Death domain containing receptor 4

[0001] The present application is a divisional of U.S. patent application Ser. No. 09/565,918, filed May 5 2000 now U.S. Pat. No. 6,433,147, which claims benefit to the filing dates of U.S. Provisional Application No. 60/132,922, filed May 6, 1999, and is a continuation-in-part of U.S. application Ser. No. 09/013,895, filed Jan. 27, 1998, now U.S. Pat. No. 6,342,363, each of which is herein incorporated by reference; said Ser. No. 09/013,895 claims priority to U.S. Provisional Application No. 60/037,829, filed Feb. 5, 1997, and U.S. Provisional Application No. 60/035,722, filed Jan. 28, 1997, each of which is herein incorporated by reference.

[0002] 1. Field of the Invention

[0003] The present invention relates to a novel member of the tumor necrosis factor family of receptors. More specifically, isolated nucleic acid molecules are provided encoding human Death Domain Containing Receptor 4, sometimes herein “DR4”. DR4 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of DR4 activity and methods for using DR4 polynucleotides and polypeptides.

[0004] 2. Related Art

[0005] Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intra-cellular response.

[0006] For example, tumor necrosis factors (TNF) alpha and beta are cytokines which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease. The TNF molecules belong to the “TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the “TNF-receptor” superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.

[0007] Among the ligands there are included TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-IBBL, OX40L and nerve growth factor (NGF). The superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-IBB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals, 22:291-295 (1994)).

[0008] Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).

[0009] Considerable insight into the essential functions of several members of the TNF receptor family has been gained from the identification and creation of mutants that abolish the expression of these proteins. For example, naturally occurring mutations in the FAS antigen and its ligand cause lymphoproliferative disease (Watanabe-Fukunaga, R., et al., Nature 356:314 (1992)), perhaps reflecting a failure of programmed cell death. Mutations of the CD40 ligand cause an X-linked immunodeficiency state characterized by high levels of immunoglobulin M and low levels of immunoglobulin G in plasma, indicating faulty T-cell-dependent B-cell activation (Allen, R. C. et al., Science 259:990 (1993)). Targeted mutations of the low affinity nerve growth factor receptor cause a disorder characterized by faulty sensory innovation of peripheral structures (Lee, K. F. et al., Cell 69:737 (1992)).

[0010] TNF and LT-α are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors). A large number of biological effects elicited by TNF and LT-α, acting through their receptors, include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation, proliferation and anti-viral responses, as well as protection against the deleterious effects of ionizing radiation. TNF and LT-α are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Von Huffel, C., Science 264:667-668 (1994)). Mutations in the p55 Receptor cause increased susceptibility to microbial infection.

[0011] Moreover, an about 80 amino acid domain near the C-terminus of TNFR1 (p55) and Fas was reported as the “death domain,” which is responsible for transducing signals for programmed cell death (Tartaglia et al., Cell 74:845 (1993)).

[0012] Apoptosis, or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H. Steller, Science 267, 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C. B. Thompson, Science 267, 1456-1462 (1995)). Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland, et al., Cell 81,479-482 (1995); A. Fraser, et al., Cell 85, 781-784 (1996); S. Nagata, et al., Science 267, 1449-56 (1995)). Both are members of the TNF receptor family which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (C. A. Smith, et al., Science 248, 1019-23 (1990); M. Tewari, et al., in Modular Texts in Molecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the “death domain”, which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et al., Cell 81, 185-6 (1995); K. White et al., Science 264, 677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified. Activation of Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J. Biol Chem 270,7795-8 (1995); F. C. Kischkel, et al., EMBO 14,5579-5588 (1995)), which in turn binds and presumably activates FLICE/MACH1, a member of the ICE/CED-3 family of pro-apoptotic proteases (M. Muzio et al., Cell 85, 817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996)). While the central role of Fas/APO-1 is to trigger cell death, TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L. A. Tartaglia, et al., Immunol Today 13, 151-3 (1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu, et al., Cell 81, 495-504 (1995); H. Hsu, et al., Cell 84, 299-308 (1996)). Through its associations with a number of signaling molecules including FADD, TRAF2, and RIP, TRADD can signal both apoptosis and NF-kB activation (H. Hsu, et al., Cell 84, 299-308 (1996); H. Hsu, et al., Immunity 4, 387-396 (1996)).

[0013] Recently a new apoptosis inducing ligand was discovered. Wiley, S. R. et al., refer to the new molecule as TNF-related apoptosis-inducing ligand or (“TRAIL”) (Immunity 3:673-682 (1995)). Pitti, R. M. et al., refer to the new molecule as Apo-2 ligand or (“Apo-2L”). For convenience, it will be referred to herein as TRAIL.

[0014] Unlike FAS ligand whose transcripts appear to be largely restricted to stimulated T-cells, significant levels of TRAIL are seen in many tissues, and it is constitutively transcribed by some cell lines. It has been shown that TRAIL acts independently from FAS ligand (Wiley, S. R., et al. (1995)), supra). Studies by Marsters, S. A. et al., have indicated that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signaling by FAS/Apo-IL but much faster than TNF-induced apoptosis (Current Biology, 6:750-752 (1996)). All work to date suggest that the receptor for TRAIL is not one of the many known TNF-receptors.

[0015] The effects of TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of such receptors and ligands that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characterize the receptor for the newly discovered TRAIL ligand.

[0016] The present invention provides for isolated nucleic acid molecules comprising, or alternatively consisting of, nucleic acid sequences encoding the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoding the cDNA clone deposited as ATCC Deposit No. 97853 on Jan. 21, 1997.

[0017] The present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as to methods of making such vectors and host cells and for using them for production of DR4 polypeptides or peptides by recombinant techniques.

[0018] The invention further provides an isolated DR4 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.

[0019] The present invention also provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of DR4 protein. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of DR4, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors.

[0020] Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes. Cellular response to TNF-family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis. Apoptosis-programmed cell death-is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes. Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers, autoimmune disorders, viral infections, inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection. Diseases associated with increased apoptosis include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock, cachexia and anorexia.

[0021] Thus, the invention further provides a method for enhancing apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the DR4 polypeptide an effective amount of an agonist capable of increasing DR4 mediated signaling. Preferably, DR4 mediated signaling is increased to treat and/or prevent a disease wherein decreased apoptosis is exhibited.

[0022] In a further aspect, the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the DR4 polypeptide an effective amount of an antagonist capable of decreasing DR4 mediated signaling. Preferably, DR4 mediated signaling is decreased to treat and/or prevent a disease wherein increased apoptosis is exhibited.

[0023] Whether any candidate “agonist” or “antagonist” of the present invention can enhance or inhibit apoptosis can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below. Thus, in a further aspect, a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand. The method involves contacting cells which express the DR4 polypeptide with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway. By the invention, a cell expressing the DR4 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.

[0030] The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a nucleic acid sequence encoding the DR4 polyeptide whose amino acid sequence is shown in SEQ ID NO:2, or a fragment of the polyeptide. The DR4 polypeptide of the present invention shares sequence homology with human TNFR-I, DR3 and Fas ligand (FIGS. 2A through C). The nucleotide sequence shown in SEQ ID NO:1 was obtained by sequencing cDNA clones such as HCUDS60, which was deposited on Jan. 21, 1997 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va., 20110-2209 and given Accession Number 97853. The deposited clone is contained in the pBK plasmid (Stratagene, LaJolla, Calif.).

Nucleic Acid Molecules

[0031] Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

[0032] By “isolated” polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0033] Using the information provided herein, such as the nucleic acid sequence set out in SEQ ID NO:1, a nucleic acid molecule of the present invention encoding a DR4 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the gene of the present invention has also been identified in cDNA libraries of the following tissues: amniotic cells, heart, liver cancer, kidney, leukocyte, activated T-cell, K562 plus PMA, W138 cells, Th2 cells, human tonsils, and CD34 depleted buffy coat (cord blood).

[0034] The DR4 gene contains an open reading frame encoding a mature protein of about 445 amino acid residues whose initiation codon is at position 19-21 of the nucleotide sequence shown in SEQ ID NO. 1, with a leader sequence of about 23 amino acid residues (i.e., a total protein length of 468 amino acids), and a deduced molecular weight of about 50 kDa. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, at either terminus or at both termini.

[0035] Of known members of the TNF receptor family, the DR4 polypeptide of the invention shares the greatest degree of homology with human TNFR1 and DR3 polypeptides shown in FIGS. 2A through C, including significant sequence homology over the multiple Cysteine Rich domains.

[0036] In addition to the sequence homology exhibited between DR4 and other death domain containing receptors, DR4 has been shown to bind to TRAIL and to induce apoptosis when transiently expressed. MCF7 human breast carcinoma cells and 293 cells were transiently transfected with a DR4 expressing construct, as described in Example 5. As shown in FIGS. 5A and 5B a substantial proportion of transfected cells underwent the morphological changes characteristic of apoptosis. As anticipated, deletion of the death domain abolished the ability of DR4 to engage the death pathway. As can be seen in FIG. 5C, DR4-induced apoptosis was efficiently blocked by inhibitors of death proteases including z-VAD-fmk, an irreversible broad spectrum caspase inhibitor and CrmA, a cowpox virus encoded serpin that preferentially inhibits apical caspases such as FLICE/MACH-1 (caspase-8). Since TNFR-1, CD-95 and DR3-induced apoptosis is also attenuated by these same inhibitors, it is likely that the downstream death effector molecules are similar in nature.

