mouse corticotropic cells. Because AtT20 cells 
possess activin receptors indistinguishable from those on 
other cell types (based on binding affinity measurements 
with activin A) , these cells were chosen to be the source 

10 of cDNA for transf ection. A cDNA library of approximately 
5xl0 6 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 

15 binding to single transf ected cells. The library was 
divided into pools of 1000 clones, DNA was prepared from 
each pool of clones and transiently transfected into COS 
cells, and the cells screened for the capacity to bind 
iodinated activin A. Binding was assessed by performing 

20 the transf ections 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 transfected with an 
activin receptor cDNA, and consequently bound radioactive 

25 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 

30 (#64) was identified by transf ecting and analyzing DNA from 
each pool of 1000 singly, and then was further fractionated 
until a single clone (pmActRl) was purified which generated 
>10 4 positive cells after transf ection (see Table 1) . 



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Table 1 

Purification of the activin receptor clone from 
the AtT20 library 

5 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 

10 pmActRl 1 >10* 



The total number of transf ected cells capable of binding I 
activin A in a field of 2xl0 5 COS cells was counted for 
15 pools of clones at each stage of the purification process. 

pmActRl contained a 1.7 kb insert, coding for a 
protein of 342 amino acids (Figure 3) ; however, it was 
incomplete on the 3 1 end, thus the last 17 amino acids were 

20 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 3). Screening 
6xl0 5 colonies yielded one additional positive clone 
(pmActR2) which had a 2.6 kb insert and contained the 

25 entire coding sequence for the mouse activin receptor 
(Figure 3) . The nucleic acid sequence and the deduced 
amino acid sequence of the insert in pmActR2 are set forth 
in Sequence ID No. 1. 

30 EXAMPLE II 

COS Cell Transfection 

Aliquots of the frozen pools of clones were grown 
overnight in 3 ml cultures of terrific broth, and mini-prep 
35 DNA prepared from 1.5 ml using the alkaline lysis method 
[Maniatis et al. Molecular Cloning (Cold Spring Harbor 
Laboratory (1982)]. 1/10 of the DNA from a mini-prap (10 



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Ml of 100 Ml) was used for each transfection. 

2xl0 5 COS cells were plated on chambered 
microscope slides (1 chamber - Nunc) that had been coated 
5 with 20 /xg /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 1 s medium (DME) containing 100 mM 
chloroquine was added to the cells. DNA was precipitated 

10 in 200 ml DME/ chloroquine containing 500 mg/ml DEAE- 
Dextran, then added to the cells. The cells were incubated 
at 37° 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 

15 3 days later. For transf ections with the purified clone, 
2.5X10 6 cells were transf ected in 100 mm dishes with 5 /zg 
purified DNA. The total transfection volume was 10 ml, and 
the DNA was precipitated in 400 /il. 

20 EXAMPLE III 

Binding Assay 

Cells were washed 2x with HEPES buffered saline 
(HDB) containing 0.1% BSA, then incubated for 90 minutes at 

25 22° in 0.5 ml HDB, 0.1% BSA containing 7xl0 5 cpm 125 I activin 
A (approximately 7 ng, 500 pM) . The cells were then washed 
3X with cold HDB, fixed for 15 minutes at 22° in 2.5% 
glutaraldehyde/HDB and washed 2X with HDB. The chambers 
were then peeled off the slides, and the slides dehydrated 

30 in 95% ethanol, dried under vacuum, dipped in NTB2 
photographic emulsion (Kodak) and exposed in the dark at 4° 
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 

35 Sciences) . The slides were analyzed under darkf ield 
illumination using a Leitz microscope. 



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EXAMPLE IV 
Subdivision of Positive Pool 

Of 300 pools screened (each pool containing about 
5 1000 cDNAs) , one positive pool (#64), which produced two 
positive cells, was identified. Bacteria from the frozen 
stock of this positive pool (#64) were replated at 
approximately 400 clones per plate, replica plates were 
made, and DNA was prepared from each subpool and analyzed 

10 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 

15 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/2xl0 5 cells. 

20 

EXAMPLE V 
Radioreceptor Assay 

10 5 COS cells transfected with either pmActRl or 
25 pmActR2, or 10 6 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 22° for 90 
minutes in 0.5 ml HDB, 0.1% BSA containing 100,000 cpm 
(approximately 1 ng, 75 pM) 125 I activin A (5 ng activin A 
30 was iodinated by chloramine T oxidation to a specific 
activity of 50-90 fiCi/fig; 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 
35 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 



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binding 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 
5 Rodbard, D. , Anal. Biochem. 107:220-259 (1980)]. 



EXAMPLE VI 
Chemical Cross-linking 

10 2x1 0 6 COS cells, or 5xl0 6 AtT20 cells, were washed 

2x with HDB, scraped off the dish, incubated for 90 minutes 
at 22° under constant rotation in 0.5 ml HDB containing 
7xl0 5 cpm (approximately 500 pM) 125 I activin A with or 
without 500 ng (37 nM) unlabeled activin A. Cells were 

15 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 7.5, 

20 100 mM NaCl, then the cells were pelleted by 
centrifugation, resuspended in 100 pi 50 mM Tris-HCl pH 
7.5, 1% Triton X-100 and incubated at 0° for 60 minutes. 
The samples were centrifuged 5 minutes at 13,000xg, and the 
Triton-soluble supernatants analyzed by SDS-PAGE using 8.5% 

25 polyacrylamide gels. The gels were dried and subjected to 
autoradiography for 4-14 days. 



EXAMPLE VII 
RNA Blot Analysis 

30 

Total RNA was purified from tissue culture cells 
and tissues using LiCl precipitation. 20 jig total RNA was 
run on 1.2% agarose, 2.2M formaldehyde gels, blotted onto 
nylon membranes (Hybond - NEN) , and hybridized with a 0.6 
35 kb Kpnl fragment (see Figure 3) which had been labeled with 
32 P by random priming using reagents from US Biochemicals. 
Hybridization was performed at 42° in 50% formamide, and 



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the filters were washed at 65° in 0.2X SSC. 

EXAMPLE VIII 
Sequence Analysis 

5 

Full length mouse activin receptor clone encodes 
a protein of 513 amino acids, with a 5' untranslated region 
of 70 bp and a 3 1 untranslated region of 951 bp. pmActR2 
does not contain a poly A tail, although it does have a 

10 potential poladenylylation 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 

15 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 1 untranslated region; however, none of these is in 
an appropriate context for initiation, and all are followed 

20 by in-frame stop codons. While this unusually long 5 1 
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) . 

25 

Hydropathy analysis using the method of Kyte and 
Doolittle [J. Mol. Biol. 152:105-132 (1982)] revealed two 
hydrophobic regions: a 10 amino acid stretch at the amino 
terminus assumed to be a single peptide, and a single 

30 putative 26 residue membrane-spanning region between amino 
acids 119-142 (see Figure 1 and Sequence ID No. 2). The 
signal peptide contains the conserved n-, h- and c- domains 
common to signal sequences; the site of cleavage of the 
signal peptide, before Ala 1 , is predicted based on rules 

35 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 



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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, 
5 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 non- 
receptor 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, 
A.M. , Meth. Enzymol. 200:38-62 (1991) and Hanks et al., 
Science 24l ;42-52 (1988)]; the activin receptor sequence 
has all of these conserved subdomains in the proper order 
(Figure 4). A conserved Gly in subdomain I is replaced by 
Ala 180 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 
25 substrate specificity [Hanks et al., (1988) supra] . The 
sequence of the mouse activin receptor in both of these 
subdomains is characteristic of serine kinases. 



