Vertebrate smoothened antibodies

Novel vertebrate homologues of Smoothened, including human and rat Smoothened, are provided. Compositions including vertebrate Smoothened chimeras, nucleic acid encoding vertebrate Smoothened, and antibodies to vertebrate Smoothened, are also provided.

 

What is claimed is:

1. An antibody which specifically binds to a vertebrate Smoothened polypeptide, wherein said Smoothened polypeptide:

(a) directly binds Patched; and

(b) is encoded by a nucleic acid which hybridizes to the complement of the nucleic acid sequence encoding residues 1 to 787 of SEQ ID NO:4 under the following conditions: hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 10.times.Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml sonicated salmon sperm; and rinsing with 2.times.SSC and washing with 0.5.times.SSC, 0.1% SDS at 42.degree. C.

2. The antibody of claim 1 which is a monoclonal antibody.

3. The antibody of claim 1 which is bispecific.

4. The antibody of claim 1 which is heteroconjugated.

5. The antibody of claim 1 which is a monoclonal antibody fragment.

6. The antibody frament of claim 5 which is an Fab fragment.

7. The antibody fragment of claim 5 which is an F(ab').sub.2 fragment.

8. The antibody fragment of claim 5 which is an Fv fragment.

9. An article of manufacture, comprising a container enclosing a composition, wherein said composition comprises the anti-Smoothened antibody of claim 1 and a pharmaceutically-acceptablc carer.

10. The article of manufacture of claim 9, further comprising instructions for using said anti-Smoothened antibody in vivo or ex vivo.

11. An antibody which specifically binds to a vertebrate Smoothened polypeptidc, wherein said Smoothened polypeptide:

(a) directly binds Patched; and

(b) is encoded by a nucleic acid which hybridizes to the complement of the nucleic acid sequence encoding residues 1 to 793 of SEQ ID NO:2 under the following conditions: hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 10.times.Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml sonicated salmon sperm; and rinsing with 2.times.SSC and washing with 0.5.times.SSC, 0.1% SDS at 42.degree. C.

12. The antibody of claim 11 which is a monoclonal antibody.

13. The antibody of claim 11 which is bispecific.

14. The antibody of claim 11 which is heteroconjugated.

15. The antibody of claim 11 which is a monoclonal antibody fragment.

16. The antibody fragment of claim 15 which is an Fab fragment.

17. The antibody fragment of claim 15 which is an F(ab')2 fragment.

18. The antibody fragment of claim 15 which is an Fv fragment.

19. An antibody which specifically binds to an ECD of the Smoothened polypeptide of SEQ ID NO:4.

20. The antibody of claim 19 which is a monoclonal antibody.

21. The antibody of claim 19 which is bispecific.

22. The antibody of claim 19 which is heteroconjugated.

23. The antibody of claim 19 which is an antibody fragment.

24. The antibody fragment of claim 23 which is an Fab fragment.

25. The antibody fragment of claim 23 which is an F(ab').sub.2 fragment.

26. The antibody fragment of claim 23 which is an Fv fragment.

27. An article of manufacture, comprising a container enclosing a composition, wherein said composition comprises the anti-Smoothened antibody of claim 19 and a pharmaceutically-acceptable carrier.

28. The article of manufacture of claim 27, further comprising instructions for using said anti-Smoothened antibody in vivo or ex vivo.

29. An antibody which specifically binds to a vertebrate Smoothened polypeptide, wherein said vertebrate Smoothened polypeptide: (1) directly binds Patched and (2) is encoded by a nucleic acid which hybridizes to the complement of the nucleic acid sequence of the human cDNA insert of ATCC Dep. No. 98162 under the following conditions: hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 10.times.Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml sonicated salmon sperm; and rinsing with 2.times.SSC and washing with 0.5.times.SSC, 0.1% SDS at 42.degree. C.

30. An antibody which specifically binds to a vertebrate Smoothened polypeptide, wherein said vertebrate Smoothened polypeptide: (1) directly binds Patched and (2) is encoded by a nucleic acid which hybridizes to the complement of the nucleic acid sequence of the human cDNA insert of ATCC Dep. No. 98163 under the following conditions: hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 10.times.Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml sonicated salmon sperm; and rinsing with 2.times.SSC and washing with 0.5.times.SSC, 0.1% SDS at 42.degree. C.