[0037] To determine if DR4 was capable of binding TRAIL, the extracellular ligand binding domain of DR4 was expressed as a fusion to the Fc region of human IgG (DR4-Fc). TRAIL selectively bound to DR4-Fc but not to corresponding extracellular domains of TNFR-1 or CD-95, also expressed as Fc fusions, data not shown. Additionally, DR4-Fc did not bind either TNF alpha or Fas ligand under conditions where both of these ligands bound their cognate receptors.

[0038] The ability of TRAIL to induce apoptosis in MCF7 cells was specifically blocked by DR4-Fc but not influenced by TNFR1-Fc, CD95-Fc or Fc alone (FIG. 6A). Further, as expected, TNF alpha-induced apoptosis was inhibited by TNFR-1-Fc but not by DR4-Fc, CD95-Fc or Fc alone (FIG. 6B).

[0039] Taken together, the data described above indicate that DR4 is a death domain containing receptor with the ability to induce apoptosis and is a receptor for TRAIL a known apoptosis inducing ligand.

[0040] As indicated, the present invention also provides the mature form(s) of the DR4 protein of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides a nucleotide sequence encoding the mature DR4 polypeptide having the amino acid sequence encoded by the cDNA contained in the host identified as ATCC Deposit No. 97853, and as shown in SEQ ID NO:2. By the mature DR4 protein having the amino acid sequence encoded by the cDNA contained in the host identified as ATCC Deposit No. 97853, is meant the mature form(s) of the DR4 protein produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human cDNA contained in the vector in the deposited host. As indicated below, the mature DR4 having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 97853, may or may not differ from the predicted “mature” DR4 protein shown in SEQ ID NO:2 (amino acids from about 24 to about 468 in SEQ ID NO:2) depending on the accuracy of the predicted cleavage site based on computer analysis. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, at either terminus or at both termini.

[0041] Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the method of McGeoch (Virus Res. 3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

[0042] In the present case, the predicted amino acid sequence of the complete DR4 polypeptide of the present invention was analyzed by a computer program (“PSORT”). (See K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis by the PSORT program predicted the cleavage sites between amino acids 23 and 24 in SEQ ID NO:2. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (−1,−3) rule of von Heine. von Heinje, supra. Thus, the leader sequence for the DR4 protein is predicted to consist of amino acid residues 1-23, underlined in SEQ ID NO:2, while the predicted mature DR4 protein consists of about residues 24-468.

[0043] As one of ordinary skill would appreciate, due to the possibility of sequencing errors, as well as the variability of cleavage sites for leaders in different known proteins, the predicted DR4 receptor polypeptide encoded by the deposited cDNA comprises about 468 amino acids, but may be anywhere in the range of 458-478 amino acids; and the predicted leader sequence of this protein is about 40 amino acids, but may be anywhere in the range of about 30 to about 50 amino acids. It will further be appreciated that, the domains described herein have been predicted by computer analysis, and accordingly, that depending on the analytical criteria used for identifying various functional domains, the exact “address” of, for example, the extracelluar domain, intracellular domain, death domain, cysteine-rich motifs, and transmembrane domain of DR4 may differ slightly. For example, the exact location of the DR4 extracellular domain in SEQ ID NO:2 may vary slightly (e.g., the address may “shift” by about 1 to about 20 residues, more likely about 1 to about 5 residues) depending on the criteria used to define the domain. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, at either terminus or at both termini. In any event, as discussed further below, the invention further provides polypeptides having various residues deleted from the N-terminus and/or C-terminus of the complete DR4, including polypeptides lacking one or more amino acids from the N-termini of the extracellular domain described herein, which constitute soluble forms of the extracellular domain of the DR4 polypeptides.

[0044] As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

[0045] By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.

[0046] However, a nucleic acid molecule contained in a clone that is a member of a mixed clone library (e.g., a genomic or cDNA library) and that has not been isolated from other clones of the library (e.g., in the form of a homogeneous solution containing the clone without other members of the library) or a chromosome isolated or removed from a cell or a cell lysate (e.g., a “chromosome spread”, as in a karyotype), is not “isolated” for the purposes of this invention. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0047] Isolated nucleic acid molecules of the present invention include DR4 DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ID NO:1 and further include DNA molecules which comprise, or alternatively consist of, a sequence substantially different than all or part of the ORF whose initiation codon is at position 19-21 of the nucleotide sequence shown in SEQ ID NO:1 but which, due to the degeneracy of the genetic code, still encode the DR4 polypeptide or a fragment thereof. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.

[0048] In another aspect, the invention provides isolated nucleic acid molecules encoding the DR4 polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97853 on Jan. 21, 1997. Preferably, these nucleic acid molecules will encode the mature polypeptide encoded by the above-described deposited cDNA. The invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:1 or the nucleotide sequence of the DR4 cDNA contained in the above-described deposited plasmid, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated DNA molecules and fragments thereof, have uses which include, but are not limited to, as DNA probes for gene mapping by in situ hybridization of the DR4 gene in human tissue by Northern blot analysis.

[0049] The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By fragments of an isolated DNA molecule having the nucleotide sequence shown in SEQ ID NO:1 or having the nucleotide sequence of the deposited cDNA (the cDNA contained in the plasmid deposited as ATCC Deposit No. 97853) are intended DNA fragments at least 20 nt, and more preferably at least 30 nt in length, and even more preferably, at least about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 nt in length, which are useful as DNA probes as discussed above. Of course, DNA fragments corresponding to most, if not all, of the nucleotide sequence shown in SEQ ID NO:1 are also useful as DNA probes. By a fragment about 20 nt in length, for example, is intended fragments which include 20 or more bases from the nucleotide sequence in SEQ ID NO:1. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

[0050] Representative examples of DR4 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively consist of, a sequence from about nucleotide 19 to 87, 88 to 732, 88 to 138, 139 to 189, 190 to 240, 241 to 291, 292 to 342, 343 to 705, 343 to 393, 394 to 444, 445 to 495, 496 to 546, 547 to 597, 598 to 648, 649 to 699, 700 to 732, 733 to 810, 733 to 771, 772 to 810, 811 to 1422, 811 to 861, 862 to 912, 913 to 963, 964 to 1014, 1015 to 1065, 1066 to 1116, 1117 to 1167, 1153 to 1284, 1153 to 1203, 1204 to 1254, 1255 to 1284, 1168 to 1218, 1219 to 1269, 1270 to 1320, 1321 to 1371, and 1372 to 1422 of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in the deposited plasmid. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

[0051] The present invention is further directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode domains of DR4. In one aspect, the invention provides polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode beta-sheet regions of DR4 protein set out in Table I. Representative examples of such polynucleotides include nucleic acid molecules which encode a polypeptide comprising, or alternatively consisting of, one, two, three, four, five or more amino acid sequences selected from the group consisting of amino acid residues from about 8 to about 17, amino acid residues from about 53 to about 60, amino acid residues from about 87 to about 103, amino acid residues from about 146 to about 155, amino acid residues from about 161 to about 166, amino acid residues from about 214 to about 221, amino acid residues from about 240 to about 252, amino acid residues from about 257 to about 264, amino acid residues from about 274 to about 283, amino acid residues from about 324 to about 329, amino acid residues from about 349 to about 354, amino acid residues from about 363 to about 369, amino acid residues from about 371 to about 376, amino acid residues from about 394 to about 399, and amino acid residues from about 453 to about 458 in SEQ ID NO:2. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

[0052] In specific embodiments, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a DR4 functional activity. By a polypeptide demonstrating a DR4 “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a complete (full-length) or mature DR4 polypeptide. Such functional activities include, but are not limited to, biological activity (e.g., ability to induce apoptosis in cells expressing the polypeptide (see, e.g., Example 5), antigenicity (ability to bind (or compete with a DR4 polypeptide for binding) to an anti-DR4 antibody), immunogenicity (ability to generate antibody which binds to a DR4 polypeptide), ability to form multimers, and ability to bind to a receptor or ligand for a DR4 polypeptide (e.g., TRAIL; Wiley et al., Immunity 3, 673-682 (1995)).

[0053] The functional activity of DR4 polypeptides, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

[0054] For example, in one embodiment where one is assaying for the ability to bind or compete with full-length (complete) DR4 polypeptide for binding to anti-DR4 antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

[0055] In another embodiment, where a DR4 ligand is identified (e.g., TRAIL), or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological correlates of DR4 binding to its substrates (signal transduction) can be assayed.

[0056] In addition, assays described herein (see Examples 5 and 6), and those otherwise known in the art may routinely be applied to measure the ability of DR4 polypeptides and fragments, variants derivatives, and analogs thereof to elicit DR4 related biological activity (e.g., ability to bind TRAIL (see e.g., Example 6), ability to induce apoptosis in cells expressing the polypeptide (see e.g., Example 5) in vitro or in vivo). For example, biological activity can routinely be measured using the cell death assays performed essentially as previously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol. Chem. 270:7795-8 (1995); Kischkel et al., EMBO 14:5579-5588 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996)) and as set forth in Example 5 below. In one embodiment involving MCF7 cells, plasmids encoding full-length DR4 or a candidate death domain containing receptor are co-transfected with the pLantern reporter construct encoding green fluorescent protein. Nuclei of cells transfected with DR4 will exhibit apoptotic morphology as assessed by DAPI staining.

[0057] Other methods will be known to the skilled artisan and are within the scope of the invention.