10 



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PCT/US92/03825 



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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 
5 and VIII f indicating that it is not a standard tyrosine 
kinase. The receptor could potentially autophosphorylate 
at Ser 333 or Thr 337 . One interesting additional possibility 
is that the activin receptor kinase may have specificity 
for serine, threonine and tyrosine residues. Several 
10 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, P., Nature 
349 :808-811 (1991)]. 

15 

Phylogenetic analysis of the activin receptor 
compared to 161 other kinase sequences revealed that the 
activin receptor and the C.elegans protein, daf-1 [Georgi 
et al., Cell 61:635-645 (1990)] may constitute a separate 

20 subfamily of kinases (see Figure 6) . daf-1 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 6) . Like the activin 
receptor, daf-1 is predicted to be a transmembrane 

25 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 

30 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, R. , Nature 345:743-746 

35 (1990)]. 



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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 
5 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 
10 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 

15 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 transfected with either pmActRl or 

20 pmActR2. Cells transfected with either construct bound 
activin A with a single high affinity component (Kd = 
180 pM; Figure 5) , indicating that a functional 
(structurally complete) intracellular kinase domain is not 
required for ligand binding. This binding affinity is 

25 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., 

30 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 

35 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 



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level of expression per cell varies considerably, though, 
based on the number of accumulated silver grains. This 
value is comparable to the expression of other transfected 
cell surface proteins in COS cells. 

5 

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; 

10 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 

15 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 

20 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 
25 analyzed by affinity cross-linking 125 I activin A to COS 
cells transfected with pmActR2 using the bifunctional 
chemical cross-1 inker, disuccinimidyl suberate (DSS) . A 
major cross-linked band of 84 kDa was observed in 
transfected, but not in untransf ected cells. Subtracting 
30 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. Cross- 
linking 125 I activin A to AtT20 cells yields a major band of 
65 kDa, with minor bands of approximately 78 and 84 kDa. 
35 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 



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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 
5 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 ; Centrella et al., Mol. Cell. Biol- 11:250-258 

10 (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 

15 Massague et al., Ann. N.Y. Acad. Sci. 593 ; 59-72 (1990)]. 

EXAMPLE X 
Expression of Activin Receptor mRNA 

20 The distribution of activin receptor mRNA was 

analyzed by Northern blot. Two mRNA species, of 6.0 and 
3.0 kb, were observed in AtT20 cells as well as a number of 
mouse tissues, including brain, testis, pancreas, liver and 
kidney. The total combined size of the inserts from 

25 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 

30 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 
35 transcripts varies depending on the source. While AtT20 
cells have approximately equal levels of both mRNAs, most 
tissues had much greater levels of the 6.0 kb transcript, 



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with little or no expression of the 3.0 Jcb transcript. 
Testis, on the other hand, had a greater amount of the 3.0 
Jcb band. Expression of activin receptor mRNA in brain, 
liver and testis is in accord with described biological 
5 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]. 

10 

EXAMPLE XI 

Tdentification of a Huma n Activin Receptor 

A human testis library (purchased from Clontech; 
15 catalog no. HLlOlOb) was probed with the full length mouse 
activin receptor gene (see Sequence ID No. 1) under the 
following conditions: 

Hybridization stringency; 

20% formamide, 6X SSC at 42 °C; 
20 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. l 1 ) . 
25 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. 

30 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- 
35 325 (1987) was probed with the full length mouse activin 
receptor gene (see Sequence ID No. 1) under the following 
conditions: 



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Hvbridization stringency: 

20% formamide, 6X SSC at 42 °C; 
Wash stringency! 

2X SSC, 0.1% SDS at 42°C. 

5 

A sequence having a substantial degree of 
homology with respect to the mouse activin receptor was 
identified (Sequence ID No. 3). The degree of overall 
amino acid homology (relative to the mouse acitvin 

10 receptor) is only about 69% (with 77% 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 

15 subclass than the mouse receptor described above. 

EXAMPLE XIII 
Functional Assays of ActRs in Xenopus embryos 

20 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 time animal caps were dissected and 

25 treated overnight with different concentrations of activin. 
The xActRIIB cDNA was cloned into rp64T [see Krieg and 
Melton in Methods in Enzymology, Abelson and Simon, Eds. 
(Academic Press, New York, 1987), vol. 155, p. 397] and 
transcribed in vitro to generate a capped, synthetic 

30 xActRIIB RNA [see Melton et al., in Nucleic Acids Res. 
12:7035 (1984) and Kintner in Neuron 1:545 (1988)]. 
Embryos at the two- to four-cell stage were injected with 
about 20 nl of RNA at concentrations of 0.02 ng/nl, or 0.1 
ng/nl, spread between four quadrants of the animal pole. 

35 At stage 9, animal caps were removed from RNA-injected 
embryos and incubated in 0.5x modified mammalian Ringer's 
(MMR) , 0.1% bovine serum albumin (BSA) with different 



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concentrations of purified, porcine activin A (six caps per 
incubation) . After 20 hours in culture, total RNA was 
prepared. 

5 The response of the caps to activin was assessed 

by quantifying muscle-specific actin RNA with a 
ribonuclease protection assay as per Blackwell and 
Weintraub, Science 250:1104 (1990). Embryos injected with 
0.4 and 2.0 ng of xActRIIB RNA were approximately 10- and 
10 100-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 probably 
a consequence of contamination of the animal cap with a 
small amount of marginal zone tissue. 

15 

The amount of muscle actin decreased with 
increasing concentration of activin in the embryos injected 
with 2 ng of xActRIIB RNA. This is consistent with the 
observation that isolated animal cap cells uniformly 

20 exposed to different concentrations of activin only form 
muscle cells in response to a narrow range of activin 
concentrations [see Blackmann and Kadesch in Genes and 
Development 5:1057 (1990)]. The present results indicate 
that the concentration of ligand and the amount of receptor 

25 are both important in determining the signal transmitted. 
Thus, the range of activin concentrations that lead to 
muscle differentiation is lower in animal cap cells from 
injected embryos, which are expressing more receptor than 
normal , than from uninjected embryos. 

30 

EXAMPLE XIV 
Analysis of kinase activity of mActRII 

A fragment of cDNA corresponding to the entire 
35 intracellular domain of mActRII (amino acids 143-494) was 
subcloned into the vector pGEX-2T [see Smith and Johnson in 
Gene 62:31-40 (1988)], creating a fusion protein between 



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glutathione S-transf erase (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 
5 Smith and Johnson. Approximately 100-200 ng of fusion 
protein, or of purified GST, were incubated with 25 /iCi 
[K- 32 P] ATPin a buffer containing 50 mM Tris, 10 mM MgCl 2 for 
30 minutes at 37 °C. The products were analyzed by SDS-PAGE 
and autoradiography. The fusion protein, but not the GST 
10 alone, became phosphorylated, indicating that the kinase 
domain of the fusion protein was functional. Phosphoamino 
acid analysis, performed according to Cooper et al. [Meth. 
Enzym. 99:387 (1983)], indicated that the predominant amino 
acid residue that became phosphorylated was threonine. 

15 

While the invention has been described in detail 
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 
20 claimed. 



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. SUMMARY OF SEQUENCES 

Sequence ID No, 1 is the nucleic acid sequence 
(and the deduced amino acid sequence) of a cDNA encoding a 
5 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 

10 Sequence ID No. 1, except that the codon for amino acid 
residue number 39 encodes lysine (i.e., nucleotides 185-187 
are AAA or AAG) , the codon for amino acid residue 92 
encodes valine (i.e., nucleotides 344-346 are GTN, wherein 
N is A, C, G or T) , and the codon for amino acid residue 

15 number 288 encodes glutamine (i.e., nucleotides 932-934 are 
CAA or CAG) . 