31. An antibody which specifically binds to a vertebrate Smoothened polypeptide, wherein said vertebrate Smoothened polypeptide: (1) directly binds Patched and (2) is encoded by a nucleic acid which hybridizes to the complement of the nucleic acid sequence of the human cDNA insert of ATCC Dep. No. 98165 under the following conditions: hybridization at 42.degree. C. in 50% formamide, 5.times.SSC, 10.times.Denhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 .mu.g/ml sonicated salmon sperm; and rinsing with 2.times.SSC and washing with 0.5.times.SSC, 0.1% SDS at 42.degree. C.

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 forms. Patched is further discussed in a recent review by Marigo et al., Development, 122:1225 (1996).

Studies in Drosophila have also led to the hypothesis that Smo could be a candidate receptor for Hh [Alcedo et al., supra; van den Heuvel and Ingham, supra]. The smoothened (smo) gene was identified as a segment polarity gene and initially named smooth [Nusslein-Volhard et al., supra]. Since that name already described another locus, though, the segment polarity gene was renamed smoothened [Lindsley and Zimm, "The Genome of Drosophila melanogaster," San Diego, Calif.:Academic Press (1992)]. As first reported by Nusslein-Volhard et al., supra, the smo gene is required for the maintenance of segmentation in Drosophila embryos.

Alcedo et al., supra, have recently described the cloning of the Drosophila smoothened gene [see also, van den Heuvel and Ingham, supra]. Alcedo et al. report that hydropathy analysis predicts that the putative Smo protein is an integral membrane protein with seven membrane spanning alpha helices, a hydrophobic segment near the N-terminus, and a hydrophilic C-terminal tail. Thus, Smo may belong to the serpentine receptor family, whose members are all coupled to G proteins. Alcedo et al., supra, also report that smo is necessary for Hh signalling and that it acts downstream of hh and ptc.

As discussed in Pennisi, Science, 272:1583-1584 (1996), certain development genes are believed to play some role in cancer because they control cell growth and specialization. Recent studies suggest that patched is a tumor suppressor, or a gene whose loss or inactivation contributes to the excessive growth of cancer cells. Specifically, Hahn et al. and other investigators have found that patched is mutated in some common forms of basal cell carcinomas in humans [Hahn et al., Cell, 85:841-851 (1996); Johnson et al., supra; Gailani et al., in Letters, Nature Genetics, 13:September, 1996]. Hahn et al. report that alterations predicted to inactivate the patched gene product were found in six unrelated patients having basal cell nevus syndrome ("BCNS"), a familial complex of cancers and developmental abnormalities. Hahn et al. also report that the ptc pathway has been implicated in tumorigenesis by the cloning of the pancreatic tumor suppressor gene, DPC4. Vertebrate homologues of two other Drosophila segment polarity genes, the murine mammary Wntl [Rijsewijk et al., Cell, 50:649 (1987)] and the human glioblastoma GLI [Kinzler et al., Science, 236:70 (1987)], have also been implicated in cancer.

SUMMARY OF THE INVENTION

Applicants have identified cDNA clones that encode novel vertebrate Smoothened proteins, designated herein as "vSmo." In particular, cDNA clones encoding rat Smoothened and human Smoothened have been identified. The vSmo proteins of the invention have surprisingly been found to be co-expressed with Patched proteins and to form physical complexes with Patched. Applicants also discovered that the vSmo alone did not bind Sonic hedgehog but that vertebrate Patched homologues did bind Sonic hedgehog with relatively high affinity. It is believed that Sonic hedgehog may mediate its biological activities through a multi-subunit receptor in which vSmo is a signalling component and Patched is a ligand binding component, as well as a ligand regulated suppressor of vSmo. Accordingly, without being limited to any one theory, pathological conditions, such as basal cell carcinoma, associated with inactivated (or mutated) Patched may be the result of constitutive activity of vSmo or vSmo signalling following from negative regulation by Patched.

In one embodiment, the invention provides isolated vertebrate Smoothened. In particular, the invention provides isolated native sequence vertebrate Smoothened, which in one embodiment, includes an amino acid sequence comprising residues 1 to 793 of FIG. 1 (SEQ ID NO:2). The invention also provides isolated native sequence vertebrate Smoothened which includes an amino acid sequence comprising residues 1 to 787 of FIG. 4 (SEQ ID NO:4). In other embodiments, the isolated vertebrate Smoothened comprises at least about 80% identity with native sequence vertebrate Smoothened comprising residues 1 to 787 of FIG. 4 (SEQ ID NO:4).

In another embodiment, the invention provides chimeric molecules comprising vertebrate Smoothened fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a vertebrate Smoothened fused to an epitope tag sequence.

