Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1993-09-21
2002-09-24
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C436S501000
Reexamination Certificate
active
06455262
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to the cloning and expression of the cellular receptor molecules that are capable of binding the TGF-&bgr; supergene family of proteins. The invention further relates to methods of production of the isolated receptor molecules and their uses.
2. Description of Related Art
Following the initial purification and characterization of transforming growth factor-beta (TGF-&bgr;) as a homodimeric, 25-Kd peptide (Frolik et al.,
Proc. Natl. Acad. Sci. USA,
80: 3676-3680 [1983]; Assoian et al.,
J. Biol. Chem.,
258: 7155-7160 [1983]; Roberts et al.,
Biochemistry,
22: 5692-5698 [1983]), there has been an exponential increase in knowledge relating to this molecule. The cloning of TGF-&bgr;1 and the resultant elucidation of its precursor structure (Derynck et al.,
Nature,
316: 701-705 [1985]) have led to the identification of at least four other forms of TGF-&bgr; and the definition of a larger gene family comprising many other structurally related, but functionally distinct, regulatory proteins.
There are now many polypeptides that belong to the TGF-&bgr; supergene family by virtue of amino acid homologies, particularly with respect to the conservation of seven of the nine cysteine residues of TGF-&bgr; among all known family members. These include the mammalian inhibins (Mason et al.,
Nature,
318: 659-663 [1985]) and activins (Ling et al.,
Nature,
321: 779-782 [1986]), and Mullerian inhibitory substance (MIS; Cate et al.,
Cell,
45: 685-698 [1986]), as well as the predicted products of both a pattern gene in Drosophila (the decapentaplegic gene complex, DPP-C; Padgett et al.,
Nature,
325: 81-84 [1987]), and an amphibian gene expressed in frog oocytes (Vg1; Weeks and Melton,
Cell,
51: 861-867 [1987]). Most recently, three new proteins, called bone morphogenetic proteins (BMPs), have been added to the family. One subset of these proteins, BMP-2A and 2B, shares about 75% sequence identity with DPP-C and may represent the mammalian equivalent of that protein. Wozney et al.,
Science,
242: 1528-1534 (1988); Wang et al.,
Proc. Natl. Acad. Sci. USA,
85: 9484-9488 (1988).
In every case where the information is available, all polypeptides belonging to this family are encoded as larger precursors. The family resemblance is limited to the C-terminus of the precursor corresponding to the processed mature TGF-&bgr; (Padgett et al., supra). With the exception of MIS, the C-terminal region is cleaved from the precursor at a pair of arginine residues. Although the position of this cleavage site varies among the family members, the C-terminus of all of the peptides is in the identical position, ending in the sequence where a cysteine residue is linked to the N-terminus of X-Cys-X (X being any amino acid), but differing in every case from the TGF-&bgr; consensus C-terminal sequence where Cys is connected to the N-terminus of Lys-Cys-Ser.
A unifying feature of the biology of these polypeptides is their ability to regulate developmental processes. MIS induces regression of the female rudiments of the developing male reproductive system. The inhibins and activins, as discussed further below, regulate reproductive functions and erythropoietic activity. The BMPs are thought to play a role in the formation of cartilage and bone in vivo. DPP-C directs dorsal-ventral patterning in the developing fly embryo. Vg1 is postulated to be involved in the process of induction of mesoderm from ectoderm during gastrulation in the amphibian embryo. In amphibians, TGF-&bgr; itself (Kimelman and Kirschner,
Cell
51: 869-877 [1987]) has been shown to augment the ability of fibroblast growth factor to induce mesoderm and plays a pivotal role in morphogenesis and organogenesis in mammalian embryos. In addition, like activin, TGF-&bgr; is reported to possess follicle-stimulating-hormone (FSH)-releasing activity. Ying et al.,
Biochem. Biophys. Res. Commun.,
135: 950-956 (1986).