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

Sequence ID No. 2' is an amino acid sequence for 
a human-derived activin receptor of the present invention. 
Sequence ID No. 2 f is substantially the same as Sequence ID 
25 No. 2, except that amino acid residue number 39 is lysine, 
amino acid residue 92 is valine, and amino acid residue 
number 288 is glutamine. 

Sequence ID No. 3 is the nucleic acid sequence 
30 (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 
35 present invention. 



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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 
5 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. 

10 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 
15 sequence. 

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

20 



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SEQUENCE LISTING 



(1} GENERAL INFORMATION: 

(i) 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: 10 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: PRETTY, SCHROEDER, BRUEGGEMANN & CLARK 

(B) STREET: 444 South Flower Street, Suite 2000 

(C) CITY: Los Angeles 

(D) STATE: California 

(E) COUNTRY: United States 

(F) ZIP: 90071-2921 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

(C) OPERATING SYSTEM: PC-DOS /MS-DOS 

(D) SOFTWARE: Patentln Release #1.0, Version #1. 25 

(vi) CURRENT APPLICATION DATA: 

(A) APPLICATION NUMBER: US 

(B) FILING DATE: 08-MAY-1992 

(C) CLASSIFICATION: 

(viii) ATTORNEY /AGENT INFORMATION: 

(A) NAME: Re iter, Mr., Stephen E. 

(B) REGISTRATION NUMBER: 31192 

(C) REFERENCE /DOCKET NUMBER: P31 9309/FP31 9291 

(ix) TELECOMMUNICATION INFORMATION: 

(A) TELEPHONE: (619) 546-4737 

(B) TELEFAX: (619) 546-9392 



(2) INFORMATION FOR SEQ ID NO:l: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2563 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 71.. 1609 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: 
CTCCGAGGAA GACCCAGGGA ACTGGATATC TAGCGAGAAC TTCCTACGGC TTCTCCGGCG 60 



CCTCGGGAAA ATG GGA GCT GCT GCA AAG TTG GCG TTC GCC GTC TTT CTT 
Met Gly Ala Ala Ala Lys Leu Ala Phe Ala Val Phe Leu 
15 10 



109 



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ATC TCT TGC TCT TCA GGT GCT ATA CTT GGC AGA TCA GAA ACT CAG GAG 157 
lie Ser Cys Ser Ser Gly Ala lie Leu Gly Arg Ser Glu Thr Gin Glu 
15 20 25 

TGT CTT TTC TTT AAT GCT AAT TGG GAA AGA GAC AGA ACC AAC CAG ACT 205 
Cys Leu Phe Phe Abxi Ala Asn Trp Glu Arg Asp Arg Thr Asn Gin Thr 
30 35 40 45 

GGT GTT GAA CCT TGC TAT GGT GAT AAA GAT AAA CGG CGA CAT TGT TTT 253 
Gly Val Glu Pro Cys Tyr Gly Asp Lye Asp Lys Arg Arg His Cys Phe 
50 55 60 

GCT ACC TGG AAG AAT ATT TCT GGT TCC ATT GAA ATA GTG AAG CAA GGT 301 
Ala Thr Trp Lys Asn lie Ser Gly Ser lie Glu lie Val Lys Gin Gly 
65 70 75 

TGT TGG CTG GAT GAT ATC AAC TGC TAT GAC AGG ACT GAT TGT ATA GAA 349 
Cys Trp Leu Asp Asp He Asn Cys Tyr Asp Arg Thr Asp Cys He Glu 
80 85 90 

AAA AAA GAC AGC CCT GAA GTG TAC TTT TGT TGC TGT GAG GGC AAT ATG 397 
Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met 
95 100 105 

TGT AAT GAA AAG TTC TCT TAT TTT CCG GAG ATG GAA GTC ACA CAG CCC 445 
Cys Asn Glu Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gin Pro 
110 115 120 125 

ACT TCA AAT CCT GTT ACA CCG AAG CCA CCC TAT TAC AAC ATT CTG CTG 493 
Thr Ser Asn Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn He Leu Leu 
130 135 140 

TAT TCC TTG GTA CCA CTA ATG TTA ATT GCA GGA ATT GTC ATT TGT GGA 541 
Tyr Ser Leu Val Pro Leu Met Leu He Ala Gly He Val He Cys Ala 
145 150 155 

TTT TGG GTG TAC AGA CAT CAC AAG ATG GCC TAC CCT CCT GTA CTT GTT 589 
Phe Trp Val Tyr Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val 
160 165 170 

CCT ACT CAA GAC CCA GGA CCA CCC CCA CCT TCC CCA TTA CTA GGG TTG 637 
Pro Thr Gin Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu 
175 180 185 

AAG CCA TTG CAG CTG TTA GAA GTG AAA GCA AGG GGA AGA TTT GGT TGT 685 
Lys Pro Leu Gin Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys 
190 195 200 205 

GTC TGG AAA GCC CAG TTG CTC AAT GAA TAT GTG GCT GTC AAA ATA TTT 733 
Val Trp Lys Ala Gin Leu Leu Asn Glu Tyr Val Ala Val Lys He Phe 
210 215 220 

CCA ATA CAG GAC AAA CAG TCC TGG CAG AAT GAA TAT GAA GTC TAT AGT 781 
Pro He Gin Asp Lys Gin Ser Trp Gin Asn Glu Tyr Glu Val Tyr Ser 
225 230 235 

CTA CCT GGA ATG AAG CAT GAG AAC ATA CTA CAG TTC ATT GGT GCA GAG 829 
Leu Pro Gly Met Lys His Glu Asn He Leu Gin Phe He Gly Ala Glu 
240 245 250 

AAA AGA GGC ACC AGT GTG GAT GTG GAC CTG TGG CTA ATC ACA GCA TTT 877 
Lys Arg Gly Thr Ser Val Asp Val Asp Leu Trp Leu He Thr Ala Phe 
255 260 265 

CAT GAA AAG GGC TCA CTG TCA GAC TTT CTT AAG GCT AAT GTG GTC TCT 925 
His Glu Lys Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser 
270 275 280 285 



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TGG AAT GAA CTT TGT CAT ATT GCA GAA ACC ATG GCT AGA GGA TTG GCA 973 
Trp Asn Glu Leu Cys His He Ala Glu Thr Met Ala Arg Gly Leu Ala 
290 295 300 

TAT TTA CAT GAG GAT ATA CCT GGC TTA AAA GAT GGC CAC AAG CCT GCA 1021 
Tyr Leu His Glu Asp He Pro Gly Leu Lys Asp Gly His Lys Pro Ala 
305 310 315 

ATC TCT CAC AGG GAC ATC AAA AGT AAA AAT GTG CTG TTG AAA AAC AAT 1069 
He Ser His Arg Asp He Lys Ser Lys Asn Val Leu Leu Lys Asn Asn 
320 325 330 

CTG ACA GCT TGC ATT GCT GAC TTT GGG TTG GCC TTA AAG TTC GAG GCT 1117 
Leu Thr Ala Cys He Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala 
335 340 345 

GGC AAG TCT GCA GGT GAC ACC CAT GGG CAG GTT GGT ACC CGG AGG TAT 1165 
Gly Lys Ser Ala Gly Asp Thr His Gly Gin Val Gly Thr Arg Arg Tyr 
350 355 360 365 

ATG GCT CCA GAG GTG TTG GAG GGT GCT ATA AAC TTC CAA AGG GAC GCA 1213 
Met Ala Pro Glu Val Leu Glu Gly Ala He Asn Phe Gin Arg Asp Ala 
370 375 380 