In another embodiment, the invention provides an isolated nucleic acid molecule encoding vertebrate Smoothened. In one aspect, the nucleic acid molecule is RNA or DNA that encodes a vertebrate Smoothened, or is complementary to such encoding nucleic acid sequence, and remains stably bound to it under stringent conditions. In one embodiment, the nucleic acid sequence is selected from:

(a) the coding region of the nucleic acid sequence of FIG. 1 (SEQ ID NO:1) that codes for residue 1 to residue 793 (i.e., nucleotides 450-452 through 2826-2828), inclusive;

(b) the coding region of the nucleic acid sequence of FIG. 4 (SEQ ID NO:3) that codes for residue 1 to residue 787 (i.e., nucleotides 13-15 through 2371-2373), inclusive; or

(c) a sequence corresponding to the sequence of (a) or (b) within the scope of degeneracy of the genetic code.

In a further embodiment, the invention provides a vector comprising the nucleic acid molecule encoding the vertebrate Smoothened. A host cell comprising the vector or the nucleic acid molecule is also provided. A method of producing vertebrate Smoothened is further provided.

In another embodiment, the invention provides an antibody which specifically binds to vertebrate Smoothened. The antibody may be an agonistic, antagonistic or neutralizing antibody.

In another embodiment, the invention provides non-human, transgenic or knock-out animals.

Another embodiment of the invention provides articles of manufacture and kits that include vertebrate Smoothened or vertebrate Smoothened antibodies.

A further embodiment of the invention provides protein complexes comprising vertebrate Smoothened protein and vertebrate Patched protein. In one embodiment the complexes further include vertebrate Hedgehog protein. The invention also provides vertebrate Patched which binds to vertebrate Smoothened. Optionally, the vertebrate Patched comprises a sequence which is a derivative of or fragment of a native sequence vertebrate Patched.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of native sequence rat Smoothened.

FIGS. 2A-2B show the primary structure of rat Smo (rSmo) (SEQ ID NO:2) and Drosophila Smo (dsmo)(SEQ ID NO:5). The signal peptide sequences are underlined, conserved amino acids are boxed, cysteines are marked with asterisks, potential glycosylation sites are marked with dashed boxes, and the seven hydrophobic transmembrane domains are shaded.

FIGS. 3A-3O show the tissue distribution of SHH, Smo and Patched in embryonic and adult rat tissues. In situ hybridization of SHH (left column); Smo (middle column) and Patched (right column, not including insets) to rat tissues. Row E15 Sag, sagittal sections through E15 rat embryos. Rows E9, E10, E12, and E15, coronal sections through E9 neural folds. E10 neural tube and somites, E12 and E15 neural tube. Insets in Row E12 show sections through forelimb bud of E12 rat embryos. Legend--ht=heart; sk=skin; bl=bladder; ts=testes; lu=lung; to=tongue; vtc=vertebral column; nf=neural fold; nc=notocord; so=somite; fp=floor plate; vh=ventral horn; vz=ventricular zone; cm=cardiac mesoderm and vm=ventral midbrain.

FIGS. 4A-4E show the nucleotide (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) for native sequence human Smoothened.

FIGS. 5A-5E show the primary structure of human Smo (hSmo) (SEQ ID NO:4) and rat Smo (rat.Smo) (SEQ ID NO:2) and homology to Drosophila Smo (dros.smo)(SEQ ID NO:5). Conserved amino acids are boxed.

FIGS. 6A-6I illustrate the results of binding and co-immunoprecipitation assays which show SHH-N binds to mPatched but not to rSmo. Staining of cells expressing the Flag tagged rSmo (a and b) or Myc tagged mPatched (c, d, and e) with (a) Flag (Smo) antibody; (c) Myc (mPatched) antibody; (b and d) IgG-SHH-N; or (e) Flag tagged SHH-N. (f) Co-immunoprecipitation of epitope tagged mPatched (Patched) or epitope tagged rSmo (Smo) with IgG-SHH-N. (g) cross-linking of .sup.125 I-SHH-N (.sup.125 I-SHH) to cells expressing mPatched or rSmo in the absence or presence of unlabeled SHH-N. (h) Co-immunoprecipitation of .sup.125 I-SHH by an epitope tagged mPatched (Patched) or an epitope tagged rSmo (Smo). (i) competition binding of .sup.125 I-SHH to cells expressing mPatched or mPatched plus rSmo.

