Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-09-20
2002-06-18
Spector, Lorraine (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S350000, C530S387100, C530S387900, C530S388100, C530S389100, C536S023100, C536S023500
Reexamination Certificate
active
06407216
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to novel Smoothened proteins which interact with Hedgehog and Patched signalling molecules involved in cell proliferation and differentiation. In particular, the invention relates to newly identified and isolated vertebrate Smoothened proteins and DNA encoding the same, including rat and human Smoothened, and to various modified forms of these proteins, to vertebrate Smoothened antibodies, and to various uses thereof.
BACKGROUND OF THE INVENTION
Development of multicellular organisms depends, at least in part, on mechanisms which specify, direct or maintain positional information to pattern cells, tissues, or organs. Various secreted signalling molecules, such as members of the transforming growth factor-beta (“TGF-beta”), Wnt, fibroblast growth factor (“FGF”), and hedgehog families, have been associated with patterning activity of different cells and structures in Drosophila as well as in vertebrates [Perrimon,
Cell,
80:517-520 (1995)].
Studies of Drosophila embryos have revealed that, at cellular blastoderm and later stages of development, information is maintained across cell borders by signal transduction pathways. Such pathways are believed to be initiated by extracellular signals like Wingless (“Wg”) and Hedgehog (“Hhf”). The extracellular signal, Hh, has been shown to control expression of TGF-beta, Wnt and FGF signalling molecules, and initiate both short-range and long-range signalling actions. A short-range action of Hh in Drosophila, for example, is found in the ventral epidermis, where Hh is associated with causing adjacent cells to maintain wingless (wg) expression [Perrimon,
Cell,
76:781-784 (1984)]. In the vertebrate central nervous system, for example, Sonic hedgehog (“SHh”; a secreted vertebrate homologue of dHh) is expressed in notocord cells and is associated with inducing floor plate formation within the adjacent neural tube in a contact-dependent manner [Roelink et al.,
Cell,
76:761-775 (1994)]. Perrimon,
Cell,
80:517-520 (1995) provide a general review of some of the long-range actions associated with Hh.
Studies of the Hh protein in Drosophila (“dHh”) have shown that Hh encodes a 46 kDa native protein that is cleaved into a 39 kDa form following signal sequence cleavage and subsequently cleaved into a 19 kDa amino-terminal form and a 26 kDa carboxy-terminal form [Lee et al.,
Science,
266:1528-1537 (1994)]. Lee et al. report that the 19 kDa and 26 kDa forms have different biochemical properties and are differentially distributed. DiNardo et al. and others have disclosed that the dHh protein triggers a signal transduction cascade that activates wg [DiNardo et al.,
Nature,
332:604-609 (1988); Hidalgo and Ingham,
Development,
110:291-301 (1990); Ingham and Hidalgo,
Development,
117:283-291 (1993)] and at least another segment polarity gene, patched (ptc) [Hidalgo and Ingham, supra; Tabata and Komberg,
Cell,
76:89-102 (1994)]. Properties and characteristics of dHh are also described in reviews by Ingham et al.,
Curr. Opin. Genet. Dev.,
5:492-498 (1995) and Luinsden and Graham et al.,
Curr. Biol.,
5:1347-1350 (1995). Properties and characteristics of the vertebrate homologue of dHh, Sonic hedgehog, are described by Echelard et al.,
Cell,
75:1417-1430 (1993); Krauss et al.,
Cell,
75:1431-1444 (1993); Riddle et al.,
Cell,
75:1401-1416 (1993); Johnson et al.,
Cell,
79:1165-1173 (1994); Fan et al.,
Cell,
81:457-465 (1995); Roberts et al.,
Development,
121:3163-3174 (1995); and Hynes et al.,
Cell,
80:95-101 (1995).