TTT CTG AGG ATA GAT ATG TAC GCC ATG GGA TTA GTC CTA TGG GAA TTG 1261 
Phe Leu Arg He Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu 
385 390 395 

GCT TCT CGT TGC ACT GCT GCA GAT GGA CCC GTA GAT GAG TAC ATG TTA 1309 
Ala Ser Arg Cys Thr Ala Ala Abp Gly Pro Val Asp Glu Tyr Met Leu 
400 405 410 

CCA TTT GAG GAA GAA ATT GGC CAG CAT CCA TCT CTT GAA GAT ATG CAG 1357 
Pro Phe Glu Glu Glu He Gly Gin His Pro Ser Leu Glu Asp Met Gin 
415 420 425 

GAA GTT GTT GTG CAT AAA AAA AAG AGG CCT GTT TTA AGA GAT TAT TGG 1405 
Glu Val Val Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp 
430 435 440 445 

CAG AAA CAT GCA GGA ATG GCA ATG CTC TGT GAA ACG ATA GAA GAA TGT 1453 
Gin Lys His Ala Gly Met Ala Met Leu Cys Glu Thr He Glu Glu Cys 
450 455 460 

TGG GAT CAT GAT GCA GAA GCC AGG TTA TCA GCT GGA TGT GTA GGT GAA 1501 
Trp Asp His Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu 
465 470 475 

AGA ATT ACT CAG ATG CAA AGA CTA ACA AAT ATC ATT ACT ACA GAG GAC 1549 
Arg He Thr Gin Met Gin Arg Leu Thr ABn He He Thr Thr Glu Asp 
480 485 490 

ATT GTA ACA GTG GTC ACA ATG GTG ACA AAT GTT GAC TTT CCT -CCC AAA 1S97 
He Val Thr Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys 
495 500 505 

GAA TCT AGT CTA TGATGGTGGC ACCGTCTGTA CACACTGAGG ACTGGGACTC 1649 

Glu Ser Ser Leu 

510 

TGAACTGGAG CTGCTAAGCT AAGGAAAGTG CTTAGTTGAT TTTCTGTGTG AAATGAGTAG 1709 

GATGCCTCCA GGACATGTAC GCAAGCAGCC CCTTGTGGAA AGCATGGATC TGGGAGATGG 1769 

ATCTGGGAAA CTTACTGCAT CGTCTGCAGC ACAGATATGA AGAGGAGTCT AAGGGAAAAG 1829 



CTGCAAACTG TAAAGAACTT CTGAAAATGT ACTCGAAGAA TGTGGCCCTC TCCAAATCAA 1889 



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GGATCTTTTG GACCTGGCTA ATCAAGTATT TGCAAAACTG ACATCAGATT TCTTAATGTC 1949 

TGTCAGAAGA CACTAATTCC TTAAATGAAC TACTGCTATT TTTTTTAAAT GAAAAACTTT 2009 

TCATTTCAGA TTTTAAAAAG GGTAACTTTT TATTGCATTT GCTGTTGTTT CTATAAATGA 2069 

CTATTGTAAT GCCAACATGA CACAGCTTGT GAATGTGTAG TGTGCTGCTG TTCTGTGTAC 2129 

ATAGTCATCA AAGTGGGGTA CAGTAAAGAG GCTTCCAAGC ATTACTTTAA CCTCCCTCAA 2189 

CAAGGTATAC CTCAGTTCCA CGGTTGTTAA ATTATAAAAT TGAAAACACT AACAGAATTT 2249 

GAATAAATCA GTCCATGTTT TATAACAAGG TTAATTACAA ATTCACTGTG TTATTTAAGA 2309 

AAAAATGGTA AGCTATGCTT AGTGCCAATA GTAAGTGGCT ATTTGTAAAG GAGTGTTTTA 2369 

GCTTTTCTTC TACTGGCTTG TAATTTAGGG AAAACAAGTG CTGTCTTTGA AATGGAAAAG 2429 

AATATGGTGT CACCCTACCC CCCATACTTA TATCAAGGTC CCAAAATATT CTTTTCCATT 2489 

TCAAAGACAG CACTTTGAAA ACCCTAAATT ACAAGCCAGT AGAAGAAAAG CTAAAACACG 2549 

CTTTACAAAT AGCC 2563 

(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 513 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO; 2: 

Met Gly Ala Ala Ala Lye Leu Ala Phe Ala Val Phe Leu lie Ser Cys 
1 5 10 15 

Ser Ser Gly Ala lie Leu Gly Arg Ser Glu Thr Gin Glu Cys Leu Phe 
20 25 30 

Phe Asn Ala Asn Trp Glu Arg Aep Arg Thr ABn Gin Thr Gly Val Glu 
35 40 45 

Pro Cys Tyr Gly Asp Lys Asp Lys Arg Arg His Cys Phe Ala Thr Trp 
50 55 60 

Lys Asn lie Ser Gly Ser lie Glu lie Val Lys Gin Gly Cys Trp Leu 
65 70 75 80 

Aep Asp lie Asn Cys Tyr Asp Arg Thr Asp Cys lie Glu Lys Lys Asp 
85 90 95 

Ser Pro Glu Val Tyr Phe Cys Cys Cys Glu Gly Asn Met Cys Asn Glu 
100 105 110 

Lys Phe Ser Tyr Phe Pro Glu Met Glu Val Thr Gin Pro Thr Ser Asn 
115 120 125 

Pro Val Thr Pro Lys Pro Pro Tyr Tyr Asn lie Leu Leu Tyr Ser Leu 
130 135 140 

Val Pro Leu Met Leu lie Ala Gly He Val He Cys Ala Phe Trp Val 
145 150 155 160 



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Tyx Arg His His Lys Met Ala Tyr Pro Pro Val Leu Val Pro Thr Gin 
1 * 165 170 175 

Asp Pro Gly Pro Pro Pro Pro Ser Pro Leu Leu Gly Leu Lys Pro Leu 
180 185 190 

Gin Leu Leu Glu Val Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys 
195 200 205 

Ala Gin Leu Leu Asn Glu Tyr Val Ala Val Lys lie Phe Pro He Gin 
210 215 220 

Asp Lys Gin Ser Trp Gin Asn Glu Tyr Glu Val Tyr Ser Leu Pro Gly 
225 230 235 240 

Met Lys His Glu Asn He Leu Gin Phe He Gly Ala Glu Lys Arg Gly 
245 250 255 

Thr Ser Val Asp Val Asp Leu Trp Leu He Thr Ala Phe His Glu Lys 
260 265 270 

Gly Ser Leu Ser Asp Phe Leu Lys Ala Asn Val Val Ser Trp Asn Glu 
275 280 285 

Leu Cys His He Ala Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His 
290 295 300 

Glu Asp He Pro Gly Leu Lys Asp Gly His Lys Pro Ala He Ser His 
305 310 315 320 

Arg Asp He Lys Ser Lys Asn Val Leu Leu Lys Asn Asn Leu Thr Ala 
325 330 335 

Cys He Ala Asp Phe Gly Leu Ala Leu Lys Phe Glu Ala Gly Lys Ser 
340 345 350 

Ala Gly Asp Thr His Gly Gin Val Gly Thr Arg Arg Tyr Met Ala Pro 
355 360 365 

Glu Val Leu Glu Gly Ala He Asn Phe Gin Arg Asp Ala Phe Leu Arg 
370 375 380 

He Asp Met Tyr Ala Met Gly Leu Val Leu Trp Glu Leu Ala Ser Arg 
385 390 395 400 

Cys Thr Ala Ala Asp Gly Pro Val Asp Glu Tyr Met Leu Pro Phe Glu 
405 410 415 

Glu Glu He Gly Gin His Pro Ser Leu Glu Asp Met Gin Glu Val Val 
420 425 430 

Val His Lys Lys Lys Arg Pro Val Leu Arg Asp Tyr Trp Gin Lys His 
435 440 445 

Ala Gly Met Ala Met Leu Cys Glu Thr He Glu Glu Cys Trp Asp His 
450 455 460 

Asp Ala Glu Ala Arg Leu Ser Ala Gly Cys Val Gly Glu Arg He Thr 
465 470 475 480 

Gin Met Gin Arg Leu Thr Asn He He Thr Thr Glu Asp He Val Thr 
485 490 495 

Val Val Thr Met Val Thr Asn Val Asp Phe Pro Pro Lys Glu Ser Ser 
500 505 510 

Leu 



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(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 2335 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(vii) IMMEDIATE SOURCE: 
(B) CLONE: XACTR 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 468.. 1997 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

