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
1999-02-25
2003-03-11
Eyler, Yvonne (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, C530S402000, C530S413000, C536S023500, C435S069100, C435S069700
Reexamination Certificate
active
06531579
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to signaling molecules, specifically to signaling and mediator molecules in the hedgehog (Hh) cascade which are involved in cell proliferation and differentiation.
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 signaling molecules, such as members of the transforming growth factor-beta (TGF-&bgr;), Wnt, fibroblast growth factors 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).
Hedgehog (Rh) was first identified as a segment-polarity gene by a genetic screen in
Drosophila melanogaster
, Nusslein-Volhard et al.,
Roux. Arch. Dev. Biol
. 193: 267-282 (1984), that plays a wide variety of developmental functions. Perrimon, supra. Although only one Drosophila Hh gene has been identified, three mammalian Hh homologues have been isolated: Sonic Hh (SHh), Desert Hh (DHh) and Indian Hh (IHh), Echelard et al.,
Cell
75: 1417-30 (1993); Riddle et al.,
Cell
75: 1401-16 (1993). SHh is expressed at high level in the notochord and floor plate of developing vertebrate embryos. In vitro explant assays as well as ectopic expression of SHh in transgenic animals show that SHh plays a key role in neuronal tube patterning, Echelard et al., supra., Krauss et al.,
Cell
75, 1431-44 (1993), Riddle et al.,
Cell
75: 1401-16 (1993), Roelink et al.,
Cell
81: 445-55 (1995). In vitro explant assays as well as ectopic expression of SHh in transgenic animals show that SHh plays a key role in neural tube patterning, Echelard et al. (1993), supra.; Ericson et al.,
Cell
81: 747-56 (1995); Marti et al.,
Nature
375: 322-5 (1995); Roelink et al. (1995), Hynes et al.,
Neuron
19: 15-26 (1997). Hh also plays a role in the development of limbs (Krauss et al.,
Cell
75: 1431-44 (1993); Laufer et al.,
Cell
79, 993-1003 (1994)), somites (Fan and Tessier-Lavigne,
Cell
79 1175-86 (1994); Johnson et al.,
Cell
79: 1165-73 (1994)), lungs (Bellusci et al.,
Develop
. 124: 53-63 (1997) and skin (Oro et al.,
Science
276: 817-21 (1997). Likewise, IHh and DHh are involved in bone, gut and germinal cell development, Apelqvist et al.,
Curr. Biol
. 7: 801-4 (1997); Bellusci et al.,
Development
. 124: 53-63 (1997); Bitgood et al.,
Curr. Biol
. 6: 298-304 (1996); Roberts et al.,
Development
121: 3163-74 (1995). SHh knockout mice further strengthened the notion that SHh is critical to many aspect of vertebrate development, Chiang et al.,
Nature
383: 407-13 (1996). These mice show defects in midline structures such as the notochord and the floor plate, absence of ventral cell types in neural tube, absence of distal limb structures, cyclopia, and absence of the spinal column and most of the ribs.