[0058] Preferred nucleic acid fragments of the present invention include a nucleic acid molecule encoding a member selected from the group: a polypeptide comprising, or alternatively consisting of, the DR4 extracellular domain (amino acid residues from about 24 to about 238 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, the DR4 cysteine rich domain (amino acid residues from about 131 to about 229 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, the DR4 transmembrane domain (amino acid residues from about 239 to about 264 in SEQ ID NO:2); a fragment of the predicted mature DR4 polypeptide, wherein the fragment has a DR4 functional activity (e.g., antigenic activity or biological activity); a polypeptide comprising, or alternatively consisting of, the DR4 intracellular domain (amino acid residues from about 265 to about 468 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, the DR4 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; a polypeptide comprising, or alternatively consisting of, DR4 receptor death domain (predicted to constitute amino acid residues from about 379 to about 422 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, one, two, three, four or more, epitope bearing portions of the DR4 receptor protein. In additional embodiments, the polynucleotide fragments of the invention encode a polypeptide comprising, or alternatively consisting of, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above-encoded polypeptide embodiments. As above, with the leader sequence, the amino acid residues constituting the DR4 receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 residues) depending on the criteria used to define the domain. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.

[0059] It is believed one or both of the extracellular cysteine rich motifs of the DR4 polypeptide disclosed in SEQ ID NO:2 is important for interactions between DR4 and its ligands (e.g., TRAIL). Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of one or both of amino acid residues 131 to 183, and/or 184 to 229 of SEQ ID NO:2. In a specific embodiment the polynucleotides encoding DR4 polypeptides of the invention comprise, or alternatively consist of both of the extracellular cysteine rich motifs disclosed in SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

[0060] In additional embodiments, the polynucleotides of the invention encode functional attributes of DR4. Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, one, two, three, four, or more of the following functional domains: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of DR4.

[0061] Certain preferred regions in these regards are set out in FIG. 3, but may, as shown in Table I, be represented or identified by using tabular representations of the data presented in FIG. 3. The DNA*STAR computer algorithm used to generate FIG. 3 (set on the original default parameters) was used to present the data in FIG. 3 in a tabular format (See Table I). The tabular format of the data in FIG. 3 may be used to easily determine specific boundaries of a preferred region.

[0062] The above-mentioned preferred regions set out in FIG. 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in FIG. 3 and in Table I, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Table I), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in Table I), Kyte-Doolittle hydrophilic regions (column VIII in Table I), Hopp-Woods hydrophobic regions (column IX in Table I), Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Table I), Karplus-Schulz flexible regions (column XII in Table I), Jameson-Wolf regions of high antigenic index (column XIII in Table I), and Emini surface-forming regions (column XIV in Table I). Among highly preferred polynucleotides in this regard are those that encode polypeptides comprising, or alternatively consisting of, regions of DR4 that combine several structural features, such as several (e.g., 1, 2, 3, or 4) of the same or different region features set out above.

[0063] The data representing the structural or functional attributes of DR4 set forth in FIG. 3 and/or Table I, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, XII and XIII of Table I can be used to determine regions of DR4 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

[0064]