CCGCCCACAC AGTGCAGTGA ATAATAGCCG GTGCGGCCCC TCCCCTCTTT CCCTGGCAGT 60 

TGTGTATCTG TCACATTGAA GTTTGGGCTC CTGTGAGTCT GAGCCTCCCC CTGTGTCTCA 120 

TGTGAAGCTG CTGCTGCAGA AGGTGGAGTC GTTGCATGAG GGTGGGGGGA GTCGCTGCTG 180 

TTTGATCTGC CTCTGCTCCC CATTCACACT CTCATTTCAT TCCCACGGAT CCA CAT T AC A 240 

ACTCGCCTTT AACCCTTTCC CTGGCGGAGC CCACGCGTCT TTCATCCCTC CTGCCGCGGC 300 

CGCTGAGCGA CCAGAGCGCG ACATTGTTGC GGCGGGGGAT TGGGCGACAT TGTTGCGAAT 360 

AATCGGAGCT GCTGGGGGGG AACTGATACA ACGTTGCGAC TGTAAAGGAA TTAACTCGGC 420 

CGAATGGGAT TTTATCTGTG TCGGTGAGAG AAGCGGATCC CAGGAGC ATG GGG GCG 476 

Met Gly Ala 
1 

TCT GTA GCG CTG ACT TTT CTA CTT CTT CTT GCA ACT TTC CGC GCA GGC 524 
Ser Val Ala Leu Thr Phe Leu Leu Leu Leu Ala Thr Phe Arg Ala Gly 
5 10 15 

TCA GGA CAC GAT GAA GTG GAG ACA AGA GAG TGC ATC TAT TAC AAT GCC 572 
Ser Gly His Asp Glu Val Glu Thr Arg Glu Cys He Tyr Tyr Asn Ala 
20 25 30 35 

AAC TGG GAA CTG GAG AAG ACC AAC CAA AGT GGG GTG GAA AGC TGC GAA 620 
Asn Trp Glu Leu Glu Lys Thr Asn Gin Ser Gly Val Glu Ser Cys Glu 
40 45 50 

GGG GAA AAG GAC AAG CGA CTC CAC TGT TAC GCG TCT TGG AGG AAC AAT 668 
Gly Glu Lys Asp Lys Arg Leu His Cys Tyr Ala Ser Trp Arg Asn Asn 
55 60 65 

TCG GGC TTC ATA GAG CTG GTG AAA AAA GGA TGC TGG CTG GAT GAC TTC 716 
Ser Gly Phe He Glu Leu Val Lys Lys Gly Cys Trp Leu Asp Asp Phe 
70 75 80 

AAC TGT TAT GAC AGA CAG GAA TGT ATT GCC AAG GAA GAA AAC CCC CAA 764 
Asn Cys Tyr Asp Arg Gin Glu Cys He Ala Lys Glu Glu Asn Pro Gin 
85 90 95 



WO 92/20793 



PCT/US92/03825 



-48- 

GTC TTT TTC TGC TGC TGC GAG GGA AAC TAG TGC AAC AAG AAA TTT ACT 812 
Val Phe Phe Cys Cys Cys Glu Gly Asn Tyr Cys Asn Lys Lys Phe Thr 
100 105 HO H5 

CAT TTG CCT GAA GTC GAA ACA TTT GAT CCG AAG CCC CAG CCG TCA GCC 860 
His Leu Pro Glu Val Glu Thr Phe Asp Pro Lys Pro Gin Pro Ser Ala 
120 125 130 

TCC GTA CTG AAC ATT CTG ATC TAT TCC CTG CTT CCA ATT GTT GGT CTT 908 
Ser Val Leu Asn lie Leu lie Tyr Ser Leu Leu Pro lie Val Gly Leu 
135 140 145 

TCC ATG GCA ATT CTC CTG GCG TTC TGG ATG TAC CGT CAT CGA AAG CCT 956 
Ser Met Ala lie Leu Leu Ala Phe Trp Met Tyr Arg His Arg Lys Pro 
150 155 160 

CCC TAC GGG CAT GTA GAG ATC AAT GAG GAC CCC GGT CTG CCC CCT CCA 1004 
Pro Tyr Gly His Val Glu lie Asn Glu Asp Pro Gly Leu Pro Pro Pro 
165 170 175 

TCT CCT CTG GTC GGG CTG AAG CCG CTG CAG TTG CTG GAG ATA AAG GCG 1052 
Ser Pro Leu Val Gly Leu Lys Pro Leu Gin Leu Leu Glu lie Lys Ala 
180 185 190 195 

CGA GGC CGT TTC GGT TGC GTC TGG AAA GCT CGT CTG CTG AAT GAA TAT 1100 
Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Arg Leu Leu Asn Glu Tyr 
200 205 210 

GTC GCA GTG AAA ATC TTC CCC GTG CAG GAT AAG CAG TCG TGG CAG TGT 1148 
Val Ala Val Lys lie Phe Pro Val Gin Asp Lys Gin Ser Trp Gin Cys 
215 220 225 

GAG AAA GAG ATC TTC ACC ACG CCG GGC ATG AAA CAT GAA AAC CTA TTG 1196 
Glu Lys Glu lie Phe Thr Thr Pro Gly Met Lys His Glu Asn Leu Leu 
230 235 240 

GAG TTC ATT GCC GCT GAG AAG AGG GGA AGC AAC CTG GAG ATG GAG CTG 1244 
Glu Phe lie Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu Met Glu Leu 
245 250 255 

TGG CTC ATC ACT GCA TTT CAT GAT AAG GGT TCT CTG ACG GAC TAC CTG 1292 
Trp Leu He Thr Ala Phe His Asp Lys Gly Ser Leu Thr Asp Tyr Leu 
260 265 270 275 

AAA GGG AAC TTG GTG AGC TGG AAT GAA CTG TGT CAC ATA ACA GAA ACA 1340 
Lys Gly Asn Leu Val Ser Trp Asn Glu Leu Cys His He Thr Glu Thr 
280 285 290 

ATG GCT CGT GGG CTG GCC TAC TTA CAT GAA GAT GTG CCC CGC TGT AAA 1388 
Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Val Pro Arg CyB Lys 
295 300 305 

GGT GAA GGG CAC AAA CCT GCA ATC GCT CAC AGA GAT TTT AAA AGT AAG 1436 
Gly Glu Gly His Lye Pro Ala He Ala His Arg Asp Phe Lys Ser Lys 
310 315 320 

AAT GTA TTG CTA AGA AAC GAC CTG ACT GCG ATA TTA GCA GAC TTC GGG 1484 
Asn Val Leu Leu Arg Asn Asp Leu Thr Ala He Leu Ala Asp Phe Gly 
325 330 335 

