US 6,031,089 AGrant

Sequences of P56, Proteins Which Affect K-Atp Channels

Issue Date:2000-02-29

•5 Claims

Abstract

This invention describes the full length sequence of human p56 protein (p56-1), a related homolog (p56-2) and the nucleic acids that code for these proteins. The sequences are provided in Charts 1, 2, 3, and 4 and the sequence listings of the application.

Metadata

Assignee

Pharmacia & Upjohn Company

Inventor

Michael J. Bienkowski

Application Information

Application Number:US 9932607

Filing Date:1997-12-18

Priority Date:1997-12-18

Art Unit:166

Classifications

IPC:

C07H 2104C12N 1512C12N 1563

Field of Search:

53653043523.5350;3007.1;325;69.1;353;250.3;254.1;255.1;320.1;235.1

Patent Drawings

This patent does not have any drawings.

Description

Field of the Invention

This invention relates to potassium channels and the p56 protein.

Information Disclosure

The disclosures appearing in PCT/US95/14124, published May 30, 1996 as WO 96/16088, particularly page 1, is hereby incorporated by reference. Additional documents in the Detailed Description of the invention should be considered as part of the Information Disclosure.

Background

The background appearing in PCT/US95/14124, published May 30, 1996 as WO 96/16088, particularly pages 1-5, are hereby incorporated by reference.

The isolation and identification of p56, a protein useful for the identification of selective drugs that will selectively open or close K channels was described in PCT/US95/14124, published May 30, 1996 as WO 96/16088. Herein the full length amino acid sequence of p56 and the nucleic acid sequence that code for p56 are described. Also described are the amino acid sequences and coding DNA that code for p56-2, a different p56 protein..

Summary of the Invention

This invention describes the full length sequence of human p56 protein (p56-1), SEQ. ID. NO. 1 a related homolog (p56-2) SEQ. ID. NO. 3 and the nucleic acids that code for these proteins SEQ. ID. NOS. 2 and 4. The sequences are provided in Charts 1, 2, 3, and 4 and the sequence listings of the application SEQ. ID. NOS. 1-4. There are two unique p56 DNA sequences and proteins disclosed in this document. For the first p56 sequence, p56-1, the protein's entire amino acid sequence of p56 is provided in Chart 1 SEQ. ID. NO. 1 and the DNA coding for p56 is provided in Chart 2 SEQ. ID. NO. 2. For the second sequence, p56-2, the DNA and amino acids are disclosed Charts 3 SEQ. ID. NO. 3 and 4 SEQ. ID. NO. 4. Equivalents and obvious homologues are disclosed. Also disclosed by reference to PCT/US95/14124, published May 30, 1996 as WO 96/16088, hereby incorporated by reference, are cloning and other useful vectors for the sequence.

Additional Description of the Invention and Description of the Preferred Embodiments

The entire document numbered PCT/US95/14124, published May 30, 1996 as WO 96/16088, particularly pages 6-11 are hereby incorporated by reference.

Utility of the Invention

The utility of this invention is disclosed and supported by the disclosure appearing in PCT/US95/14124, published May 30, 1996 as WO 96/16088, particularly ages 11-12 are hereby incorporated by reference.

Detailed Description of the Invention

A detailed description of the invention may be found by reference to PCT/US95/14124, published May 30, 1996 as WO 96/16088, particularly page 12, lines 33-35--page 20, hereby incorporated by reference. In addition to the PCT/WO publication the following additional remarks and materials disclose this invention.

Type I ATP-sensitive K channels (I.sub.K-ATP) were first described in cardiac muscle (1) and have subsequently been characterized in skeletal muscle (2) vascular smooth muscle (3) and the .beta.-cell of the pancreas (4-6). These type I channels are inhibited by micomolar concentrations of intracellular ATP and are insensitive to voltage and Ca.sup.2+ (7). The .beta.-cell I.sub.K-ATP channel is the most well understood in functional terms and it serves as a metabolic sensor that controls the release of insulin. Glucose stimulation of the .beta.-cell results in an increase in the intracellular ATP/ADP ratio via glycolysis and the increase in ATP inhibits channel conductance. Because I.sub.K-ATP channels dominate the resting membrane potential of the .beta. cell, inhibition of channel conductance results in a depolarization that activates voltage-dependent Ca.sup.2+ channels and the resultant increase in intracellular Ca.sup.2+ triggers insulin release. Pharmacological agents like sulfonylureas [I.sub.K-ATP blocker] or diazoxide [I.sub.K-ATP opener] stimulate or inhibit insulin release, respectively (8). In cardiac and skeletal muscle, the I.sub.K-ATP channels serve a similar function, coupling cell metabolism and electrical activity. These channels have a low open probability under resting conditions and are activated by a decline in the intracellular ATP/ADP ratio in response to either ischemic injury and/or exercise (2,9).