At the cell surface, the Hh signals is thought to be relayed by the 12 transmembrane domain protein Patched (Ptch) [Hooper and Scott,
Cell
59: 751-65 (1989); Nakano et al.,
Nature
341: 508-13 (1989)] and the G-protein coupled like receptor Smoothened (Smo) [Alcedo et al.,
Cell
86: 221-232 (1996); van den Heuvel and Ingham,
Nature
382: 547-551 (1996)]. Both genetic and biochemical evidence support a receptor model where Ptch and Smo are part of a multicomponent receptor complex, Chen and Struhl,
Cell
87: 553-63 (1996); Marigo et al.,
Nature
384: 176-9 (1996); Stone et al.,
Nature
384: 129-34 (1996). Upon binding of Hh to Ptch, the normal inhibitory effect of Ptch on Smo is relieved, allowing Smo to transduce the Hh signal across the plasma membrane. Loss of function mutations in the Ptch gene have been identified in patients with the basal cell nevus syndrome (BCNS), a hereditary disease characterized by multiple basal cell carcinomas (13CCs). Disfunctional Ptch gene mutations have also been associated with a large percentage of sporadic basal cell carcinoma tumors, Chidambaram et al.,
Cancer Research
56: 4599-601 (1996); Gailani et al.,
Nature Genet
. 14: 78-81 (1996); Hahn et al.,
Cell
85: 841-51 (1996); Johnson et al.,
Science
272: 1668-71 (1996); Unden et al.,
Cancer Res
. 56: 4562-5 (1996); Wicking et al.,
Am. J. Hum. Genet
. 60: 21-6 (1997). Loss of Ptch function is thought to cause an uncontrolled Smo signaling in basal cell carcinoma Similarly, activating Smo mutations have been identified in sporatic BCC tumors (Xie et al.,
Nature
391: 90-2 (1998)), emphasizing the role of Smo as the signaling subunit in the receptor complex for SHh. However, the exact mechanism by which Ptch controls Smo activity still has yet to be clarified and the signaling mechanisms by which the Hh signal is transmitted from the receptor to downstream targets also remain to be elucidated. Genetic epistatic analysis in Drosophila has identified several segment-polarity genes which appear to function as components of the Hh signal transduction pathway, Ingham,
Curr. Opin. Genet. Dev
. 5: 492-8 (1995); Perrimon, supra. These include a kinesin-like molecule, Costal-2 (Cos-2) [Robbins et al.,
Cell
90: 225-34 (1997); Sisson et al.,
Cell
90: 235-45 (1997)], a protein designated fused [Preat et al.,
Genetics
135: 1047-62 (1993); Therond et al., Proc. Natl Acad Sci. USA 93: 4224-8 (1996)], a novel molecule with unknown function designated Suppressor of fused [Pham et al.,
Genetics
140: 587-98 (1995); Preat,
Genetics
132: 725-36 (1992)] and a zinc finger protein Ci. [Alexandre et al.,
Genes Dev
. 10: 2003-13 (1996); Dominguez et al.,
Science
272: 1621-5 (1996); Orenic et al.,
Genes Dev
. 4: 1053-67 (1990)]. Additional elements implicated in Hh signaling include the transcription factor CBP [Akimaru et al.,
Nature
386: 735-738 (1997)], the negative regulator slimb [Jiang and Struhl,
Nature
391: 493-496 (1998)] and the SHh response element COUP-TFII [Krishnan et al.,
Science
278: 1947-1950 (1997)].
Mutants in Cos-2 are embryonicly lethal and display a phenotype similar to Hh over expression, including duplications of the central component of each segment and expansion domain of Hh responsive genes. In contrast, mutant embryos for fused and Ci show a phenotype similar to Hh loss of function including deletion of the posterior part of each segment and replacement of a mirror-like image duplication of the anterior part or each segment and replacement of a mirror-like duplication of the anterior part, Busson et al.,
Roux. Arch. Dev. Biol
. 197: 221-230 (1988). Molecular characterizations of Ci suggested that it is a transcription factor which directly activates Hh responsive genes such as Wingless and Dpp, Alexandre et al., (1996) supra, Dominguez et al., (1996) supra. Likewise, molecular analysis of fused reveals that it is structurally related to serine threonine kinases and that both intact N-terminal kinase domain and a C-terminal regulatory region are required for its proper function, Preat et al.,
Nature
347: 87-9 (1990); Robbins et al., (1997), supra; Therond et al.,
Proc. Natl. Acad. Sci. USA
93: 4224-8 (1996). Consistent with the putative opposing functions of Cos-2 and fused, fused mutations are suppressed by Cos-2 mutants and also by Suppressor of fused mutants, Preat et al.,
Genetics
135: 1047-62 (1993). However, whereas fused null mutations and N-terminal kinase domain mutations can be fully suppressed by Suppressor of fused mutations, C-terminus mutations of fused display a strong Cos-2 phenotype in a Suppressor of fused background. This suggests that the fused kinase domain can act as a constitutive activator of SHh signaling when Suppressor of Fused is not present. Recent studies have shown that the 92 kDa Drosophila fused, Cos-2 and Ci are present in a microtubule associated multiprotein complex and that Hh signaling leads to dissociation of this complex from microtubules, Robbins et al.,
Cell
90: 225-34 (1997); Sisson et al.,
Cell
de Sauvage Frederic
Rosenthal Arnon
Eyler Yvonne
Genentech Inc.
O'Hara Eileen B.
Svoboda Craig G.
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