CTG GCC GTA CGA TTT GAG CCT GGA AAA CCT CCG GGA GAT ACA CAC GGG 1532 
Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp Thr His Gly 
340 345 350 355 

CAG GTT GGC ACC AGG AGG TAT ATG GCT CCT GAG GTT CTA GAG GGA GCA 1S80 
Gin Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu Glu Gly Ala 
360 365 370 



WO 92/20793 



PCT/US92/0382S 



-49- 

ATT AAC TTT CAG CGA GAT TCC TTT CTC AGG ATA GAT ATG TAT GCC ATG 1628 

lie Asn Phe Gin Arg Asp Ser Phe Leu Arg He Asp Met Tyr Ala Met 

375 380 385 

GGA CTG GTA CTC TGG GAA ATA GTA TCC CGA TGT ACA GCA GCA GAT GGG 1676 
Gly Leu Val Leu Trp Glu He Val Ser Arg Cys Thr Ala Ala Asp Gly 
390 395 400 

CCA GTA GAT GAG TAT CTG CTC CCA TTC GAA GAA GAG ATT GGG CAA CAT 1724 
Pro Val Asp Glu Tyr Leu Leu Pro Phe Glu Glu Glu He Gly Gin His 
405 410 415 

CCT TCC CTA GAG GAT CTG CAA GAA GTT GTC GTT CAC AAG AAG ATA CGC 1772 
Pro Ser Leu Glu Asp Leu Gin Glu Val Val Val His Lys Lys He Arg 
420 425 430 435 

CCT GTA TTC AAA GAC CAC TGG CTG AAA CAC CCT GGT CTG GCC CAA CTG 1820 
Pro Val Phe Lys Asp His Trp Leu Lys His Pro Gly Leu Ala Gin Leu 
440 445 450 

TGC GTC ACC ATT GAA GAA TGC TGG GAC CAT GAT GCG GAA GCA CGG CTT 1868 
Cys Val Thr lie Glu Glu Cys Trp Asp His Asp Ala Glu Ala Arg Leu 
455 460 465 

TCG GCA GGC TGC GTA GAG GAG CGT ATT TCC CAA ATC CGT AAA TCA GTG 1916 
Ser Ala Gly Cys Val Glu Glu Arg He Ser Gin He Arg Lys Ser Val 
470 475 480 

AAC GGC ACT ACC TCG GAC TGC CTT GTA TCC ATT GTT ACA TCT GTC ACC 1964 
Asn Gly Thr Thr Ser Asp Cys Leu Val Ser He Val Thr Ser Val Thr 
485 490 495 

AAT GTG GAC TTG CCG CCC AAA GAG TCC AGT ATC TGAGGTTTCT TTGGTCTTTC 2017 
Asn Val Asp Leu Pro Pro Lys Glu Ser Ser He 
500 505 510 



CAGACTCAGT 


GACTTTTAAA 


AAAAAAACTC 


ACGAATGCAG 


CTGCTATTTT 


ATCTTGACTT 


2077 


TTTAATATTT 


TTTTTCTTGG 


ATTTTACTTG 


GATCGGATCA 


ATTTACCAGC 


ACGTCATTCG 


2137 


AAAGTATTAA 


AAAAAAAAAA 


CAAAACAAAA 


AAGCAAAAAC 


AGACATCTCA 


GCAAGCATTC 


2197 


AGGTGCCGAC 


TTATGAATGC 


CAATAGGTGC 


AGGAACTTCA 


GAACCTCAAC 


AAACTCATTT 


2257 


CTAGAGAATG 


TTCTCCTGGT 


TTCCTTTATC 


TCAGAAGAGG 


ACCCATAGGA 


AAACACCTAA 


2317 


GTCAAGCAAA 


TGCTGCAG 










2335 



(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 510 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 

Met Gly Ala Ser Val Ala Leu Thr Phe Leu Leu Leu Leu Ala Thr Phe 
15 10 15 

Arg Ala Gly Ser Gly His Asp Glu Val Glu Thr Arg Glu Cys He Tyr 
20 25 30 



WO 92/20793 PCT/US92/03825 



-50- 

Tyr Asn Ala Asn Trp Glu Leu Glu Lys Thr Asn Gin Ser Gly Val Glu 
35 40 45 

Ser Cye Glu Gly Glu Lys Asp Lys Arg Leu His Cys Tyr Ala Ser Trp 
50 55 60 

Arg Asn Asn Ser Gly Phe He Glu Leu Val Lys Lys Gly Cys Trp Leu 
65 70 75 80 

Asp Asp Phe Asn Cys Tyr Asp Arg Gin Glu Cys He Ala Lys Glu Glu 
85 90 95 

Asn Pro Gin Val Phe Phe Cys Cys CyB Glu Gly Asn Tyr Cys Asn Lys 
100 105 110 

Lys Phe Thr His Leu Pro Glu Val Glu Thr Phe Asp Pro Lys Pro Gin 
115 120 125 

Pro Ser Ala Ser Val Leu Asn He Leu lie Tyr Ser Leu Leu Pro He 
130 135 140 

Val Gly Leu Ser Met Ala He Leu Leu Ala Phe Trp Met Tyr Arg His 
145 150 155 160 

Arq Lys Pro Pro Tyr Gly His Val Glu He Asn Glu Asp Pro Gly Leu 
165 170 175 

Pro Pro Pro Ser Pro Leu Val Gly Leu Lys Pro Leu Gin Leu Leu Glu 
180 185 190 

He Lys Ala Arg Gly Arg Phe Gly Cys Val Trp Lys Ala Arg Leu Leu 
195 200 205 

Asn Glu Tyr Val Ala Val Lys He Phe Pro Val Gin Asp Lys Gin Ser 
210 215 220 

Trp Gin Cys Glu Lys Glu He Phe Thr Thr Pro Gly Met Lys His Glu 
225 230 235 240 

Asn Leu Leu Glu Phe He Ala Ala Glu Lys Arg Gly Ser Asn Leu Glu 
245 250 255 

Met Glu Leu Trp Leu He Thr Ala Phe His Asp Lys Gly Ser Leu Thr 
260 265 270 

Asp Tyr Leu Lys Gly Asn Leu Val Ser Trp Asn Glu Leu Cys His He 
275 280 285 

Thr Glu Thr Met Ala Arg Gly Leu Ala Tyr Leu His Glu Asp Val Pro 
290 295 300 

Arg Cys Lys Gly Glu Gly His Lys Pro Ala He Ala His Arg Asp Phe 
305 310 315 320 

Lys Ser Lys Asn Val Leu Leu Arg Asn Asp Leu Thr Ala He Leu Ala 
325 330 335 

Asp Phe Gly Leu Ala Val Arg Phe Glu Pro Gly Lys Pro Pro Gly Asp 
340 345 350 

Thr His Gly Gin Val Gly Thr Arg Arg Tyr Met Ala Pro Glu Val Leu 
355 360 365 

Glu Gly Ala He Asn Phe Gin Arg Asp Ser Phe Leu Arg He Asp Met 
370 375 380 



WO 92/20793 PCT/US92/03825 



-51- 

Tyr Ala Met Gly Leu Val Leu Trp Glu He Val Ser Arg Cys Thr Ala 
385 390 395 400 

Ala Asp Gly Pro Val Asp Glu Tyr Leu Leu Pro Phe Glu Glu Glu He 
405 410 415 

Gly Gin Hie Pro Ser Leu Glu Asp Leu Gin Glu Val Val Val His Lys 
420 425 430 

LyB He Arg Pro Val Phe Lys Asp His Trp Leu Lys His Pro Gly Leu 
435 440 445 

Ala Gin Leu Cys Val Thr He Glu Glu Cye Trp Asp His Asp Ala Glu 
450 455 460 

Ala Arg Leu Ser Ala Gly Cys Val Glu Glu Arg He Ser Gin lie Arg 
465 470 475 480 

Lys Ser Val Asn Gly Thr Thr Ser Asp CyB Leu Val Ser He Val Thr 
485 490 495 

Ser Val Thr Asn Val Asp Leu Pro Pro Lys Glu Ser Ser He 
500 505 510 



SEQ ID NO.: 5 

DLKPEN 
SEQ ID NO. : 6 

G(T/S)XX(Y/F)X 
SEQ ID NO.: 7 

DIKSKN 
SEQ ID NO. : 8 

GTRRYM 
SEQ ID NO.: 9 

DLAAHN 
SEQ ID NO.: 10 

XP(I/V) (K/R)W(T/M) 