I.sub.K-ATP channels are unique among potassium channels because their activity can be regulated by a wide variety of structurally diverse pharmacological agents (10-15). These include both channel blockers (eg. sulfonylureas, guanidines and cyanoguanidines) and channel openers (eg. diazoxide, pinacidil, chromakalim, and minoxidil sulfate). This rich pharmacology translates into many opportunities for therapeutic intervention such as antidiabetics (sulfonylureas blockers), diuretics (guanidine and cyanoguanidine blockers), antihypertensives (openers like pinacidil and minoxidil sulfate) and agents that promote hair growth (minoxidil sulfate). Given the structural diversity among the chemical classes of agents that modify the activity of I.sub.K-ATP channels in various cell types, it is likely that the structure of these channels are complex. This diversity, coupled to the key role that I.sub.K-ATP channels play in linking cell excitability to metabolism, offers the hope that tissue selective modulators of I.sub.K-ATP channel activity can provide additional opportunities for the management of human disease.

Elucidation of the minimal structural components necessary to form functional I.sub.K-ATP channels was the result of two divergent efforts, namely the cloning and charaterization of either inward rectifier potassium channels or the high affinity sulfonylurea receptor (16,17). The first two members of the inward rectifier potassium channel gene family were isolated using expression cloning paradigms (18,19) and to date, homology cloning efforts have defined a total of 13 distinct genes in this class. Heterologous expression of the inward rectifier referred to as K.sub.ir 6.1 in human embryonic kidney cells led to the synthesis of a channel that displayed some of the properties of native I.sub.K-ATP channels (16). Subsequent cloning studies have defined a second member of the K.sub.ir 6 family, referred to as K.sub.ir 6.2 (19, 21-22). Molecular identification of the high affinity sulfonylurea receptor was facilitated by the use of sulfonylurea photoprobes to `tag` the polypeptide responsible for high affinity sulfonylurea binding. Using either [.sup.3 H]-glyburide (23-25), [.sup.125 I]-glyburide analogs (26-28), or [.sup.125 I]/azido analogs of glyburide (29,30), multiple investigators groups have identified a high affinity 140-150 kDa sulfonylurea binding protein in both .beta. cells and in brain. Purification of photolabeled 140 kDa sulfonylurea receptor from .beta.-cell lines followed by protein microsequence analysis and cDNA cloning has resulted in the molecular definition of the high affinity sulfonylurea receptor [SUR-1] (17). Subsequent cloning efforts have resulted in the identification of a paralog of SUR-1 referred to as SUR-2 (31-33). The primary structure of SUR-1 and SUR-2 resembles ABC cassette transporter proteins and heterologous expression of either SUR-1 or SUR-2 does not led to the synthesis of functional I.sub.K-ATP channels. Alternatively, co-expression of K.sub.ir 6.1/SUR-2 or K.sub.ir 6.2/SUR-1 combinations leads to the reconstitution of functional I.sub.K-ATP channels that display many of the electrophysiological and pharmacological signatures of native I.sub.K-ATP channels (31-33).