FIGS. 7A-7E illustrate (a) Double immunohistochemical staining of Patched (red) and Smo (green) in transfected cells. Yellow indicates co-expression of the two proteins; (b and c) Detection of Patched-Smo Complex by immunoprecipitation; (b) immunoprecipitation with antibodies to the epitope tagged Patched and analysis on a Western blot with antibodies to epitope tagged Smo; (c) immunoprecipitation with antibodies to the epitope tagged Smo and analysis on a Western blot with antibodies to epitope tagged Patched; (d and e) co-immunoprecipitation of .sup.125 I-SHH bound to cells expressing both Smo and Patched with antibodies to either Smo (d) or Patched (e) epitope tags.

FIG. 8 shows a Western blot from a SDS-gel depicting the expression level of a wildtype (WT) and mutated Patched (mutant).

FIG. 9 shows a model describing the putative SHH receptor and its proposed activation by SHH. As shown in the model, Patched is a ligand binding component and vSmo is a signalling component in a multi-subunit SHH receptor.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms "vertebrate Smoothened", "vertebrate Smoothened protein" and "vSmo" when used herein encompass native sequence vertebrate Smoothened and vertebrate Smoothened variants (each of which is defined herein). These terms encompass Smoothened from a variety of animals classified as vertebrates, including mammals. In a preferred embodiment, the vertebrate Smoothened is rat Smoothened (rSmo) or human Smoothened (hSmo). The vertebrate Smoothened may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.

A "native sequence vertebrate Smoothened" comprises a protein having the same amino acid sequence as a vertebrate Smoothened derived from nature. Thus, a native sequence vertebrate Smoothened can have the amino acid sequence of naturally occurring human Smoothened, rat Smoothened, or Smoothened from any other vertebrate. Such native sequence vertebrate Smoothened can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence vertebrate Smoothened" specifically encompasses naturally-occurring truncated forms of the vertebrate Smoothened, naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the vertebrate Smoothened. In one embodiment of the invention, the native sequence vertebrate Smoothened is a mature native sequence Smoothened comprising the amino acid sequence of SEQ ID NO:4. In another embodiment of the invention, the native sequence vertebrate Smoothened is a mature native sequence Smoothened comprising the amino acid sequence of SEQ ID NO:2.

"Vertebrate Smoothened variant" means a vertebrate Smoothened as defined below having less than 100% sequence identity with vertebrate Smoothened having the deduced amino acid sequence shown in SEQ ID NO:4 for human Smoothened or SEQ ID NO:2 for rat Smoothened. Such vertebrate Smoothened variants include, for instance, vertebrate Smoothened proteins wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the sequences of SEQ ID NO:4 or SEQ ID NO:2; wherein about one to thirty amino acid residues are deleted, or optionally substituted by one or more amino acid residues; and derivatives thereof, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid. Ordinarily, a vertebrate Smoothened variant will have at least about 80% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identity with the sequence of SEQ ID NO:4 or SEQ ID NO:2.

The term "epitope tag" when used herein refers to a tag polypeptide having enough residues to provide an epitope against which an antibody thereagainst can be made, yet is short enough such that it does not interfere with activity of the vertebrate Smoothened. The tag polypeptide preferably also is fairly unique so that the antibody thereagainst does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8-50 amino acid residues (preferably between about 9-30 residues).

"Isolated," when used to describe the various proteins disclosed herein, means protein that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In preferred embodiments, the protein will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated protein includes protein in situ within recombinant cells, since at least one component of the vSmo natural environment will not be present. Ordinarily, however, isolated protein will be prepared by at least one purification step.

An "isolated" vSmo nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the vSmo nucleic acid. An isolated vSmo nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated vSmo nucleic acid molecules therefore are distinguished from the vSmo nucleic acid molecule as it exists in natural cells. However, an isolated vSmo nucleic acid molecule includes vSmo nucleic acid molecules contained in cells that ordinarily express vSmo where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The term "antibody" is used in the broadest sense and specifically covers single anti-vSmo monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-vSmo antibody compositions with polyepitopic specificity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an anti-vSmo antibody with a constant domain (e.g. "humanized" antibodies), or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab').sub.2, and Fv), so long as they exhibit the desired activity. See, e. g. U.S. Pat. No. 4,816,567 and Mage et al., in Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The "monoclonal antibodies" may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), for example.

"Humanized" forms of non-human (e.g. murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

The term "vertebrate" as used herein refers to any animal classified as a vertebrate including certain classes of fish, reptiles, birds, and mammals. The term "mammal" as used herein refers to any animal classified as a mammal, including humans, cows, rats, mice, horses, dogs and cats.