TABLE I
Res	Pos.	I	II	III	IV	V	VI	VII	VIII	IX	X	XI	XII	XIII
Met	1	.	.	B	.	.	.	.	0.12	.	.	.	−0.10	0.90
Ala	2	.	.	.	.	.	.	C	−0.08	*	*	.	0.25	1.08
Pro	3	.	.	.	.	.	.	C	0.42	*	*	.	0.10	0.86
Pro	4	.	.	.	.	.	T	C	−0.04	*	*	.	1.05	1.69
Pro	5	A	.	.	.	.	T	.	0.31	.	*	F	1.00	1.24
Ala	6	A	.	.	.	.	T	.	0.10	.	*	F	1.00	1.10
Arg	7	A	.	.	.	.	T	.	0.34	.	*	.	0.10	0.58
Val	8	.	.	B	B	.	.	.	−0.03	.	*	.	−0.30	0.37
His	9	.	.	B	B	.	.	.	−0.52	.	*	.	−0.30	0.37
Leu	10	.	.	B	B	.	.	.	−1.12	.	*	.	−0.60	0.17
Gly	11	.	.	B	B	.	.	.	−1.12	.	*	.	−0.60	0.18
Ala	12	.	.	B	B	.	.	.	−2.09	.	*	.	−0.60	0.14
Phe	13	.	.	B	B	.	.	.	−1.54	.	*	.	−0.60	0.12
Leu	14	.	.	B	B	.	.	.	−1.72	.	.	.	−0.60	0.18
Ala	15	.	.	B	B	.	.	.	−0.91	.	.	.	−0.60	0.27
Val	16	.	.	B	B	.	.	.	−0.78	.	.	.	−0.60	0.51
Thr	17	.	.	B	B	.	.	.	−0.53	.	.	F	−0.45	0.95
Pro	18	.	.	.	B	.	.	C	−0.13	.	.	F	0.05	0.93
Asn	19	.	.	.	.	.	T	C	0.09	.	.	F	0.60	1.69
Pro	20	.	.	.	.	.	T	C	0.09	.	.	F	0.60	1.18
Gly	21	.	.	.	.	T	T	.	0.64	.	.	F	0.65	0.77
Ser	22	.	.	.	.	.	T	C	0.61	.	.	F	0.45	0.64
Ala	23	.	.	.	.	.	.	C	0.51	.	.	F	0.25	0.41
Ala	24	.	.	.	.	.	T	C	0.51	.	.	F	0.45	0.60
Ser	25	.	.	B	.	.	T	.	0.13	.	.	F	0.85	0.78
Gly	26	A	.	.	.	.	T	.	−0.11	.	.	F	0.85	0.78
Thr	27	A	.	.	.	.	T	.	−0.40	.	.	F	0.85	0.78
Glu	28	A	A	.	.	.	.	.	−0.40	.	.	F	0.45	0.58
Ala	29	A	A	.	.	.	.	.	−0.12	.	.	.	0.30	0.60
Ala	30	A	A	.	.	.	.	.	−0.03	.	.	.	0.30	0.60
Ala	31	A	A	.	.	.	.	.	0.01	.	.	.	0.30	0.53
Ala	32	A	A	.	.	.	.	.	0.37	.	.	.	−0.30	0.71
Thr	33	A	.	.	.	.	T	.	−0.49	*	.	F	1.00	1.40
Pro	34	A	.	.	.	.	T	.	−0.19	.	.	F	1.00	1.03
Ser	35	.	.	B	.	.	T	.	0.06	.	.	F	0.40	1.07
Lys	36	.	.	B	.	.	T	.	0.34	.	.	F	0.25	0.73
Val	37	.	.	B	B	.	.	.	0.63	.	.	F	−0.15	0.64
Trp	38	.	.	B	B	.	.	.	0.36	.	.	F	−0.15	0.64
Gly	39	.	.	B	B	.	.	.	0.22	*	*	F	−0.15	0.32
Ser	40	.	.	.	.	.	.	C	0.63	*	*	F	−0.05	0.43
Ser	41	.	.	.	.	.	T	C	−0.30	*	*	F	0.45	0.80
Ala	42	.	.	.	.	.	T	C	0.56	*	*	F	1.05	0.57
Gly	43	.	.	.	.	.	T	C	0.63	*	*	F	1.35	0.73
Arg	44	.	.	B	.	.	T	.	1.09	*	*	F	1.49	0.84
Ile	45	.	.	B	.	.	.	.	1.04	*	*	F	1.78	1.63
Glu	46	.	.	B	.	.	.	.	1.00	*	*	F	2.12	1.63
Pro	47	.	.	B	.	.	T	.	1.24	*	*	F	2.51	0.83
Arg	48	.	.	.	.	T	T	.	1.70	*	*	F	3.40	1.17
Gly	49	.	.	.	.	T	T	.	1.24	*	*	F	3.06	1.32
Gly	50	.	.	.	.	T	T	.	1.54	*	*	F	2.57	0.84
Gly	51	.	.	.	.	.	T	C	0.73	*	*	F	2.03	0.44
Arg	52	.	.	.	.	.	T	C	0.73	*	*	F	1.39	0.36
Gly	53	.	.	B	.	.	T	.	0.31	*	*	F	0.85	0.57
Ala	54	.	.	B	.	.	T	.	0.36	.	*	F	0.85	0.83
Leu	55	.	.	B	.	.	.	.	0.10	.	*	F	0.65	0.57
Pro	56	.	.	B	.	.	.	.	0.10	.	*	F	−0.25	0.57
Thr	57	.	.	B	.	.	.	.	−0.01	.	*	F	−0.25	0.55
Ser	58	.	.	B	.	.	T	.	0.30	.	.	F	0.10	1.16
Met	59	.	.	B	.	.	T	.	0.54	.	.	F	0.40	1.02
Gly	60	.	.	B	.	.	T	.	1.14	.	.	F	0.25	0.70
Gln	61	.	.	.	.	T	T	.	1.06	.	.	F	0.65	0.81
His	62	.	.	.	.	.	.	C	0.78	.	*	F	0.40	1.10
Gly	63	.	.	.	.	.	T	C	1.19	.	*	F	0.60	1.12
Pro	64	.	.	.	.	.	T	C	1.20	.	*	F	1.20	1.27
Ser	65	.	.	.	.	.	T	C	1.66	.	*	F	1.05	0.94
Ala	66	.	.	B	.	.	T	.	1.07	.	*	F	1.30	1.86
Arg	67	.	.	B	.	.	.	.	0.76	*	*	.	1.29	1.22
Ala	68	.	.	B	.	.	.	.	1.21	*	*	.	1.48	0.90
Arg	69	.	.	B	.	.	T	.	0.83	.	*	.	2.17	1.74
Ala	70	.	.	B	.	.	T	.	0.92	.	*	F	2.51	0.90
Gly	71	.	.	.	.	T	T	.	1.17	.	*	F	3.40	1.37
Arg	72	.	.	.	.	.	T	C	0.84	.	*	F	2.71	0.69
Ala	73	.	.	.	.	.	T	C	1.54	*	.	F	2.48	1.06
Pro	74	.	.	.	.	.	T	C	1.22	*	.	F	2.70	2.10
Gly	75	.	.	.	.	.	T	C	1.22	*	.	F	2.62	1.66
Pro	76	.	.	.	.	.	T	C	1.68	*	*	F	2.24	1.66
Arg	77	.	.	.	.	.	.	C	1.57	*	.	F	2.60	2.10
Pro	78	.	A	B	.	.	.	.	1.57	*	.	F	1.94	3.68
Ala	79	.	A	B	.	.	.	.	1.48	*	.	F	1.68	2.40
Arg	80	.	A	B	.	.	.	.	1.61	*	*	F	1.42	1.64
Glu	81	.	A	B	.	.	.	.	1.93	*	*	F	1.16	1.64
Ala	82	A	A	.	.	.	.	.	1.01	*	*	F	0.90	3.19
Ser	83	A	.	.	.	.	T	.	1.33	*	*	F	1.30	1.34
Pro	84	A	.	.	.	.	T	.	1.07	*	*	F	1.30	1.52
Arg	85	A	.	.	.	.	T	.	0.92	*	*	F	1.00	1.12
Leu	86	A	.	.	.	.	T	.	0.97	.	*	.	0.85	1.13
Arg	87	A	.	.	B	.	.	.	1.24	.	*	.	0.75	1.46
Val	88	A	.	.	B	.	.	.	0.84	*	*	.	0.75	1.08
His	89	A	.	.	B	.	.	.	1.10	.	*	.	−0.15	1.13
Lys	90	A	.	.	B	.	.	.	0.29	*	*	F	0.90	1.16
Thr	91	.	.	B	B	.	.	.	0.24	*	*	F	0.00	1.35
Phe	92	.	.	B	B	.	.	.	−0.72	*	*	.	−0.30	0.74
Lys	93	.	.	B	B	.	.	.	−0.72	*	*	.	−0.30	0.27
Phe	94	.	.	B	B	.	.	.	−1.03	*	.	.	−0.60	0.14
Val	95	.	.	B	B	.	.	.	−1.93	*	.	.	−0.60	0.16
Val	96	.	.	B	B	.	.	.	−2.43	.	*	.	−0.60	0.06
Val	97	.	.	B	B	.	.	.	−2.54	.	*	.	−0.60	0.06
Gly	98	.	.	B	B	.	.	.	−2.59	.	*	.	−0.60	0.06
Val	99	.	.	B	B	.	.	.	−2.74	.	.	.	−0.60	0.15
Leu	100	.	.	B	B	.	.	.	−2.74	*	.	.	−0.60	0.15
Leu	101	.	.	B	B	.	.	.	−2.10	*	.	.	−0.60	0.11
Gln	102	.	.	B	B	.	.	.	−1.54	*	.	.	−0.60	0.23
Val	103	.	.	B	B	.	.	.	−1.50	.	.	.	−0.60	0.37
Val	104	.	.	B	.	.	T	.	−1.23	.	.	.	−0.20	0.61
Pro	105	.	.	B	.	.	T	.	−1.01	*	.	F	0.25	0.35
Ser	106	A	.	.	.	.	T	.	−0.51	*	.	F	−0.05	0.48
Ser	107	A	.	.	.	.	T	.	−1.40	*	*	F	0.25	0.94
Ala	108	A	.	.	.	.	.	.	−0.50	.	*	F	0.05	0.43
Ala	109	A	.	.	.	.	.	.	−0.46	.	*	.	0.50	0.63
Thr	110	A	.	.	.	.	.	.	−0.28	.	*	.	−0.10	0.39
Ile	111	A	.	.	.	.	.	.	0.02	.	*	.	−0.10	0.53
Lys	112	.	.	B	.	.	.	.	0.32	.	*	.	0.50	0.87
Leu	113	.	.	B	.	.	.	.	0.61	.	*	F	1.05	1.04
His	114	.	.	B	.	.	.	.	0.31	.	*	F	1.30	1.99
Asp	115	.	.	.	.	.	T	C	0.28	*	*	F	1.80	0.70
Gln	116	.	.	.	.	T	T	.	0.86	.	*	F	1.65	0.84
Ser	117	.	.	.	.	T	T	.	0.81	.	.	F	2.50	0.89
Ile	118	.	.	.	.	T	T	.	1.62	.	.	F	2.25	0.92
Gly	119	.	.	.	.	.	.	C	1.37	.	.	F	1.00	0.92
Thr	120	.	.	.	.	.	.	C	1.37	.	.	F	0.45	0.72
Gln	121	.	.	B	.	.	.	C	1.33	.	.	F	0.65	1.79
Gln	122	.	.	B	.	.	.	.	1.33	.	.	F	0.20	2.46
Trp	123	.	.	B	.	.	.	.	2.01	.	.	.	0.05	2.28
Glu	124	.	.	.	.	.	.	C	1.54	.	.	.	0.25	2.04
His	125	.	.	.	.	.	.	C	1.51	.	.	.	0.10	0.97
Ser	126	.	.	.	.	.	T	C	1.51	.	.	F	0.45	0.91
Pro	127	.	.	.	.	T	T	.	0.70	.	.	F	1.55	0.91
Leu	128	.	.	.	.	T	T	.	0.32	.	.	F	0.65	0.55
Gly	129	.	.	.	.	T	T	.	0.11	.	.	F	0.65	0.22
Glu	130	.	.	.	.	T	.	.	−0.07	.	.	F	0.45	0.22
Leu	131	.	.	B	.	.	.	.	−0.11	*	.	.	0.18	0.42
Cys	132	.	.	B	.	.	.	.	−0.20	*	.	F	1.21	0.42
Pro	133	.	.	B	.	.	T	.	0.58	*	*	F	1.69	0.32
Pro	134	.	.	.	.	T	T	.	1.03	.	*	F	1.47	0.53
Gly	135	.	.	.	.	T	T	.	0.73	.	*	F	2.80	1.94
Ser	136	.	.	.	.	.	T	C	1.54	*	.	F	2.32	1.68
His	137	.	.	.	.	.	.	C	2.32	*	.	F	2.48	1.88
Arg	138	.	.	B	.	.	.	.	2.32	*	.	F	2.34	3.72
Ser	139	.	.	B	.	.	.	.	2.19	*	.	F	2.40	4.29
Glu	140	.	.	.	.	T	.	.	1.94	*	.	F	2.86	3.12
Arg	141	.	.	.	.	T	T	.	1.58	*	.	F	3.40	1.61
Pro	142	.	.	.	.	T	T	.	1.61	.	*	F	2.91	0.64
Gly	143	.	.	.	.	T	T	.	1.61	.	*	F	2.57	0.60
Ala	144	.	.	.	.	T	T	.	1.24	.	*	.	2.08	0.60
Cys	145	.	.	.	.	T	.	.	0.93	.	*	.	1.41	0.21
Asn	146	.	.	B	.	.	.	.	0.82	.	*	.	0.84	0.30
Arg	147	.	.	B	.	.	.	.	0.69	*	.	.	1.01	0.52
Cys	148	.	.	B	.	.	T	.	0.18	*	.	F	1.83	0.96
Thr	149	.	.	B	.	.	T	.	0.42	*	.	F	1.70	0.44
Glu	150	.	.	B	.	.	T	.	0.84	*	.	F	1.53	0.22
Gly	151	.	.	B	.	.	T	.	0.53	*	.	F	0.76	0.65
Val	152	.	.	B	B	.	.	.	0.42	.	*	F	0.19	0.65
Gly	153	.	.	B	B	.	.	.	0.50	.	.	.	−0.13	0.61
Tyr	154	.	.	B	B	.	.	.	0.51	.	.	.	−0.60	0.62
Thr	155	.	.	B	B	.	.	.	0.51	.	.	F	−0.30	1.12
Asn	156	.	.	.	B	.	.	C	0.86	.	.	F	0.20	1.81
Ala	157	.	.	.	.	T	T	.	0.90	.	.	F	0.80	1.86