Inhibin is a glycoprotein produced by diverse tissues, including the gonads, pituitary, brain, bone marrow, placenta, and adrenal gland. It was initially identified by its ability to inhibit the secretion of FSH by the pituitary. De Jong and Sharpe,
Nature,
263: 71-72 (1976); Schwartz and Channing,
Proc. Natl. Acad. Sci. USA,
74: 5721-5724 (1977). Such preferential regulation of the gonadotropin secretion has generated a great deal of interest and prompted many laboratories in the past fifty years to attempt to isolate and characterize this substance from extracts of testis, spermatozoa, rete testis fluid, seminal plasma, and ovarian follicular fluid using various bioassays. Rivier et al.,
Biochem. Biophys. Res. commun.,
133: 120 (1985); Ling et al.,
Proc. Natl. Acad. Sci. USA,
82: 7217 (1985); Fukuda et al.,
Mol. Cell Endocrinol.,
44: 55 (1985). The structure of inhibin, characterized from several species, consists, of two disulfide-linked subunits: an &agr; and either a &bgr;
A
or a &bgr;
B
chain, designated “inhibin A” and “inhibin B,” respectively.
After the identification of inhibin, activin was shown to exist in follicular fluid as a naturally occurring substance. Activin was found to be capable of stimulating FSH release by rat anterior pituitary cells. Vale et al.,
Nature,
321: 776-779 (1986); Ling et al.,
Nature,
(1986), supra. Activin consists of a homodimer or heterodimer of inhibin &bgr; subunits, which may be &bgr;
A
or &bgr;
B
subunits. Vale et al.,
Recent Prog. Horm. Res.,
44: 1-34 (1988). There is 95-100% amino acid conservation of &bgr; subunits among human, porcine, bovine, and rat activins. The &bgr;
A
and &bgr;
B
subunits within a given species are about 64-70% homologous. The activin &bgr;
A
and &bgr;
B
homodimers (“activin A” and “activin B,” respectively) have been identified in follicular fluid, and both molecules have been cloned and their genes expressed. Mason et al.,
Biochem. Biophys. Res. Commun.,
135: 957 (1986); U.S. Pat. No. 4,798,885 issued Jan. 17, 1989; Mason et al.,
Molecular Endocrinol.,
3: 1352-1358 (1989). The complete sequence of the &bgr;
B
subunit is published in Sarono Symposium Publications,
Inhibin-Non-Steroidal Regulation of Follicle Stimulating Hormone Secretion,
eds. H. G. Burger et al., abstract by A. J. Mason et al., vol. 42, pp. 77-88 (Raven Press: New York, 1987), entitled “Human Inhibin and Activin: Structure and Recombinant Expression in Mammalian Cells.”
Both activin A and activin AB (the &bgr;
A
&bgr;
B
heterodimer), but thus far not activin B, have been isolated from natural sources. Activin mRNA (&bgr;
A
and &bgr;
B
subunits), bioactivity, and immunoactivity have been reported to be produced by testicular Leydig cells from immature rat and pig. Lee et al.,
Science,
243: 396-398 (1989); Lee et al., in Serono Symposium Publications,
The Molecular and Cellular Endocrinology of the Testis,
eds. Cooke and Sharpe, vol. 50, pp. 21-27 (Raven Press: New York, 1988). Activin A has been found recently to have erythropoietic-stimulating activity as well as FSH-releasing activity. EP Publ. No. 210,461 published Feb. 4, 1987 (where the protein is called BUF-3): Eto et al.,
Biochem Biophys. Res. Commun.,
142: 1095-1103 (1987); Murata et al.,
Proc. Natl. Acad. Sci. U.S.A.,
85: 2434-2438 (1988) (where the activin is called EDF); Yu et al.,
Nature,
330: 765-767 (1987) (where the activin is called FRP). In these systems, inhibin antagonized the actions of activin.
Recently, the expression of inhibin subunits, each encoded by a separate gene, was demonstrated in several tissues in addition to ovary and testis. Inhibin &agr;, &bgr;
A
, and &bgr;
B
mRNAs were detected in placental, pituitary, adrenal, bone marrow, and brain tissues. Meunier et al.,
Proc. Natl. Acad. Sci. USA,
85: 247-251 (1988). The expression of the inhibin subunit mRNAs varied by several-fold in a tissue-specific manner, suggesting different functions for these proteins depending on their pattern of association
Cox Edward T.
Mather Jennie P.
Sliwkowski Mary B.
Woodruff Teresa K.
DeBerry Regina M.
Genentech Inc.
Hasak Janet E.
Kemmerer Elizabeth
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