A parallel strategy for the identification of cyanoguanidine binding proteins was initiated with the synthesis and characterization of the azido photoprobe [.sup.3 H]-probe 1. Because the value of this photoprobe was reduced by its relatively low specific activity, a second generation cyanoguanidine analog, [.sup.125 I]-probe 2 was prepared and characterized. Photolabeling of intact rat aortic smooth muscle cells (A10) with .sup.125 I-probe 2, identified both high affinity (p56) and a low affinity (p47) binding sites that were displaced by homologous competition. Purification of .sup.125 I-p56 from A10 cells and protein microsequence analysis identified a unique twelve amino acid residue sequence tag derived from the NH.sub.2 terminus of .sup.125 I-p56 and this identification was substantiated by the preparation and characterization of an antipeptide antibody that recognizes this sequence tag. This amino acid sequence tag was used to query expressed sequence tag (EST) databases to identify EST clones that potentially encode the human ortholog of the p56 sequence. Complete sequence analysis of multiple EST clones revealed a full-length cDNA that encoded the human ortholog of rat p56. Subsequent queries of an EST database with the full-length sequence of human p56 and sequence analysis revealed a second full-length human cDNA p56 paralog, referred to as p56-2 SEQ. ID. NO. 4. Human p56-1 SEQ. ID. NO. 2 and p56-2 SEQ. ID. NO. 4 were characterized by determining their tissue distribution of expression, their glycosylation in vitro and their ability to reconstitute the .sup.125 I-probe 2 binding site following transient expression in COS cells.

References by number above are reported here, all are incorporated by reference.

1. Noma, A. "ATP-regulated K.sup.+ channels in cardiac muscle" Nature 305: 147-148 (1983).

2. Spruce, A. E., Standen, N. B. and Standfield, P. R. "Voltage-dependent ATP-sensitive potassium channels of skeletal muscle membrane" Nature 316: 736-738 (1985).

3. Standen, N. B., Quayle, J. M., Davies, N. W., Brayden, J. E., Huang, Y. and Nelson, M. T. "Hyperpolarizing vasodialators activate ATP-sensitive K.sup.+ channels in arterial smooth muscle" Science 245: 177-180 (1989).

4. Cook, D. L. and Hales, C. N. "Intracellular ATP directly blocks K.sup.+ channels in pancreatic beta cells" Nature 311: 271-273 (1984).

5. Sturgess, N. C., Ashford, M. L., Cook, D. L., and Hales, C. N. "The sulfonylurea receptor may be an ATP-sensitive potassium channel" Lancet 1:474-475 (1985).

6. Rorsman, P. and Trube, G. "Glucose-dependent K.sup.+ channels in pancreatic beta cells are regulated by intracellular ATP" Pflugers Arch 405: 305-309 (1985).

7. Ashcroft, S. J. H. and Ashcroft F. M. "Properties and function of ATP-sensitive K.sup.+ channels" Cell Signal 2:197-214 (1990).

8. Trube, G., Rorsman, P. and Ohno-Shosaku, T. "Opposite effects of tolbutamide and diazoxide on the ATP-dependent K.sup.+ channel in mouse pancreatic .beta.-cells" Pflugers Arch 407:493-499 (1986).

9. Escande, D. and Cavero, I. "K.sup.+ channel openers and `natural` cardioprotection" Trends Pharmacol. Sci. 13:269-271 (1992).

10. Gopalakrishnan, M., Janis, R. A. and Triggle, D. J. "ATP-sensitive K.sup.+ channels: Pharmacologic properties, regulation and therapeutic potential" Drug Dev. Res. 28: 95-127 (1993).

11. Atwal, K. S. "Advances in the structure-activity relationships, mechanisms of action, and therapeutic utilities of ATP-sensitive potassium channel openers" Drug Dev. Res. 33:250-262 (1994).

12. Edwards, G. and Weston, A. H. "The pharmacology of ATP-sensitive potassium channels" Ann.Rev. Pharmacol. Toxicol. 33: 597-637 (1993).

13. de Weille, J. R. "Modulation of ATP sensitive potassium channels" Cardiovascular Res. 26: 1017-1020 (1992).

14. Atwal, K. S. "Modulation of potassium channels by organic molecules" Medicinal Research Rev. 12:569-591 (1992).

15. Robertson, D. W. and Steinberg, M. I. "Potassium channel modulators: Scientific applications and therapeutic promise" J. Med. Chem. 33: 1530-1541 (1990).

16. Inagaki, N., Tsuura, Y., Namba, N., Masuda, K, Gonoi, T., Horie, M., Seino, Y., Mizuta, M. and Seino, S. "Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle and heart" J. Biol. Chem. 270: 5691-5694 (1995).

17. Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E., Gonzalez, G., Herrera-Sosa, H., Nguy, K, Bryan, J. and Nelson, D. A. "Cloning of the .beta.-cell high affinity sulfonylurea receptor; a regulator of insulin secretion" Science 268:423426 (1995).

18. Ho, K, Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. S, Kanazirska, M. V., and Hebert, S. C. "Cloning and expression of an inwardly rectifying ATP-regulated potassium channel" Nature 362:31-38 (1993).

19. Dascal, N., Schreibmayer, W., Lim, N. F., Wang, W., Chavkin, C., DiMagno, L., Labarca, C., Kieffer, B. L., Gaveriaux-Ruff, C., Trollinger, D., Lester, H. A., and Davidson, N. "Atrial G-protein activated K.sup.+ channel: Expression cloning and molecular properties" Proc. Natl. Acad. Sci. USA 90:10235-10239 (1993).

21. Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S. and Bryan, J. "Reconstitution of I.sub.K-ATP : an inward rectifier subunit plus the sulfonylurea receptor" Science 270: 1166-170 (1995).

22. Tokuyama, Y., Fan, Z., Furuta, H., Makielski, J. C., Polonsky, K. S., Bell, G. I. and Yano, H. "Rat inwardly rectifying potassium channel Kir 6.2: Cloning, electrophysiological characterization, and decreased expression in pancreatic islets of male Zucker diabetic fatty rats" Biochem. Biophys. Res. Comm. 220: 532-538 (1996).

23. Kramer, W., Oekonomopulos, R., Punter, J., and Summ, H. D. "Direct photolabeling of the putative sulfonylurea receptor in rat .beta.-cell tumor membranes by [.sup.3 H]- glybenclamide" FEBS Let 229:355-359 (1988).

24. Bernardi, H., Fosset, M., and Lazdunski, M. "Characterization, purification and affinity labeling of the brain [.sup.3 H]-glybenclamide-binding protein, a putative neuronal ATP-regulated K.sup.+ channel" Proc. Natl. Acad. Sci. USA 85:9816-9820 (1988).

25. Benardi, H., Fosset, M., and Lazdunski, M. "ATP/ADP Binding sites are present in the sulfonylurea binding protein associated with brain ATP-sensitive K.sup.+ channels" Biochemistry 31:6328-6332 (1992).

26. Aguilar-Bryan, L., Nelson, D. A., Vu, Q., Humphrey, M. B., and Boyd III, A. E. "Photoaffinity labeling and partial purification of the .beta.-cell sulfonylurea receptor using a novel, biologically active glyburide analog" J. Biol. Chem. 265:8218-8224 (1990).

27. Nelson, D.A., Aguilar-Bryan, L., and Bryan, J. "Specficity of photolabeling of .beta.-cell membrane proteins with an .sup.125 I-labeled glyburide analog" J. Biol. Chem. 267:14928-14933 (1992).

28. Bernardi, H., De Weille, J. R, Epelbaum, J., Mourre, C., Amoroso, S., Slama, A., Fosset, M., and Lazdunski, M. "ATP-modulated K.sup.+ channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release" Proc. Natl. Acad. Sci. USA 90:1340-1344 (1993).

29. Schwanstecher, M., Loser, S., Chudziak, F., and Panten, U. "Identification of a 38 kDa high affinity sulfonylurea-binding peptide in insulin-secreting cells and cerebral cortex" J. Biol. Chem. 269:17768-17771 (1994).

30. Schwanstecher, M., Loser, S., Chudziak, F., Bachmann, C., and Panten, U. "Photoaffinity labeling of cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue" J. Neurochem. 63:698-708 (1994).

31. Inagaki, N., Gonoi, T., Clement, J. P., Wang, C.-Z., Aguilar-Bryan, L., Bryan, J. and Seino, S. "A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K.sup.+ channels" Neuron 16: 1011-1017 (1996).

32. Chutkow, W. A., Simon, M. C., Le Beau, M. M. and Burant, C. F. "Cloning, tissue expression and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle and vascular K.sub.ATP channels" Diabetes 45: 1439-1445 (1996).