Ser	158	.	.	.	.	T	T	.	0.54	.	.	F	0.80	1.06
Asn	159	.	.	.	.	T	T	.	0.20	.	.	F	0.35	0.57
Asn	160	.	.	.	.	T	T	.	−0.16	*	.	F	0.35	0.57
Leu	161	.	A	B	.	.	.	.	−0.97	*	.	.	−0.60	0.23
Phe	162	.	A	B	.	.	.	.	−0.59	.	.	.	−0.60	0.12
Ala	163	.	A	B	.	.	.	.	−0.96	.	.	.	−0.60	0.11
Cys	164	.	A	B	.	.	.	.	−1.27	*	.	.	−0.60	0.07
Leu	165	.	.	B	.	.	T	.	−1.86	.	.	.	−0.20	0.12
Pro	166	.	.	B	.	.	T	.	−1.71	*	.	.	−0.20	0.12
Cys	167	.	.	.	.	T	T	.	−0.97	*	.	.	0.20	0.12
Thr	168	A	.	.	.	.	T	.	−0.68	.	.	.	0.10	0.30
Ala	169	A	.	.	.	.	.	.	−0.01	.	.	.	0.50	0.26
Cys	170	A	.	.	.	.	T	.	0.80	.	.	.	0.70	0.80
Lys	171	A	.	.	.	.	T	.	1.01	.	.	F	1.15	0.96
Ser	172	A	.	.	.	.	T	.	1.68	.	*	F	1.30	1.65
Asp	173	A	.	.	.	.	T	.	2.10	.	*	F	1.30	5.33
Glu	174	A	A	.	.	.	.	.	2.39	.	*	F	0.90	5.22
Glu	175	A	A	.	.	.	.	.	2.84	.	*	F	1.24	5.22
Glu	176	A	A	.	.	.	.	.	2.13	.	*	F	1.58	4.83
Arg	177	.	A	.	.	T	.	.	2.12	.	.	F	2.32	1.50
Ser	178	.	.	.	.	.	T	C	1.81	.	.	F	2.86	1.25
Pro	179	.	.	.	.	T	T	.	1.50	*	.	F	3.40	1.04
Cys	180	.	.	.	.	T	T	.	1.61	*	.	F	2.61	0.77
Thr	181	.	.	.	.	T	T	.	1.61	*	.	F	2.67	1.12
Thr	182	.	.	.	.	T	.	.	1.19	*	*	F	2.38	1.16
Thr	183	.	.	.	.	T	T	.	0.90	.	.	F	2.49	3.13
Arg	184	.	.	.	.	T	T	.	0.44	.	.	F	2.40	2.19
Asn	185	.	.	.	.	T	T	.	1.11	.	.	F	2.50	0.81
Thr	186	.	.	.	.	T	T	.	0.76	*	.	F	2.25	0.98
Ala	187	.	.	.	.	T	.	.	1.11	*	.	.	1.65	0.27
Cys	188	.	.	.	.	T	.	.	1.21	*	.	.	1.40	0.33
Gln	189	.	.	B	.	.	.	.	0.76	*	.	.	0.75	0.36
Cys	190	.	.	B	.	.	.	.	0.44	.	.	.	0.50	0.35
Lys	191	.	.	B	.	.	T	.	0.06	.	*	F	0.85	0.94
Pro	192	.	.	.	.	T	T	.	0.76	.	.	F	0.65	0.47
Gly	193	.	.	.	.	T	T	.	1.42	.	*	F	1.74	1.72
Thr	194	.	.	B	.	.	T	.	1.42	.	*	F	1.68	1.38
Phe	195	.	.	B	.	.	.	.	2.09	.	*	F	1.82	1.49
Arg	196	.	.	.	.	T	.	.	1.74	.	*	F	2.56	2.42
Asn	197	.	.	.	.	T	T	.	1.37	.	*	F	3.40	2.25
Asp	198	.	.	.	.	T	T	.	1.71	.	*	F	3.06	2.63
Asn	199	.	.	.	.	.	T	C	1.42	.	*	F	2.52	2.32
Ser	200	A	.	.	.	.	T	.	1.46	.	*	F	1.98	1.43
Ala	201	A	.	.	.	.	.	.	1.46	.	*	.	1.14	0.46
Glu	202	A	.	.	.	.	.	.	1.50	*	.	.	0.80	0.56
Met	203	A	.	.	.	.	.	.	0.83	*	.	.	1.11	0.83
Cys	204	A	.	.	.	.	T	.	0.53	*	.	.	1.62	0.44
Arg	205	.	.	.	.	T	T	.	0.52	*	.	.	2.33	0.34
Lys	206	.	.	.	.	T	T	.	0.77	*	.	F	2.49	0.50
Cys	207	.	.	.	.	T	T	.	0.10	*	.	F	3.10	0.92
Ser	208	.	.	.	.	T	.	.	0.49	*	*	F	2.59	0.25
Thr	209	.	.	.	.	T	.	.	1.27	*	*	F	1.98	0.19
Gly	210	.	.	.	.	T	.	.	0.81	*	.	F	1.67	0.71
Cys	211	.	.	B	.	.	T	.	0.17	*	*	F	1.16	0.53
Pro	212	.	.	.	.	T	T	.	−0.02	*	*	F	1.25	0.36
Arg	213	.	.	.	.	T	T	.	0.32	*	*	F	0.65	0.27
Gly	214	.	.	B	.	.	T	.	−0.22	*	*	.	0.85	1.01
Met	215	.	.	B	B	.	.	.	0.17	*	*	.	0.30	0.48
Val	216	.	.	B	B	.	.	.	0.83	*	*	.	0.79	0.49
Lys	217	.	.	B	B	.	.	.	0.38	*	*	.	0.98	0.83
Val	218	.	.	B	B	.	.	.	−0.04	*	*	F	1.32	0.45
Lys	219	.	.	B	B	.	.	.	0.09	.	*	F	1.51	0.88
Asp	220	.	.	B	.	.	.	.	0.40	.	*	F	1.90	0.68
Cys	221	.	.	B	.	.	.	.	0.96	.	*	F	0.81	0.96
Thr	222	.	.	.	.	.	T	C	0.91	.	*	F	1.62	0.65
Pro	223	.	.	.	.	T	T	.	0.88	.	*	F	1.63	0.65
Trp	224	.	.	.	.	T	T	.	0.83	.	*	F	0.54	0.84
Ser	225	A	.	.	.	.	T	.	0.17	.	.	F	1.00	1.01
Asp	226	A	A	.	.	.	.	.	−0.02	.	.	F	0.45	0.35
Ile	227	A	A	.	.	.	.	.	0.26	*	.	.	−0.30	0.25
Glu	228	A	A	.	.	.	.	.	0.51	*	.	.	0.30	0.25
Cys	229	.	A	B	.	.	.	.	0.80	*	.	.	0.60	0.30
Val	230	A	A	.	.	.	.	.	0.80	*	*	.	0.60	0.74
His	231	A	A	.	.	.	.	.	0.46	*	*	.	0.60	0.58
Lys	232	A	A	.	.	.	.	.	1.34	*	.	F	0.60	1.06
Glu	233	.	A	.	.	T	.	.	1.00	*	.	F	1.30	2.30
Ser	234	.	.	.	.	T	T	.	1.63	*	.	F	1.70	1.68
Gly	235	.	.	.	.	T	T	.	2.49	*	.	F	1.70	1.14
Asn	236	.	.	.	.	T	T	.	1.63	*	.	F	1.40	1.06
Gly	237	.	.	.	.	.	T	C	1.30	*	.	F	0.45	0.55
His	238	.	.	.	B	.	.	C	0.44	.	.	.	−0.40	0.59
Asn	239	.	.	.	B	.	.	C	−0.14	.	.	.	−0.40	0.27
Ile	240	.	.	B	B	.	.	.	−0.61	.	.	.	−0.60	0.19
Trp	241	.	.	B	B	.	.	.	−1.47	.	.	.	−0.60	0.12
Val	242	.	.	B	B	.	.	.	−1.98	.	.	.	−0.60	0.05
Ile	243	.	.	B	B	.	.	.	−2.26	.	.	.	−0.60	0.06
Leu	244	.	.	B	B	.	.	.	−3.07	.	.	.	−0.60	0.08
Val	245	.	.	B	B	.	.	.	−3.03	.	.	.	−0.60	0.09
Val	246	.	.	B	B	.	.	.	−3.60	.	.	.	−0.60	0.09
Thr	247	.	.	B	B	.	.	.	−2.96	.	.	.	−0.60	0.08
Leu	248	.	.	B	B	.	.	.	−2.88	.	.	.	−0.60	0.17
Val	249	.	.	B	B	.	.	.	−2.88	.	*	.	−0.60	0.19
Val	250	.	.	B	B	.	.	.	−2.83	.	.	.	−0.60	0.11
Pro	251	.	.	B	B	.	.	.	−2.83	.	.	.	−0.60	0.11
Leu	252	.	.	B	B	.	.	.	−3.11	.	.	.	−0.60	0.11
Leu	253	A	.	.	B	.	.	.	−3.16	.	.	.	−0.60	0.15
Leu	254	A	.	.	B	.	.	.	−3.11	.	.	.	−0.60	0.07
Val	255	A	.	.	B	.	.	.	−3.14	.	.	.	−0.60	0.07
Ala	256	A	.	.	B	.	.	.	−3.79	.	.	.	−0.60	0.06
Val	257	.	.	B	B	.	.	.	−3.64	.	.	.	−0.60	0.05
Leu	258	.	.	B	B	.	.	.	−3.50	.	.	.	−0.60	0.04
Ile	259	.	.	B	B	.	.	.	−3.36	.	.	.	−0.60	0.02
Val	260	.	.	B	B	.	.	.	−3.39	.	.	.	−0.60	0.02
Cys	261	.	.	B	B	.	.	.	−3.14	.	.	.	−0.60	0.01
Cys	262	.	.	B	B	.	.	.	−2.59	.	.	.	−0.60	0.02
Cys	263	.	.	B	B	.	.	.	−2.12	.	.	.	−0.60	0.03
Ile	264	.	.	B	B	.	.	.	−1.90	.	.	.	−0.60	0.06
Gly	265	.	.	.	.	T	T	.	−1.39	.	.	F	0.35	0.06
Ser	266	.	.	.	.	T	T	.	−1.07	.	.	F	0.35	0.11
Gly	267	.	.	.	.	T	T	.	−0.40	.	.	F	0.65	0.16
Cys	268	.	.	.	.	T	T	.	0.06	.	.	F	1.25	0.27
Gly	269	.	.	.	.	T	.	.	0.99	.	*	F	1.39	0.31
Gly	270	.	.	.	.	T	.	.	0.67	.	.	F	2.03	0.62
Asp	271	.	.	.	.	.	T	C	0.37	.	.	F	2.37	0.62
Pro	272	.	.	.	.	T	T	.	0.71	*	*	F	2.91	0.62
Lys	273	.	.	.	.	T	T	.	1.49	*	*	F	3.40	1.05
Cys	274	.	.	B	.	.	T	.	0.98	*	*	.	2.51	1.23
Met	275	.	.	B	B	.	.	.	0.66	*	*	.	1.62	0.59
Asp	276	.	.	B	B	.	.	.	−0.04	*	*	.	1.28	0.16
Arg	277	.	.	B	B	.	.	.	−0.12	.	*	.	0.04	0.26
Val	278	.	.	B	B	.	.	.	−0.06	.	*	.	−0.60	0.27
Cys	279	.	.	B	B	.	.	.	−0.20	.	.	.	0.30	0.32
Phe	280	.	.	B	B	.	.	.	0.06	.	*	.	−0.60	0.13
Trp	281	.	.	B	B	.	.	.	−0.76	.	.	.	−0.60	0.18
Arg	282	.	.	B	B	.	.	.	−1.68	.	.	.	−0.60	0.28
Leu	283	.	.	B	B	.	.	.	−0.71	.	.	.	−0.60	0.26
Gly	284	.	.	.	B	T	.	.	−0.39	.	*	.	−0.20	0.49
Leu	285	.	.	.	B	.	.	C	0.10	.	*	.	0.50	0.25
Leu	286	.	.	.	B	.	.	C	0.04	.	*	.	0.20	0.46
Arg	287	.	.	.	B	.	.	C	−0.66	.	.	F	0.65	0.46
Gly	288	.	.	.	.	.	T	C	0.16	.	.	F	1.35	0.57
Pro	289	.	.	.	.	.	T	C	0.50	.	*	F	2.70	1.19
Gly	290	.	.	.	.	.	T	C	1.31	*	*	F	3.00	1.01
Ala	291	A	.	.	.	.	T	.	1.53	.	*	F	2.50	1.65
Glu	292	A	.	.	.	.	.	.	1.39	.	.	F	2.00	1.08
Asp	293	A	.	.	.	.	.	.	1.73	.	.	F	1.70	1.48
Asn	294	A	.	.	.	.	T	.	1.94	.	*	.	1.45	2.36
Ala	295	A	.	.	.	.	T	.	1.40	.	.	.	1.15	2.36
His	296	A	.	.	.	.	T	.	1.18	*	.	.	1.00	0.99
Asn	297	A	.	.	.	.	T	.	0.88	.	.	.	0.10	0.51
Glu	298	A	.	.	.	.	.	.	0.88	*	.	.	−0.10	0.67
Ile	299	A	.	.	.	.	.	.	0.29	*	*	.	−0.10	0.80
Leu	300	A	.	.	.	.	.	.	0.88	*	*	.	−0.10	0.50
Ser	301	A	.	.	.	.	.	.	0.61	*	.	F	0.65	0.48
Asn	302	A	.	.	.	.	T	.	−0.20	*	.	F	0.25	0.92
Ala	303	A	.	.	.	.	T	.	−0.50	*	.	F	0.25	0.92
Asp	304	A	.	.	.	.	T	.	0.08	*	.	F	0.85	0.92
Ser	305	.	.	.	.	.	T	C	0.19	*	.	F	1.05	0.83
Leu	306	.	.	.	B	.	.	C	−0.37	*	.	F	0.05	0.71
Ser	307	.	.	B	B	.	.	.	−0.67	*	.	F	−0.15	0.31
Thr	308	.	.	B	B	.	.	.	−0.08	*	.	.	−0.60	0.31
Phe	309	.	.	B	B	.	.	.	−0.08	*	.	.	−0.30	0.66
Val	310	A	.	.	B	.	.	.	0.22	.	.	F	−0.15	0.85
Ser	311	A	A	.	.	.	.	.	0.43	.	.	F	0.00	1.03
Glu	312	A	A	.	.	.	.	.	0.73	.	.	F	0.00	1.17
Gln	313	A	A	.	.	.	.	.	0.74	.	.	F	0.90	2.73