WO 92/20793 



PCT/US92/0382S 



-52- 

That which is claimed is: 

1. A novel receptor protein characterized by 
having the following domains, reading from the N-terminal 
end of said protein: 

an extracellular, ligand-binding domain, 
5 a hydrophobic, trans -membrane domain, and 

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

2 . A protein according to Claim 1 , further 
10 comprising a second hydrophobic domain at the amino 

terminus thereof. 

3» A protein according to Claim 1, wherein said 
protein is further characterized by having sufficient 
15 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 of said 
receptor protein. 

20 

4. A protein according to Claim 3, wherein said 
protein is further characterized by: 

having a greater binding affinity for activins than 
for inhibins, 

25 having substantially no binding affinity for 

transforming growth factors-B, and 

having substantially no binding affinity for 
non-activin-like proteins. 

3 0 5. A protein according to Claim 1 having an 

amino acid sequence substantially the same as set forth in 
Sequence ID No . 2 , Sequence ID No . 2 1 , or Sequence ID No . 
4. 



WO 92/20793 



PCT/US92/0382S 



-53- 

6. A soluble, extracellular, ligand-binding 
protein, further characterized by: 

having a sufficient binding affinity for at least one 
member of the activin/TGF-B superfamily of polypeptide 
5 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: 

10 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 
15 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. 

20 7. A protein according to Claim 6, further 

characterized by: 

having a greater binding affinity for activins than 
for inhibins, 

having . substantially no binding affinity for 
25 transforming growth factors-B, and 

having substantially no binding affinity for 
non-activin-like proteins. 

8. A protein according to Claim 6 wherein said 
3 0 protein comprises in the range of about 114-118 amino 

acids. 

9. A DNA encoding a mature protein according to 

Claim 1. 

35 

10. A DNA encoding a mature protein according to 

Claim 3. 



WO 92/20793 



PCT/US92/0382S 



-54- 

11. A DNA encoding a precursor-form of the 
protein of Claim l. 

12. A DNA encoding a protein according to 

5 Claim 2. 

13. A DNA encoding a soluble protein according 
to Claim 6. 

10 14. A DNA encoding a soluble protein according 

to Claim 8. 

15 . A DNA encoding a precursor-form of the 
protein of Claim 6. 

15 

16. A DNA according to Claim 9 having a 
contiguous nucleotide sequence substantially the same as: 

nucleotides 128 - 1609 of Sequence ID No. 1; 

variations of nucleotides 128 - 1609 of Sequence 
20 * 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; 
25 nucleotides 528 - 1997 of Sequence ID No. 3; 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. 

30 17. A DNA according to Claim 9 having a 

contiguous nucleotide sequence substantially the same as: 
nucleotides 71 - 1609 of Sequence ID No. 1; 
variations of nucleotides 71 - 1609 of Sequence 
ID No. 1, wherein the codon for residue number 39 of 
35 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 



WO 92/20793 



PCT/US92/03825 



-55- 

encoded amino acid encodes g lut amine; 

nucleotides 468 - 1997 of Sequence ID No. 3; or 
variations of any of the above sequences which 

encode the same amino acid sequences, but employ 
5 different codons for some of the amino acids* 

18. A DNA according to Claim 9 having a 
contiguous nucleotide sequence substantially the same as 
set forth in Sequence ID No. 1, Sequence ID No. 1' or 

10 Sequence ID No. 3. 

19. A DNA according to Claim 13 having a 
contiguous nucleotide sequence substantially the same as 
nucleotides 71 - 127 of Sequence ID No. 1, or nucleotides 

15 468-527 of Sequence ID No. 3. 

20. A method for the recombinant production of 
activin receptor (s) , said method comprising 

expressing the DNA of Claim 9 in a suitable host cell. 

20 

21. A method for the recombinant production of 
soluble activin receptor (s) , said method comprising 

expressing the DNA of Claim 13 in a suitable host 

cell. 

25 

22. A DNA fragment useful as a hybridization 
probe , wherein said DNA fragment comprises at least a 
portion of the DNA according to Claim 9, and wherein said 
DNA fragment is labeled with a readily detectable 

3 0 substituent . 

23. A DNA fragment according to Claim 22 wherein 
said readily detectable substituent is selected from a 
radiolabeled molecule, a fluorescent molecule, an enzyme, 

35 or a ligand. 



I 



WO 92/20793 



PCT/US92/03825 



-56- 

24. A method to identify clones encoding 
receptors of the activin/TGF-£ superf amily, said method 
comprising: 

screening a genomic or cDNA library with a DNA 
5 fragment according to Claim 22 under low stringency 

hybridization conditions, and 

identifying those clones which display a 
substantial degree of hybridization to said DNA 
fragment. 

10 

25. A method for screening a collection of 
compounds to determine those compounds which bind to 
receptors of the activin/TGF-6 superfamily, said method 
comprising employing the receptor of claim 1 in a 

15 competitive binding assay. 



26. A bioassay for evaluating whether compounds 
are agonists for receptor protein (s) according to Claim 1, 
20 or functional modified forms of said receptor protein (s), 
said bioassay comprising: 

(a) culturing cells containing: 

DNA which expresses said receptor 
protein (s) or functional modified forms of 
25 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 
30 to induce transcription activation activity of 

said receptor protein is sought to be determined; 
and thereafter 

(b) monitoring said cells for expression of said 
reporter gene. 



35 



WO 92/20793 



PCT/US92/0382S 



-57- 

27. A bioassay for evaluating whether compounds 
aire antagonists for receptor protein (s) according to Claim 
1, or functional modified forms of said receptor 
protein(s), said bioassay comprising: 
5 (a) 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 
10 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 
15 activation of said receptor protein (s) is sought 

to be determined, and 

a f ixed concentration of at least one 
agonist for said receptor protein (s), or 
functional modified forms of said receptor 
2 0 pr ot e in ( s ) ; and thereafter 

(b) 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 
25 to inhibit activation of transcription. 

28. A method for modulating the transcription 
trans-activation of activin receptor ( s) , said method 
comprising: 

30 contacting said receptor with an effective, modulating 

amount of the protein of Claim 6. 

29. An antibody generated against the protein of 

Claim 6. 

35 

30. An antibody according to Claim 29, wherein 
said antibody is a monoclonal antibody. 



WO 92/20793 



PCT/US92/03825 



-58- 

31. A method for modulating the transcription 
trans-activation of activin receptor (s), said method 
comprising: 

contacting said receptor with a modulating, effective 
5 amount of the antibody of Claim 29. 