33. Isomoto, S., Kondo, C., Yamada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y. and Kurachi, Y. "A novel sulfonylurea receptor forms with BIR (K.sub.ir 6.2) a smooth muscle type ATP-sensitive K.sup.+ channel" J. Biol. Chem. 271:24321-24324 (1996).

The NH2-terminal amino acid sequence was established, by the method described above in PCT/US95/14124, then various data bases and DNA libraries were repeatedly searched with no positive result. Finally, after numerous searches, the p56-1 NH2-terminal amino acid sequence (EPRAPPEKIAIGAG), disclosed in PCTIUS95/14124, published 30 May 1996 as WO 96/16088, led to the discovery of a human expressed sequence tag (EST) that displayed a significant match to the sequence disclosed in that PCT publication. A clone containing this sequence, identified as number 56-1, was obtained by searching various data bases and DNA libraries and the complete DNA sequence was determined.

Clone 56-1 contained a 1515 bp open-reading frame that encoded a 505 amino acid polypeptide with a predicted Mr for the mature protein of 52 kDa, close to the expected p56. The predicted sequence contained a signal peptide followed by a mature NH2-terminus that showed 11/14 exact matches with the rat p56 NH2-terminal sequence disclosed in PCT/US95/14124, published 30 May 1996 as WO 96/16088.

Consistent with biochemical data for rat p56, which demonstrated that p56 is a glycoprotein, the predicted polypeptide for clone 56-1 SEQ. ID. NO. 2 also contained 3 canonical acceptor sites for Asn-linked glycosylation. The predicted amino acid sequence has no significant homology to known sequences.

The predicted amino acid from clone 56-1 SEQ. ID. NO. 2 was analyzed using a variety of secondary structure prediction algorithms. A Rossman fold was detected near the NH2-terminus, indicating that this protein was likely to bind nucleotides like ATP. In contrast to the biochemical data on rat p56, no predicted transmembrane segments were detected but an endoplasmic reticulum retention signal (KEL-versus-the canonical KDEL) near the COOH-terminus was scored. A .beta.-turn-.beta. membrane association motif, similar to the P (pore)-region of voltage-gated-K.sup.+ -channels, was also detected. It appears that this polypeptide may associate with other K-channel pore-forming polypeptides (e.g. Kir 6.1 or 6.2) and regulate K channel activity. See, Inagaki, N., Tsuura, Y., Namba, N., Masuda, K., Gonoi, T., Horie, M., Seino, Y., Mizuta, M. and Seino, S. "Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle and heart" J. Biol. Chem. 270: 5691-5694 (1995). AND. Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar- Bryan, L., Seino, S. and Bryan, J. "Reconstitution of I.sub.K-ATP : an inward rectifier subunit plus the sulfonylurea receptor" Science 270: 1166-170 (1995). Both articles incorporated by reference, and in particular aspects describing functional channel systems.

Using the sequence information disclosed herein one ordinarily skilled in the art should be able to use known PCT techniques to create, fashion, or produce clones having and or expressing the desired sequences described herein. The sequences can also be used in screens and assays for the detection of biologically active compounds. Additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in case 6001. NCP, Ser. No. 08/709,923 filed Sep. 9, 1996, hereby incorporated by reference. In particular page 16 relating to procedures for preparing and using clones in assays especially relevant and incorporated by reference. In a similar manner additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in WO 94/19464, PCT/US94/01210, published Sep. 1, 1994, hereby incorporated by reference. In particular, the pages relating to procedures for preparing and using clones in assays is especially relevant and incorporated by reference.

The second p56 sequence SEQ. ID. NOS. 3 and 4 was discovered according to the following procedures. Various data bases were queried with the amino acid sequence of human p56 using the FASTA search tool. In addition to identifying the known EST matches to p56, three additional ESTs that shared approximately 38-48% identity with p56 were also scored [447210, 2607571 and 2663551]. Alignment of the 5' EST sequence reads for these 3 clones with the p56 sequence showed that it was unlikely that the EST sequences overlapped and that clone 2607571 was most likely to be full-length. These clones were obtained and clone 2607571 was completely sequenced. This clone contained a 2.6 kb insert complete with a 1482 bp open reading frame that showed 41% shared identity with human p56. Motifs that were common to both predicted amino acid sequences include (1) a signal sequence, (2) Rossman fold, (3) canonical acceptor sites for Asn-linked glycosylation [3 in p56-1 and 6 in p56-2] and (4) an ER retention signal at the COOH-terminus [KTEL].