Gln	314	A	A	.	.	.	.	.	1.44	.	.	F	0.90	2.73
Met	315	A	A	.	.	.	.	.	2.30	.	.	F	0.90	2.73
Glu	316	A	A	.	.	.	.	.	2.39	.	.	F	0.90	2.73
Ser	317	A	A	.	.	.	.	.	1.80	.	*	F	0.90	2.44
Gln	318	A	A	.	.	.	.	.	1.80	.	*	F	0.90	2.49
Glu	319	A	A	.	.	.	.	.	0.99	.	*	F	0.90	2.40
Pro	320	A	A	.	.	.	.	.	1.28	.	*	F	0.90	1.48
Ala	321	A	A	.	.	.	.	.	0.93	.	.	F	0.60	1.23
Asp	322	A	A	.	B	.	.	.	0.38	.	.	F	0.45	0.70
Leu	323	A	A	.	B	.	.	.	0.07	.	.	F	−0.15	0.34
Thr	324	.	A	B	B	.	.	.	−0.79	.	.	F	−0.15	0.48
Gly	325	.	A	B	B	.	.	.	−0.58	.	.	.	−0.30	0.21
Val	326	.	.	B	B	.	.	.	−0.29	.	.	.	−0.60	0.45
Thr	327	.	.	B	B	.	.	.	−0.50	.	.	.	−0.60	0.42
Val	328	.	.	B	B	.	.	.	−0.03	.	*	F	−0.17	0.65
Gln	329	.	.	B	B	.	.	.	0.28	.	*	F	0.11	0.87
Ser	330	.	.	.	.	.	T	C	0.03	.	*	F	2.04	1.05
Pro	331	.	.	.	.	.	T	C	0.89	.	*	F	2.32	1.42
Gly	332	.	.	.	.	T	T	.	0.53	.	*	F	2.80	1.42
Glu	333	A	.	.	.	.	T	.	0.58	.	*	F	1.97	0.57
Ala	334	.	.	B	.	.	.	.	−0.23	.	*	.	0.74	0.30
Gln	335	.	.	B	.	.	.	.	−0.28	.	.	.	0.46	0.25
Cys	336	.	.	B	.	.	.	.	−0.28	.	.	.	0.18	0.14
Leu	337	.	.	B	.	.	.	.	−0.52	.	*	.	−0.40	0.22
Leu	338	.	.	B	.	.	.	.	−0.52	.	*	.	−0.40	0.13
Gly	339	.	A	.	.	.	.	C	−0.52	.	*	F	0.05	0.42
Pro	340	A	A	.	.	.	.	.	−0.52	.	*	F	−0.15	0.51
Ala	341	A	A	.	.	.	.	.	−0.20	.	*	F	0.60	1.07
Glu	342	A	A	.	.	.	.	.	0.31	.	*	F	0.90	1.07
Ala	343	A	A	.	.	.	.	.	1.12	*	*	F	0.75	0.93
Glu	344	A	A	.	.	.	.	.	1.58	.	*	F	0.90	1.60
Gly	345	A	A	.	.	.	.	.	1.90	.	*	F	0.90	1.80
Ser	346	A	.	.	.	.	T	.	2.60	.	*	F	1.30	3.50
Gln	347	A	.	.	.	.	T	.	1.79	.	*	F	1.30	3.96
Arg	348	A	.	.	.	.	T	.	1.57	.	*	F	1.30	3.30
Arg	349	.	.	B	.	.	T	.	0.71	.	*	F	1.30	2.03
Arg	350	.	.	B	B	.	.	.	0.84	.	*	F	0.75	0.87
Leu	351	.	.	B	B	.	.	.	0.56	.	*	.	0.60	0.69
Leu	352	.	.	B	B	.	.	.	0.56	.	*	.	0.30	0.35
Val	353	.	.	B	B	.	.	.	0.10	*	*	.	−0.30	0.29
Pro	354	.	.	B	.	.	T	.	−0.60	*	.	.	−0.20	0.35
Ala	355	.	.	.	.	T	T	.	−0.71	.	*	.	0.50	0.43
Asn	356	.	.	.	.	.	T	C	−0.11	.	.	F	1.65	0.96
Gly	357	.	.	.	.	.	T	C	0.39	.	.	F	1.95	0.96
Ala	358	.	.	.	.	.	.	C	1.24	.	.	F	2.20	1.37
Asp	359	.	.	.	.	.	T	C	1.14	.	.	F	3.00	1.48
Pro	360	A	.	.	.	.	T	.	0.92	*	.	F	2.50	2.16
Thr	361	A	.	.	.	.	T	.	0.32	.	.	F	1.90	1.76
Glu	362	A	.	.	.	.	T	.	−0.14	.	.	F	1.60	1.04
Thr	363	A	.	.	B	.	.	.	−0.26	.	.	F	0.15	0.56
Leu	364	A	.	.	B	.	.	.	−0.96	*	.	.	−0.60	0.33
Met	365	A	.	.	B	.	.	.	−0.74	*	.	.	−0.60	0.17
Leu	366	A	.	.	B	.	.	.	−0.39	*	.	.	−0.60	0.19
Phe	367	A	.	.	B	.	.	.	−1.09	*	.	.	−0.60	0.47
Phe	368	A	.	.	B	.	.	.	−1.37	*	.	.	−0.60	0.41
Asp	369	A	.	.	B	.	.	.	−0.56	*	.	.	−0.60	0.50
Lys	370	A	A	.	.	.	.	.	−0.84	*	.	.	−0.30	0.93
Phe	371	A	A	.	B	.	.	.	−0.89	*	.	.	−0.30	0.75
Ala	372	A	A	.	B	.	.	.	−0.40	*	.	.	−0.30	0.34
Asn	373	.	A	B	B	.	.	.	−0.40	*	.	.	−0.60	0.26
Ile	374	.	A	B	B	.	.	.	−0.40	*	.	.	−0.60	0.26
Val	375	.	A	B	B	.	.	.	−0.74	.	.	.	−0.60	0.43
Pro	376	.	A	.	B	.	.	C	−0.33	.	.	.	−0.10	0.36
Phe	377	.	.	.	.	T	T	.	0.26	.	.	.	0.20	0.54
Asp	378	.	.	.	.	T	T	.	0.26	.	.	F	0.80	1.21
Ser	379	.	.	.	.	T	T	.	0.33	.	.	F	1.40	1.35
Trp	380	A	.	.	.	.	T	.	0.59	*	*	F	0.40	1.29
Asp	381	A	A	.	.	.	.	.	0.91	*	.	F	−0.15	0.76
Gln	382	A	A	.	.	.	.	.	1.61	*	.	.	−0.15	1.11
Leu	383	A	A	.	.	.	.	.	0.80	*	.	.	−0.15	1.84
Met	384	A	A	.	.	.	.	.	1.10	*	.	.	0.30	0.91
Arg	385	A	A	.	.	.	.	.	0.58	*	.	.	0.30	0.87
Gln	386	A	A	.	.	.	.	.	0.27	*	.	.	−0.30	0.87
Leu	387	A	A	.	.	.	.	.	0.31	*	.	.	0.45	1.27
Asp	388	A	A	.	.	.	.	.	1.12	*	.	.	0.75	1.30
Leu	389	A	A	.	.	.	.	.	1.72	*	.	F	0.60	1.21
Thr	390	A	.	.	.	.	T	.	0.72	*	.	F	1.30	2.54
Lys	391	A	.	.	.	.	T	.	0.72	.	*	F	1.30	1.07
Asn	392	A	.	.	.	.	T	.	0.68	*	*	F	1.30	2.16
Glu	393	A	.	.	.	.	T	.	−0.18	*	.	F	1.30	1.11
Ile	394	.	.	B	B	.	.	.	0.74	*	.	F	0.75	0.41
Asp	395	.	.	B	B	.	.	.	0.47	*	*	.	0.60	0.50
Val	396	.	.	B	B	.	.	.	0.08	*	*	.	0.60	0.29
Val	397	.	.	B	B	.	.	.	−0.23	.	.	.	0.51	0.41
Arg	398	.	.	B	.	.	T	.	−0.82	*	.	.	1.12	0.36
Ala	399	.	.	B	.	.	T	.	−0.28	*	.	.	0.73	0.49
Gly	400	.	.	.	.	T	T	.	−0.49	*	.	F	2.09	0.65
Thr	401	.	.	.	.	.	T	C	0.02	*	*	F	2.10	0.51
Ala	402	.	.	.	.	.	.	C	0.88	*	*	F	1.09	0.50
Gly	403	.	.	.	.	.	T	C	0.18	*	*	F	1.68	0.85
Pro	404	.	.	.	.	.	T	C	−0.04	.	.	F	1.47	0.59
Gly	405	.	.	.	.	.	T	C	0.06	.	.	F	1.26	0.48
Asp	406	A	.	.	.	.	T	.	−0.22	.	.	F	0.25	0.76
Ala	407	A	A	.	.	.	.	.	−0.23	.	.	.	−0.30	0.50
Leu	408	A	A	.	.	.	.	.	−0.70	.	.	.	−0.60	0.50
Tyr	409	A	A	.	.	.	.	.	−1.09	*	.	.	−0.60	0.25
Ala	410	A	A	.	.	.	.	.	−0.70	*	.	.	−0.60	0.24
Met	411	A	A	.	.	.	.	.	−0.99	*	.	.	−0.60	0.59
Leu	412	A	A	.	.	.	.	.	−1.26	*	.	.	−0.60	0.39
Met	413	A	A	.	.	.	.	.	−0.44	*	.	.	−0.60	0.29
Lys	414	A	A	.	B	.	.	.	−0.16	*	.	.	−0.60	0.47
Trp	415	A	A	.	B	.	.	.	0.12	*	.	.	0.15	1.14
Val	416	A	A	.	B	.	.	.	0.38	*	*	.	0.45	1.66
Asn	417	A	.	.	.	.	T	.	1.30	*	.	F	1.75	0.82
Lys	418	A	.	.	.	.	T	.	1.90	*	.	F	2.20	1.53
Thr	419	.	.	.	.	.	T	C	1.27	*	.	F	3.00	3.32
Gly	420	.	.	.	.	.	T	C	1.26	*	.	F	2.70	2.08
Arg	421	.	.	.	.	T	.	.	1.22	*	.	F	2.40	1.40
Asn	422	.	.	.	.	.	T	C	1.19	*	.	F	1.65	0.68
Ala	423	.	.	B	.	.	T	.	0.83	.	.	.	1.00	0.93
Ser	424	.	.	B	.	.	T	.	0.33	.	.	.	0.70	0.69
Ile	425	.	.	B	.	.	T	.	−0.13	.	*	.	−0.20	0.35
His	426	.	A	B	.	.	.	.	−0.24	.	*	.	−0.60	0.29
Thr	427	.	A	B	.	.	.	.	−0.83	*	*	.	−0.60	0.36
Leu	428	A	A	.	.	.	.	.	−1.06	*	*	.	−0.60	0.52
Leu	429	A	A	.	.	.	.	.	−0.76	*	*	.	−0.60	0.31
Asp	430	A	A	.	.	.	.	.	0.24	*	*	.	−0.30	0.38
Ala	431	A	A	.	.	.	.	.	−0.32	*	*	.	0.30	0.89
Leu	432	A	A	.	.	.	.	.	−0.01	*	*	.	0.75	1.07
Glu	433	A	A	.	.	.	.	.	0.80	*	*	.	0.75	1.11
Arg	434	A	A	.	.	.	.	.	1.72	*	*	F	0.90	1.90
Met	435	A	A	.	.	.	.	.	1.69	*	*	F	0.90	4.52
Glu	436	A	A	.	.	.	.	.	1.69	*	*	F	0.90	3.55
Glu	437	A	A	.	.	.	.	.	2.54	*	.	F	0.90	1.83
Arg	438	A	A	.	.	.	.	.	2.54	*	*	F	0.90	3.70
His	439	A	A	.	.	.	.	.	2.48	*	*	F	0.90	3.70
Ala	440	A	A	.	.	.	.	.	2.19	*	*	F	0.90	4.28
Lys	441	A	A	.	.	.	.	.	2.19	*	*	F	0.90	1.53
Glu	442	A	A	.	.	.	.	.	2.19	*	.	F	0.90	1.95
Lys	443	A	A	.	.	.	.	.	1.27	*	*	F	0.90	3.22
Ile	444	A	A	.	.	.	.	.	0.49	*	*	F	0.90	1.33
Gln	445	A	A	.	.	.	.	.	0.22	*	*	F	0.75	0.63
Asp	446	A	A	.	.	.	.	.	0.18	*	*	F	−0.15	0.23
Leu	447	A	A	.	.	.	.	.	−0.12	*	.	.	−0.30	0.56
Leu	448	A	A	.	.	.	.	.	−0.51	*	.	.	0.55	0.43
Val	449	A	A	.	.	.	.	.	0.42	*	.	F	0.95	0.26
Asp	450	A	.	.	.	.	T	.	−0.28	*	.	F	1.60	0.62
Ser	451	.	.	.	.	T	T	.	−1.17	*	.	F	2.25	0.65
Gly	452	.	.	.	.	T	T	.	−0.60	*	.	F	2.50	0.62
Lys	453	.	.	B	.	.	T	.	−0.60	.	.	F	1.25	0.58
Phe	454	.	A	B	.	.	.	.	0.26	.	.	.	0.15	0.36
Ile	455	.	A	B	.	.	.	.	0.26	.	.	.	0.20	0.62
Tyr	456	.	A	B	.	.	.	.	0.21	.	.	.	0.55	0.52
Leu	457	.	A	B	.	.	.	.	0.24	.	.	.	−0.03	0.59
Glu	458	.	A	B	.	.	.	.	−0.14	.	.	F	0.54	1.22
Asp	459	.	A	.	.	T	.	.	0.26	.	.	F	1.66	0.77
Gly	460	.	.	.	.	T	T	.	0.56	.	.	F	2.78	1.26
Thr	461	.	.	.	.	.	T	C	−0.06	*	.	F	2.70	0.73
Gly	462	.	.	.	.	.	T	C	0.46	*	.	F	2.13	0.33
Ser	463	.	.	.	.	.	T	C	−0.36	.	.	F	1.26	0.44
Ala	464	A	.	.	.	.	.	.	−0.36	.	.	.	0.14	0.25
Val	465	.	.	B	.	.	.	.	−0.40	.	.	.	0.17	0.44
Ser	466	.	.	B	.	.	.	.	−0.48	.	.	.	−0.10	0.42
Leu	467	.	.	B	.	.	.	.	−0.52	.	.	.	−0.10	0.53
Glu	468	A	.	.	.	.	.	.	−0.61	.	.	.	0.50	0.92