In Perrimon,
Cell,
80:517-520 (1995), it was reported that the biochemical mechanisms and receptors by which signalling molecules like Wg and. Hh regulate the activities, transcription, or both, of secondary signal transducers have generally not been well understood. In Drosophila, genetic evidence indicates that Frizzled (“Fz”) functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. Fz rat homologues which have structural similarity with members of the G-protein-coupled receptor superfamily have been described by Chan et al.,
J. Biol. Chem.,
267:25202-25207 (1992). Specifically, Chan et al. describe isolating two different cDNAs from a rat cell library, the first cDNA encoding a predicted 641 residue protein, Fz-1, having 46% homology with Drosophila Fz, and a second cDNA encoding a protein, Fz-2, of 570 amino acids that is 80% homologous with Fz-1. Chan et al. state that mammalian fz may constitute a gene family important for transduction and intercellular transmission of polarity information during tissue morphogenesis or in differentiated tissues. Recently, Bhanot et al. did describe the identification of a Drosophila gene, frizzled2 (Dfz2), and predicted Dfz2 protein, which can function as a wg receptor in cultured cells [Bhanot et al.,
Nature,
382:225-230 (1996)]. Bhanot et al. disclose, however, that there is no in vivo evidence that shows Dfz2 is required for Wg signalling.
Although some evidence suggests that cellular responses to dHh are dependent on the transmembrane protein, smoothened (dSmo), [Nusslein-Volhard et al.,
Wilhelm Roux's Arch. Dev. Biol.,
193:267-282 (1984); Jurgens et al.,
Wilhelm Roux's Arch. Dev. Biol.,
193:283-295 (1984); Alcedo et al.,
Cell,
86:221-232 (Jul. 26, 1996); van den Heuvel and Ingham,
Nature,
382:547-551 (Aug. 8, 1996)], and are negatively regulated by the transmembrane protein, “Patched” [(Hooper and Scott,
Cell,
59:751-765 (1989); Nakano et al.,
Nature,
341:508-513 (1989); Hidalgo and Ingham, supra; Ingham et al.,
Nature,
353:184-187 (1991)], the receptors for Hh proteins have not previously been biochemically characterized. Various gene products, including the Patched protein, the transcription factor cubitus interruptus, the serine/threonine kinase “fused”, and the gene products of Costal-2, smoothened (smo) and Suppressor of fused (Su(fu)), have been implicated as putative components of the Hh signalling pathway.
Prior studies in Drosophila led to the hypothesis that ptc encoded the Hh receptor [Ingham et al.,
Nature,
353:184-187 (1991)]. The activity of the ptc product, which is a multiple membrane spanning cell surface protein referred to as Patched [Hooper and Scott, supra], represses the wg and ptc genes and is antagonized by the Hh signal. Patched was proposed by Ingham et al. to be a constitutively active receptor which is inactivated by binding of Hh, thereby permitting transcription of Hh-responsive genes. As reported by Bejsovec and Wieschaus,
Development,
119:501-517 (1993), however, Hh has effects in ptc null Drosophila embryos and thus cannot be the only Hh receptor. Accordingly, the role of Patched in Hh signalling has not been fully understood.
Goodrich et al. have isolated a murine patched gene [Goodrich et al., Genes Dev., 10:301-312 (1996)]. Human patched homologues have also been described in recently published literature. For instance, Hahn et al.,
Cell,
85:841-851 (1996) describe isolation of a human homolog of Drosophila ptc. The gene displays up to 67% sequence identity at the nucleotide level and 60% similarity at the amino acid level with the Drosophila gene [Hahn et al., supra]. Johnson et al. also provide a predicted amino acid sequence of a human Patched protein [Johnson et al.,
Science,
272:1668-1671 (1996)]. Johnson et al. disclose that the 1447 amino acid protein has 96% and 40% identity to mouse and Drosophila Patched, respectively. The human and mouse data from these investigators suggest that patched is a single copy gene in mammals. According to Hahn et al.,
Cell,
85:841-851 (1996), analyses revealed the presence of three different 5′ ends for their human ptc gene. Hahn et al. postulate there may be at least three different forms of the Patched protein in mammalian cells: the ancestral form represented by the murine sequence, and the two human
de Sauvage Frederick J.
Rosenthal Arnon
Stone Donna M.
Genentech Inc.
Kaufman Claire M.
Spector Lorraine
Svoboda Craig G.
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