Comparison of the biochemical properties of p56-1

Biochemical characterization of .sup.125 I-p56-1 from A10 cells revealed a Triton X-100 soluble 56 kDa glycoprotein that could be de-glycosylated with N-glycanase to yield a 52 kDa form. These data are consistent with the M.sub.r of human p56-1 predicted from the cDNA sequence and the presence of canonical acceptor sites for Asn-linked glycosylation in the sequence. To establish these biochemical parameters experimentally for p56-1 SEQ. ID. NO. 2 and to investigate the glycosylation pattern of p56-2 SEQ. ID. NO. 4, both polypeptides were prepared by in vitro translation. Each expression plasmid was linearized by digestion with NotI and capped cRNA synthesized using T7-RNA polymerase. These cRNAs were then used to direct the synthesis of .sup.35 S-methionine labeled protein using rabbit reticulocyte lysates.+-.canine pancreatic microsomes. The radiolabeled proteins were fractionated by SDS-PAGE and visualized by fluorography of the dried gel. P56-1 and p56-2 SEQ. ID. NOS. 2 and 4 have unglycosylated M.sub.r values of 52 kDa and 50 kDa, respectively. In the presence of canine pancreatic microsomes, multiple bands with higher M.sub.r values, including 56kDa, were also detected, indicating the addition of Asn-linked oligosaccharides to p56-1 SEQ. ID. NO. 2. In contrast to p56-1 SEQ. ID. NO. 2, p56-2 SEQ. ID. NO. 4 did not appear to be glycosylated under these conditions.

Tissue distribution of expression of human p56-1 and p56-2 SEQ. ID. NOS. 2 and 4

Comparison of Northern analysis and transcript imaging-The expression pattern of p56-1 and p56-2 SEQ. ID. NO. 4 transcripts was determined using both classical Northern blot analysis and by BLAST searching of EST database. For Northern blot analysis, poly A.sup.+ RNAs isolated from various peripheral tissues and various brain regions were fractionated by electrophoresis under denaturing conditions and displayed on a nylon membrane. The blots were then visualized by hybridization to .sup.32 P-labeled coding sequence DNA probes prepared from either human p56-1 or human p56-2 SEQ. ID. NOS. 2 and 4. The p56-1 probe visualized a 6.0 kb transcript that was expressed at the highest level in skeletal muscle and lower levels detected in heart and pancreas. Minor signal was also detected in brain, placenta and liver while no transcript was detected in either lung or kidney under these conditions. Alternatively, BLAST searching revealed 20 ESTs derived from 11 different tissues matching the query with the complete p56-1 DNA sequence. These included the following ESTs; 5 from breast (4 normal/l tumor), 3 from prostate (1 normal/2 tumor), 2 from brain, 2 from colon tumors, 2 from kidney and single ESTs from stomach, uterus, pituitary, nasal polyp, thyroid and mononuclear cells.

The human p56-2 probe visualized a 2.8 kb transcript that was expressed at the highest level in heart, brain, pancreas and placenta. No signal for p56-2 SEQ. ID. NO. 4 was observed in lung, liver, skeletal muscle or kidney under these conditions. p56-2 SEQ. ID. NO. 4 transcripts were widely expressed in brain regions, with the highest levels in the amygdala and lower levels in the hippocampus, the caudate nucleus, the corpus callosum, the substantia nigra and the subthalamic nucleus. Twenty ESTs matched the p56-2 query sequence and these were derived from 13 different tissues including 3 from pancreas, 3 from lung tumors, 2 from synovial membrane, 2 from leukocytes, 2 from hippocampus and single EST matches from heart, kidney, bladder, small intestine, adrenal, breast, prostate and nasal polyp.