[0065] Preferred nucleic acid fragments of the invention encode a full-length DR4 polypeptide lacking the nucleotides encoding the amino-terminal methionine (nucleotides 19-21 in SEQ ID NO:1) as it is known that the methionine is cleaved naturally and such sequences maybe useful in genetically engineering DR4 expression vectors. Polypeptides encoded by such polynucleotides are also contemplated by the invention.

[0066] Among highly preferred fragments in this regard are those that comprise, or alternatively consist of, regions of DR4 that combine several structural features, such as several of the features set out above. Preferred nucleic acid fragments of the present invention further include nucleic acid molecules encoding a polypeptide comprising, or alternatively consisting of, one, two, three, four, five, or more epitope-bearing portions of the DR4 protein. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 35 to about 92 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 114 to about 160 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 169 to about 240 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 267 to about 298 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 330 to about 364 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 391 to about 404 in SEQ ID NO:2; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 418 to about 465 in SEQ ID NO:2. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, at either terminus or at both termini. The inventors have determined that the above polypeptide fragments are antigenic regions of the DR4 protein. Methods for determining other such epitope-bearing portions of the DR4 protein are described in detail below. Polypeptides encoded by these nucleic acids are also encompassed by the invention.

[0067] In specific embodiments, the polynucleotides of the invention are less than 100000 kb, 50000 kb, 10000 kb, 1000 kb, 500 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5 kb, or 5 kb in length.

