Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds hormone or other secreted growth regulatory factor,...
Reexamination Certificate
2000-12-15
2004-02-17
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds hormone or other secreted growth regulatory factor,...
C530S387900, C530S388100, C530S389100, C530S350000
Reexamination Certificate
active
06692744
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of endocrinology, reproductive biology and cell biology, especially in regard to hormone/growth factor signaling. More specifically, the present invention relates to the identification of an inhibin receptor and uses thereof.
2. Description of the Related Art
Inhibins and activins were initially recognized as protein hormones of gonadal origin that reciprocally modulate follicle stimulating hormone (FSH) production by the anterior pituitary (1). These proteins are disulfide-linked dimers of related polypeptides. Activins consist of two &bgr; chains, while inhibins possess a &bgr; chain linked to a related but divergent &agr; chain (2). Activins are now known to exert important endocrine, paracrine and autocrine actions in both reproductive and non-reproductive tissues. These actions regulate processes including hormone secretion as well as cell proliferation and differentiation, both during development and in adult animals (1,3). Inhibin generally opposes the actions of activin (4) although there are systems where inhibin is unable to block activin responses (5,6).
Inhibins and activins belong to the transforming growth factor-&bgr; (TGF-&bgr;) superfamily of growth and differentiation factors (7). Like other characterized members of this family, activins have been shown to signal through two classes of transmembrane serine/threonine kinase receptors (8). In 1991, the Type II receptor for activin, designated ActRII, was cloned and characterized (9). ActRII was the first vertebrate receptor serine kinase (RSK) to be characterized as well as the first receptor to be described in molecular detail for any member of the TGF-&bgr; superfamily. Over a dozen receptor serine kinase family members have now been identified including a second Type II activin receptor (ActRIIB) (10,11), the Type II TGF-&bgr; receptor (12) and Type I receptors for both activin (13,14) and TGF-&bgr; (15).
The broad spectrum of critical biological actions of inhibin, activin and related factors as well as their connection to potential applications for the treatment of reproductive, developmental, bone, hepatic, hematopoietic and central nervous system disorders together form a compelling rationale for the exploration of their receptors, signaling mechanisms, and regulation. Collectively, this work involves the identification of multiple novel molecular targets and should therefore provide the basis for new therapeutic approaches aimed at treating endocrine disorders and neoplastic diseases.
Activins (&bgr;-&bgr;) and inhibins (&agr;-&bgr;) are structurally related by virtue of a common 14-kDa &bgr; subunit while the inhibin dimer also has a dissimilar 18-kDa &agr; subunit. Isoforms of activin and inhibin have been isolated from follicular fluid. These include activin A (&bgr;A-&bgr;A), activin B (&bgr;B-&bgr;B), activin AB (&bgr;A-&bgr;B), inhibin A (&agr;-&bgr;A), and inhibin B (&agr;-&bgr;B). Based on sequence alignment and locations of conserved cysteine residues, these polypeptides are thought to be structurally similar to other TGF-&bgr; family members for which crystal structure information is available (16,17).
To date, inhibin has been shown to have activity only in the context of activin responses where it typically antagonizes the activin signal (5,18-20) although there are recent reports in abstract form of activin-independent inhibin effects in bone. It has been shown that inhibin can compete with activin for binding to its target cells and that inhibin can prevent activin-induced receptor heteromerization (5,19). Unlabeled inhibin directly competes with labeled activin for binding to type II activin receptors although its potency as a displacing agent is approximately ten-fold lower than that of unlabeled activin (9,10).
The &bgr; subunits present in both activin and inhibin are proposed to mediate binding to type II activin receptors. After activin binds ActRII, the activin-ActRII complex subsequently promotes the recruitment and phosphorylation of the type I activin receptor serine kinase ALK4 (5,8,14). This results in phosphorylation of the cognate type I receptor and the activation of downstream Smad proteins (21,22). Inhibins also bind to type II activin receptors, but the &agr; subunit of the inhibin molecule does not support the recruitment of type I receptors (i.e. ALK4). This suggests that inhibins block signaling through direct competition for receptor access (5,18,19), thus preventing activin binding to type II activin receptors (23). However, inhibins fail to antagonize activin in some tissues and cells consistent with the hypothesis that additional components are required for inhibin action (5,24,25).
Previous findings indicate that an additional receptor component may be required for inhibin to successfully compete with activin for access to the type II activin receptor and to thereby functionally antagonize activin responses. It is likely that simple, direct competition for access to the activin type II receptor between activin and inhibin is not solely sufficient to explain the effects of inhibin on activin responses. Indeed, the ability of activin to suppress pituitary ACTH secretion is not antagonized even by a large molar excess of inhibin (6). In addition, in K562 erythroleukemic cells engineered to overexpress ActRII (KAR6 cells), increased ActRII expression blocks the ability of inhibin to antagonize activin signaling even in the presence of a substantial molar excess of inhibin (5).
In an effort to identify putative inhibin-specific receptor components, cross-linking experiments were performed using [
125
I]-labeled activin and inhibin to label both wild type K562 erythroleukemic cells and KAR6 cells overexpressing ActRII. The results showed that activin binds to type I and type II receptors in both cell lines and that binding of labeled activin to the two receptors was displaced by an excess of unlabeled activin or unlabeled inhibin (5). As expected, the labeled inhibin is capable of binding the type II receptor but not the type I receptor in both cell lines. Inhibin's binding to the type II receptor can be displaced by addition of either unlabeled activin or inhibin. Interestingly, a high molecular weight protein cross-linked to labeled inhibin was also evident in these experiments that could be competitively displaced by addition of excess unlabeled inhibin but not activin (5).
Together, these results suggest that in addition to binding to ActRII, inhibin also binds another putative co-receptor of higher molecular weight that might serve to stabilize the inhibin-ActRII interaction and therefore prevent ActRII from binding activin and mediating activin responses. The lack of inhibin antagonism of activin responses in certain tissues can therefore be explained by the absence of this or a similar inhibin binding co-receptor component. The presence of a similar high molecular weight inhibin-binding component in the ovarian tumor cell line KK-1 has been subsequently confirmed. High affinity inhibin binding to unidentified high molecular weight proteins has also been reported (24).
Betaglycan is the type III TGF-&bgr; receptor and was originally identified as the largest of three cell surface receptors shown to bind TGF-&bgr; with high affinity (26). The rat betaglycan cDNA encodes a protein of 853 amino acids containing a large extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain that lacks clearly identifiable signaling motifs (27,28). Betaglycan binds all three TGF-&bgr; isoforms with high affinity and is thought to play an accessory role in facilitating access of TGF-&bgr; to its signaling receptors (22,29).
Mature betaglycan is a proteoglyean which contains both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains yielding a molecule that migrates between 250 kDa and 350 kDa on SDS-PAGE gels. The betaglycan core polypeptide without attached glycosaminoglycan chains retains TGF-&bgr; binding
Bilezikjian Louise M.
Blount Amy L.
Gray Peter C.
Lewis Kathy A.
Vale Wylie
Adler Benjamin Aaron
Andres Janet L.
Eyler Yvonne
Research Development Foundation
LandOfFree
Betaglycan as an inhibin receptor and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Betaglycan as an inhibin receptor and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Betaglycan as an inhibin receptor and uses thereof will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3339302