Reconstitution of p56 photolabeling by transient expression of human p56-1 and p56-2 SEQ. ID. NOS. 2 and 4

Photolabeling of p56-1 SEQ. ID. NO. 2 with a cyanoguanidine opener photoprobe was reconstituted by transient heterologous expression of the p56-1 cDNA SEQ. ID. NO. 2 in COS7 cells. The coding sequence of human p56-1 SEQ. ID. NO. 2 was placed under the control of the SV40 immediate early promotor in the vector pSVL. This construct was introduced into COS7 cells using cationic liposomes and 48 hr post-infection, the transfected cells were photolabeled with .sup.125 I. Wild-type COS7 cells showed minimal photolabeling of a 52 kDa band and no p56. Alternatively, transient expression of human p56-1 in these same cells led to photolabeling of a polypeptide that migrated with a M.sub.r of 56 kDa.

Charts

The following charts disclose the full length human p56-1 and p56-2 SEQ. ID. NOS. 1 and 2 proteins and the DNA that code for those proteins. Chart 1 provides the sequences for the full length of the p56-1 protein SEQ. ID. NO. 1. The sequences in Chart 1 include the signal or leader sequence. The sequences in Chart 1 are also in sequence listing no. 1 SEQ. ID. NO. 1. Chart 2 provides the cDNA residues that code for the p56-1 protein SEQ. ID. NO. 2 and it includes untranslated sequences. All of the sequences in Chart 2 are provided for in sequence listing no. 2 SEQ. ID. NO. 2.

The p56-1 DNA SEQ. ID. NO. 2 sequence shown in Chart 2 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence. This full-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-39), 2) a coding sequence (alignment positions 40-1554), 3) a stop codon "TGA" (alignment position 1555-1557) and 4) a 3' untranslated sequence (alignment positions 1555-1724 with poly A). The coding sequence beginning with ATG at position 40 starts with a signal sequence that ends at position 123 while the mature sequence begins at position 124. These positions are noted on Charts 1 and 2 below.

The signal sequence or leader sequence is a hydrophobic region, usually about 20-25 amino acids, here 28 aa, at the N-terminus that `signals` attachment of the ribosome to the endoplasmic reticulum and aids in the extrusion of the nacent polypeptide chain into the lumen of the ER. This signal sequence is cleaved off in the lumen by the signal peptidase. The untranslated sequences may have important regulatory functions such as governing MRNA stability and the like. The poly A track is added after transcription, the length is variable, often from 10-200 A's, here we show a 27 track poly A.

Charts 3 and 4 disclose the p56-2 proteins SEQ. ID. NO. 3 and the DNA that code for those proteins. Chart 3 provides the sequences for the full length of the p56-2 protein SEQ. ID. NO. 3. The sequences in Chart 3 include the signal or leader sequence. The sequences in Chart 3 are also in sequence listing no. 3 SEQ. ID. NO. 3. Chart 4 provides the cDNA residues that code for the p56-2 protein SEQ. ID. NO. 3 and it includes untranslated sequences. All of the sequences in Chart 4 are provided for in sequence listing no. 4 SEQ. ID. NO. 4.

The p56-2 DNA sequence SEQ. ID. NO. 4 shown in Chart 4 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence. This fill-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-35), 2) a coding sequence (alignment positions 36-1517), 3) a stop codon "TGA" (alignment position 1518-1520) and 4) a 3' untranslated sequence (alignment positions 1518-2567 with poly A). The coding sequence beginning with ATG at position 36 starts with a signal sequence. These positions are noted on Charts 3 and 4 below.

Claims

What is claimed is:

1. An isolated protein selected from the proteins comprising the proteins disclosed in SEQ. ID. NOS. 1 and 3.

2. An isolated protein of claim 1 comprising the protein disclosed in SEQ. ID. NO. 1.

3. An isolated protein of claim 1 comprising the protein disclosed in SEQ. ID. NO. 3.

4. A nucleic acid molecule encoding the proteins selected from the proteins comprising the proteins disclosed in SEQ. ID. NOS. 1 and 3, incorporated into a vector selected from a cloning vector, a shuttle vector or an expression vector, where the vectors are plasmids.

5. The plasmid of claim 4 adapted for expression in a bacterial cell, a mammalian cell, and a yeast cell.

Patent Citations (4)

Patent	Date	Inventor
US5525724	1996-06-01	Gadwood et al.
WOXWO94/194645	1994-09-01
WOXWO94/19464	1994-09-01
WOXWO96/16088	1996-05-01

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