[0068] In further embodiments, polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of DR4 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5′ or 3′ coding nucleotide set forth in SEQ ID NO:1. In further embodiments, polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of DR4 coding sequence, but do not comprise all or a portion of any DR4 intron. In another embodiment, the nucleic acid comprising, or alternatively consisting of, DR4 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the DR4 gene in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

[0069] In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:1 as follows: HTOIY07R (SEQ ID NO:6) and HTXEY80R (SEQ ID NO:7) both shown in FIG. 4.

[0070] Further, the invention includes a polynucleotide comprising, or alternatively consisting of, any portion of at least about 30 nucleotides, preferably at least about 50 nucleotides, of SEQ ID NO:1 from residue 365 to 1,422. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

[0071] In another embodiment, the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the sequence complementary to the coding and/or noncoding (i.e., transcribed, untranslated) sequence depicted in SEQ ID NO:1, the cDNA contained in ATCC Deposit No. 97853, and the sequence encoding a DR4 domain or a polynucleotide fragment as described herein. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising, or alternatively consisting of: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

[0072] Polypeptides encoded by these nucleic acids are also encompassed by the invention.

[0073] By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. By a portion of a polynucleotide of “at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1. These have uses, which include, but are not limited to, as diagnostic probes and primers as discussed above and in more detail below.

[0074] Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3 terminal poly(A) tract of the DR4 cDNA shown in SEQ ID NO:1), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA).

[0075] As indicated, nucleic acid molecules of the present invention which encode the DR4 polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretary sequence, such as a pre-, or pro- or prepro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing including splicing and polyadenylation signals, for example—ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.

[0076] The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode for fragments, analogs or derivatives of the DR4 polypeptide. Variants may occur naturally, such as an allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

[0077] Such variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and functional activities of the DR4 receptor or portions thereof. Also especially preferred in this regard are conservative substitutions.

[0078] Further embodiments of the invention include isolated nucleic acid molecules that are at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical, to (a) a nucleotide sequence encoding the full-length DR4 polypeptide having the complete amino acid sequence in SEQ ID NO:2, including the predicted leader sequence; (b) nucleotide sequence encoding the full-length DR4 polypeptide having the complete amino acid sequence in SEQ ID NO:2, including the predicted leader sequence but lacking the amino terminal methionine; (c) a nucleotide sequence encoding the mature DR4 polypeptide (full-length polypeptide with the leader removed) having the amino acid sequence at positions about 24 to about 468 in SEQ ID NO:2; (d) a nucleotide sequence encoding the full-length DR4 polypeptide having the complete amino acid sequence including the leader encoded by the cDNA contained in ATCC Deposit No. 97853; (e) a nucleotide sequence encoding the full-length DR4 polypeptide having the complete amino acid sequence including the leader but lacking the amino terminal methionine encoded by the cDNA contained in ATCC Deposit No. 97853; (f) a nucleotide sequence encoding the mature DR4 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 97853; (g) a nucleotide sequence that encodes the DR4 extracellular domain having the amino acid sequence at positions about 24 to about 238 in SEQ ID NO:2, or the DR4 extracellular domain encoded by the cDNA contained in ATCC Deposit No. 97853; (h) a nucleotide sequence that encodes the DR4 transmembrane domain having the amino acid sequence at positions about 239 to about 264 in SEQ ID NO:2, or the DR4 transmembrane domain encoded by the cDNA contained in ATCC Deposit No. 97853; (i) a nucleotide sequence that encodes the DR4 intracellular domain having the amino acid sequence at positions about 265 to about 468 in SEQ ID NO:2, or the DR4 intracellular domain encoded by the cDNA contained in ATCC Deposit No. 97853; (j) a nucleotide sequence that encodes the DR4 death domain having the amino acid sequence at positions about 379 to about 422 in SEQ ID NO:2, or the DR4 death domain encoded by the cDNA contained in ATCC Deposit No.97853; (k) a nucleotide sequence that encodes the DR4 cysteine rich domain having the amino acid sequence at positions about 131 to about 229 in SEQ ID NO:2, or the DR4 cysteine rich domain encoded by the cDNA contained in ATCC Deposit No. 97853; (l) a nucleotide sequence that encodes the DR4 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; (m) a nucleotide sequence that encodes a fragment of the polypeptide of (c) having DR4 functional activity (e.g., antigenic or biological activity); or (n) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), or (m) above. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) amino acid residues, at either terminus or at both termini. Polypeptides encoded by these nucleic acids are also encompassed by the invention.

[0079] By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence encoding a DR4 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the DR4 polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mismatches of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire DR4 nucleotide sequence shown in SEQ ID NO:1 or any fragment (e.g., a polynucleotide encoding the amino acid sequence of a DR4 N- and/or C-terminal deletion described herein) as described herein.

[0080] As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO:1 or to the nucleotide sequences of the deposited cDNA can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0081] In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.

[0082] The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:1 or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having DR4 functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having DR4 functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having DR4 functional activity include, inter alia, (1) isolating the DR4 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the DR4 gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting DR4 mRNA expression in specific tissues.

[0083] Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:1 or to the nucleic acid sequence of the deposited cDNAs which do, in fact, encode a polypeptide having DR4 protein functional activity. By “a polypeptide having DR4 protein functional activity” is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the DR4 protein of the invention (either the full-length protein (i.e. complete) or, preferably, the mature protein), as measured in a particular functional and/or biological assay. For example, DR4 polypeptide functional activity can be measured by the ability of a polypeptide sequence described herein to form multimers (e.g., homodimers and homotrimers) with complete DR4, and to bind a DR4 ligand (e.g., TRAIL). These functional assays can be routinely performed using techniques described herein and otherwise known in the art.

[0084] For example, DR4 protein functional activity (e.g., biological activity) can routinely be measured using the cell death assays performed essentially as previously described (A. M. Chinnaiyan, et al., Cell 81, 505-12 (1995); M. P. Boldin, et al., J Biol Chem 270,7795-8 (1995); F. C. Kischkel, et al., EMBO 14, 5579-5588(1995); A. M. Chinnaiyan, et al, J Biol Chem 271,4961-4965 (1996)) or as set forth in Example 5, below. In MCF7 cells, plasmids encoding full-length DR4 or a candidate death domain containing receptors are co-transfected with the pLantern reporter construct encoding green fluorescent protein. Nuclei of cells transfected with DR4 will exhibit apoptotic morphology as assessed by DAPI staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzio, et al., Cell 85, 817-827 (1996); M. P. Boldin, et al., Cell 85, 803-815 (1996); M. Tewari, et al., J Biol Chem 270, 3255-60 (1995)), DR4-induced apoptosis is blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk.

[0085] Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown in SEQ ID NO:1 will encode “a polypeptide having DR4 protein functional activity.” In fact, since degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having DR4 protein functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.

[0086] For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.

Polynucleotide Assays

[0087] This invention is also related to the use of the DR4 polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of DR4 associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of DR4 or a soluble form thereof, such as, for example, tumors or autoimmune disease.

[0088] Individuals carrying mutations in the DR4 gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al., Nature 324:163-166 (1986)). RNA orcDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid encoding DR4 can be used to identify and analyze DR4 expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled DR4 RNA or alternatively, radiolabeled DR4 antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

[0089] Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.

[0090] Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230:1242 (1985)).

[0091] Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).

[0092] Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms (“RFLP”) and Southern blotting of genomic DNA.

[0093] In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.

Vectors and Host Cells

[0094] The present invention also relates to vectors which include DNA molecules of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

[0095] Host cells can be genetically engineered to incorporate nucleic acid molecules and express polypeptides of the present invention. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.

[0096] In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.

[0097] Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

[0098] A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.

[0099] The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s)), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. In general, expression constructs will contain sites for transcription, initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0100] In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.

[0101] Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.

[0102] The vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention.

[0103] Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors available to those of skill in the art.

[0104] Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art.

[0105] The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.

[0106] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).

[0107] In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., DR4 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with DR4 polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous DR4 polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous DR4 polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number WO 96/29411, published Sep. 26, 1996; International Publication Number WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

[0108] The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Such a fusion protein can be made by ligating polynucleotides of the invention and the desired nucleic acid sequence encoding the desired amino acid sequence to each other, by methods known in the art, in the proper reading frame, and expressing the fusion protein product by methods known in the art. Alternatively, such a fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. For example, in one embodiment, polynucleotides encoding DR4 polypeptides of the invention may be fused to the pelB pectate lyase signal sequence to increase the efficiency to expression and purification of such polypeptides in Gram-negative bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents of which are herein incorporated by reference in their entireties.

[0109] The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, hIL-5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16:9459-9471 (1995).