WO 92/20793 



PCT/US92/0382S 



1/6 



w 

c 

e 
u 

0) 
I 

u 



0) c 

c m 
C B 
•H O 
^T3 



>4 

o c 

0) 6 
U O 
(0 U 

■P 
G 
M 



0) 
C 

t 10 c 
W fc-H 
C JQ «J 
* fi fl 
U 0) O 



0) 

u 



c 

*o 
c 

I 

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C-H 
ftt A3 

•rj o 



I 

5C 



43 

O 

•C 
T3 OiC 
C O-H 
O U * 
O T5 e 
CJ >,0 
w x: 73 



SUBSTITUTE SHEET 



WO 92/20793 



PCT/US92/0382S 



2/6 



Divide a cDNA library in a 
mammalian expression vector into 
pools of 1000 clones, prepare DNA 
from each pool 



Transfect COS cells directly on 
microscope slides 



Bind [1251] activin A, wash 
cells, fix, dip in photographic 
emulsion 



Subdivide bacteria from 
positive pool and rescreen; 
repeat until receptor clone is 
pure 




FIG. 2 



SUBSTITUTE SHEET" 



WO 92/20793 



PCT/US92/03825 



3/6 



o 



— CM 



E 

CO 



Pst I - 



Pst I 
Kpn I, 
Pst I' 



Kpn I - 



Nar I - 



I — o 



I 



Sac 



T 



o 
o 



o 
o 

CO 



o 
o 

CM 



O 
O 



ro 

CD 
Li. 



JO 



< 
E 



X2 
CD 

cm 

t 

CM 

eg 

o 

< 
£ 



SUBSTITUTE SHEET 



WO 92/20793 



PCIYUS92/03825 



4/6 



00 00 








u 



CD 



SUBSTITUTE SHEET 



WO 92/20793 PCT/US92/03825 

5/6 




o .01 .1 i io ioo 



nM competitor 



SUBSTITUTE SHEET 



WO 92/20793 



PCT/US92/03825 



6/6 




, SUBSTITUTE SHEET 



INTERNATIONAL SEARCH REPORT 

International Application No 



PCT/US 92/03825 



I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply* indicate all)' 



According to International Patent Classification (IPC) or to both National Classification ana* IPC 

Int.Cl. 5 C12N15/12; C07K15/00; C12Q1/68; 

C12N15/62; A61K37/02; C12P21/08; 



G01N33/53 
A61K39/395 



n. FIELDS SEARCHED 



Minimum Documentation Searched' 



Classification System 



Classification Symbols 



Int.Cl. 5 



C12N ; 
G01N ; 



C07K ; 
A61K 



C12P ; 



C12Q 



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included in the Fields Searched 1 



HI. DOCUMENTS CONSIDERED TO BE RELEVANT 9 



Category 0 



Citation of Document, 11 with indication, where appropriate, of the relevant passages 12 



Relevant to Claim No. u 



CELL 

vol. 61, no. 3, 18 May 1990, CAMBRIDGE, MA, US 
pages 635 - 645; 

L. GEORGI ET AL. : 'daf-l, a C. elegans gene 
controlling Dauer larva development, encodes a 
novel receptor protein kinase. 1 
cited 1n the application 
see abstract; figures 4,6 

BIOCHEMICAL AND BIOPHYSICAL RESEARCH 
COMMUNICATIONS* 

vol. 157, no. 2, 15 December 1988, DULUTH, MN, 
US \ 
pages 844 - 849; 

C. CAMPEN ET AL.: 'Characterization of actlvln A 
binding sites on the human leukemia cell line 
K562. ' 

see the whole document 



I, 2,9, 

II, 12 



1-4,25 



° Special categories of cited documents : 10 

*A* document defining the general state of the art which is not 

considered to be of particular relevance 
*E* earlier document but published on or after the International 

filing date 

*L' document which may throw doubts on priority daim(s) or 
which is dted to establish the publication date of another 
citation or other spedal reason (as specified) 

"O* document referring to an oral disclosure, use, exhibition or 
other means 

*P" document published prior to the international filing date hut 
later than the priority date dalmed 



*T* later document published after the international filing date 
or priority date and not in conflict with the application but 
dted to understand the principle or theory underlying the 
invention 

tr X m document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step 

*Y* document of particular relevance; the claimed Invention 
cannot be considered to involve an inventive step when the 
document Is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
m the art. 

*&* document member of -the same patent family 



IV. CERTIFICATION 



Date of the Actual Completion of the International Search 

16 SEPTEMBER 1992 



Date of Mailing of this International Search Report 

29.09.92 



International Searching Authority 

EUROPEAN PATENT OFFICE 



Signature of Authorized Officer 

NOOIJ F.J.M. 




AST* 



turn PCT/I5A/210 {tec—i fttotf) (Jeunry IMS) 



PCT/US 92/03825 

International AppUcatioa No 



m. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SH£LI> 




Category 0 


atation of Document, with indication, where appropriate, of the relevant passages 




P,X 


SCIENCE 

vol. 255, no. 5052, 27 March 1992, WASHINGTON 

n/« I to 

DC, US 

pages 1702 - 1705; 

L. MATHEWS ET AL. : "Cloning of a second type of 
activln receptor and functional characterization 
1n Xenopus embryos. ' 
see abstract; figure 1 


1-5, 

9-12,20, 
25 


P,X 


BIOCHEMICAL AND BIOPHYSICAL RESEARCH 
COMMUNICATIONS. 

vol. 184, no. 1, 15 April 1992, DULUIH, m, 
pages 310 - 316; 

C. DONALDSON ET AL. : 'Molecular cloning and 
binding properties of the human type II activln 
receptor. 1 

see abstract; figure 2 


1-5, 
9-12, 
16-20, 
22-24 


P,X 


CELL 

vol. 68, no. 4, 21 February 1992, CAMBRIDGE, MA, 
US 

pages 775 - 785; 

H. LIN ET AL. : 'Expression cloning of the 
TGF-beta type II receptor, a functional 
transmembrane serine/threonine kinase. 1 
see abstract; figures 2,5,6 


1-3, 

9-12, 

22-24 


p,x 


BIOCHEMICAL AND BIOPHYSICAL RESEARCH 
COMMUNICATIONS. _ UM 
vol. 181, no. 2, 16 December 1991, DULUTH, MN, 

US 

pages 684 - 690; 

M. K0ND0 ET AL.: 'Activln receptor raRNA 1s 
expressed early 1n Xenopus embryogenesls and the 
level of the expression affects the body axis 
formation. 1 

see abstract; figure 1 


1-5, 
9-12, 
16-18, 
20,22-24 



hn PCr/ISA/ZtO HXSn iM) (J«nry IttS) 



^ ■nternattonai application No. 

INTERNA » <ON AL SEARCH REPORT 1 , pcT/us ^ ^ 



Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) 



This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons: 
1. [T) Claims Nos^ 

because they relate to subject matter not required to be searched by this Authority, namely; 

Although claims 28 and 31 (both practically, as far as an 1n vivo method 1s 
concerned) are directed to a method of treatment of the human/animal body, 
the search has been carried out and based on the alleged effects of the 
compound/composl t 1 on . 



□ 



Claims Nosj 

because they relate to parts of the international application that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out, specifically: 



□ 



Claims Nos^ 

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box II Observations where unity of invention is lacking (Continuation of hem 2 of first sheet) 



This International Searching Authority found multiple inventions in this international application, as follows: 



As all required additional search fees were timely paid by the applicant, this international search report covers all 
searchable claims. 

Q As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. r jtuw«. 



3 - I I As °nly of the required additional search fees were timely paid by the applicant, this international search report 
covers only those claims for which fees were paid, specifically claims Nos.: 



4 * D No required additional search fees were timely paid by the applicant. Consequently, this international search report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos^ 



Remark on Protest | [ The additional search fees were accompanied by the applicant's protest. 

| | No protest accompanied the payment of additional search fees. 



Form PCT/IS A/310 (continuation of first sheet (1)) (July 1992) 



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