II. Modes For Carrying Out the Invention

The present invention is based on the discovery of vertebrate homologues of Smoothened. In particular, Applicants have identified and isolated human and rat Smoothened. The properties and characteristics of human and rat Smoothened are described in further detail in the Examples below. Based upon the properties and characteristics of human and rat Smoothened disclosed herein, it is Applicants' present belief that vertebrate Smoothened is a signalling component in a multi-subunit Hedgehog (particularly Sonic Hedgehog "SHH") receptor.

A description follows as to how vertebrate Smoothened may be prepared.

A. Preparation of vSmo

Techniques suitable for the production of vSmo are well known in the art and include isolating vSmo from an endogenous source of the polypeptide, peptide synthesis (using a peptide synthesizer) and recombinant techniques (or any combination of these techniques). The description below relates primarily to production of vSmo by culturing cells transformed or transfected with a vector containing vSmo nucleic acid. It is of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare vSmo.

1. Isolation of DNA Encoding vSmo

The DNA encoding vSmo may be obtained from any cDNA library prepared from tissue believed to possess the vSmo mRNA and to express it at a detectable level. Accordingly, human Smo DNA can be conveniently obtained from a cDNA library prepared from human tissues, such as the library of human embryonic lung cDNA described in Example 3. Rat Smo DNA can be conveniently obtained from a cDNA library prepared from rat tissues, such as described in Example 1. The vsmo-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the vSmo or oligonucleotides or polypeptides as described in the Examples) designed to identify the gene of interest or the protein encoded by it. The probes are preferably labeled such that they can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like .sup.32 P-labeled ATP, biotinylation or enzyme labeling. Screening the cDNA or genomic library with a selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding vSmo is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer:A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

Nucleic acid having all the protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

vSmo variants can be prepared by introducing appropriate nucleotide changes into the vSmo DNA, or by synthesis of the desired vSmo polypeptide. Those skilled in the art will appreciate that amino acid changes (compared to native sequence vSmo) may alter post-translational processes of the vSmo, such as changing the number or position of glycosylation sites.

Variations in the native sequence vSmo can be made using any of the techniques and guidelines for conservative and non-conservative mutations set forth in U.S. Pat. No. 5,364,934. These include oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.

2. Insertion of Nucleic Acid Into a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding vSmo may be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, each of which is described below.

(i) Signal Sequence Component

The vSmo may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous amino acid sequence or polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the vSmo DNA that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses.

Most expression vectors are "shuttle" vectors, i.e., they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA. Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of vSmo DNA.

(iii) Selection Gene Component

Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin [Southern et al., J. Molec. Appl. Genet., 1:327 (1982)], mycophenolic acid (Mulligan et al., Science, 209:1422 (1980)] or hygromycin [Sugden et al., Mol. Cell. Biol., 5:410-413 (1985)]. The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the vSmo nucleic acid, such as DHFR or thymidine kinase. The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker. Selection pressure is imposed by culturing the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes vSmo. Amplification is the process by which genes in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells.

Cells transformed with the DHFR selection gene may first be identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). The transformed cells are then exposed to increased levels of methotrexate. This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA comprising the expression vectors, such as the DNA encoding vSmo.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the vSmo nucleic acid sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence, such as the vSmo nucleic acid sequence, to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known. These promoters are operably linked to vSmo encoding DNA by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector.

Promoters suitable for use with prokaryotic hosts include the .beta.-lactamase and lactose promoter systems [Chang el al., Nature, 275:617-624 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CXCAAT region where X may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:12073-12080 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

vSmo transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication [Fiers et al., Nature, 273:113 (1978); Mulligan and Berg, Science, 209:1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad. Sci. USA, 78:7398-7402 (1981)]. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment [Greenaway et al., Gene, 18:355-360 (1982)]. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978 [See also Gray et al., Nature, 295:503-508 (1982) on expressing cDNA encoding immune interferon in monkey cells; Reyes et al., Nature, 297:598-601 (1982) on expression of human .beta.-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79:5166-5170 (1982) on expression of the human interferon .beta.1 gene in cultured mouse and rabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA, 79:6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter].

(v) Enhancer Element Component

Transcription of a DNA encoding the vSmo by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' [Laimins et al., Proc. Natl. Acad. Sci. USA, 78:464-468 (1981]) and 3' [Lusky el al., Mol. Cell Bio., 3:1108 (1983]) to the transcription unit, within an intron [Banerji el al., Cell. 33:729 (1983)], as well as within the coding sequence itself [Osborne et al., Mol. Cell Bio., 4:1293 (1984)]. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also typically contain sequences necess