AU6445394A - Cloned human alphaic adrenergic receptor - Google Patents

Description

TTTLE OF THE INVENTION

CLONED HUMAN ALPHA1C ADRENERGIC RECEPTOR

CROSS REFERENCES TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. Application serial number 08/032,849, filed on March 15, 1993, pending.

BACKGROUND OF THE INVENTION

i. Field of the Invention:

This invention relates to a method for defining the potency and selectivity of compounds for use as human alphalC adrenergic receptor antagonists using cloned human alpha 1 receptors. The invention also relates to the cloned receptors themselves, to compounds identified according to the method of this invention, and to methods of use of such compounds, alone or in combination with other agents. Particularly preferred are combinations of compounds identified according to this invention and testosterone 5-alpha reductase inhibitors, to alleviate pathologic conditions, particularly benign prostatic hyperplasia (also known as benign prostatic hypertrophy, BPH).

ii. Background:

Human adrenergic receptors are integral membrane proteins which have been classified into two broad classes, the alpha and the beta adrenergic receptors. Both types mediate the action of the peripheral sympathetic nervous system upon binding of catecholamines, norepinephrine and epinephrine.

Norepinephrine is produced by adrenergic nerve endings, while epinephrine is produced by the adrenal medulla. The binding affinity of adrenergic receptors for these compounds forms one basis of the classification: alpha receptors bind norepinephrine more strongly than epinephrine and much more strongly than the synthetic compound isoproterenol. The binding affinity of these hormones is reversed for the beta receptors. In many tissues, the functional responses, such as smooth muscle contraction, induced by alpha receptor activation are opposed to responses induced by beta receptor binding.

Subsequently, the functional distinction between alpha and beta receptors was further highlighted and refined by the pharmacological characterization of these receptors from various animal and tissue sources. As a result, alpha and beta adrenergic receptors were further subdivided into oq, cc2, Bl, and B2 subtypes. Functional differences between c and α2 receptors have been recognized, and compounds which exhibit selective binding between these two subtypes have been developed. Thus, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to selectively bind to adrenergic receptors of the alpha 1 subtype was reported. The αi/α2 selectivity of this compound was disclosed as being significant because agonist stimulation of the OA∑ receptors was said to inhibit secretion of epinephrine and norepinephrine, while antagonism of the o 2 receptor was said to increase secretion of these hormones. Thus, the use of non- selective alpha-adrenergic blockers, such as phenoxybenzamine and phentolamine, is limited by their α2 adrenergic receptor mediated induction of increased plasma catecholamine concentration and the attendant physiological sequelae (increased heart rate and smooth muscle contraction).

For a general background on the α-adrenergic receptors, the reader's attention is directed to Robert R. Ruffolo, Jr., α- Adrenoreceptors: Molecular Biology. Biochemistry and Pharmacology. (Progress in Basic and Clinical Pharmacology series, Karger, 1991), wherein the basis of αi/oc2 subclassification, the molecular biology, signal transduction (G-protein interaction and location of the significant site for this and ligand binding activity away from the 3'-terminus of alpha adrenergic receptors), agonist structure-activity relationships, receptor functions, and therapeutic applications for compounds exhibiting -adrenergic receptor affinity was explored.

The cloning, sequencing and expression of alpha receptor subtypes from animal tissues has led to the subclassification of the i receptors into eel A, (Lomasney, et al., J. Biol. Chem.. 266:6365-6369 (1991), rat αiA; Bruno et al, BBRC. 179:1485-1490 (1991), human αiA), αiB (Cotecchia, et al, PNAS. 85;7159-7163 (1988), hamster αlβ; Libert, et al., Science. (1989), dog αiB; Ramarao, et al., J. Biol. Chem.. 267:21936-21945 (1992), human αiB), and most recently, in a study using bovine brain, a new ocic subtype was proposed (Schwinn, et al.. J. Biol. Chem.. 265:8183-8189, 1990; Hirasawa et al., BBRC 195:902-909 (1993), described the cloning, functional expression and tissue distribution of a human αic adrenergic receptor; Hoehe et al., Human Mol. Genetics 1(5):349 (8/92) noted the existence of a two-allele Pstl restriction fragment polymoφhism in the αic adrenergic receptor gene; another study suggests that there may even be an alpha- ID receptor subtype, see Perez et al., Mol. Pharm... 40:876-883, 1992). Each αi receptor subtype exhibits its own pharmacologic and tissue specificities. Schwinn and coworkers noted that the cloned bovine αic receptor exhibited pharmacological properties proposed for the αi A subtype. Nonetheless, based on its non-expression in tissues where the αiA subtype is expressed, and its sensitivity to chloroethylclonidine, the receptor was given a new designation.

The differences in the α-adrenergic receptor subtypes have relevance in pathophysiologic conditions. Benign prostatic hypertrophy, BPH, is an illness typically affecting men over fifty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limted to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hypertrophy, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra. Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.

The mechanism of prostatic hypertrophy is well understood. The male hormone, 5α-dihydrotestosterone has been identified as the principal culprit. The continual production of 5α- dihydrotestosterone by the male testes induces incremental growth of the prostate gland throughout the life of the male. Beyond the age of about fifty years, in many men, this enlarged gland begins to obstruct the urethra with the pathologic symptoms noted above.

The elucidation of the mechanism summarized above has resulted in the recent development of effective agents to control, and in many cases reverse, the pernicious advance of BPH. In the forefront of these agents is Merck & Co., Inc.s' product PROSCAR® (finasteride). The effect of this compound is to inhibit the enzyme testosterone 5- alpha reductase, which converts testosterone into 5α-dihydrotesterone, resulting in a reduced rate of prostatic enlargement, and often reduction in prostatic mass.

The development of such agents as PROSCAR® bodes well for the long-term control of BPH. However, as may be appreciated from the lengthy development of the syndrome, its reversal also is not immediate. In the interim, those males suffering with BPH continue to suffer, and may in fact lose hope that the agents are working sufficiently rapidly.

In response to this problem, one solution is to identify pharmaceutically active compounds which complement slower-acting therapeutics by providing acute relief. Agents which induce relaxation of the urethral smooth muscle, by binding to alpha- 1 adrenergic receptors, thus reducing the increased adrenergic tone due to the disease, would be good candidates for this activity. Thus, one such agent is alfύzosin, which is reported in EP 0 204597 to induce urination in cases of prostatic hypertrophy. Likewise, in WO 92/0073, the selective ability of the R(+) enantiomer of terazosin to bind to adrenergic receptors of the αi subtype was reported. In addition, in WO 92/161213, hereby incorporated by reference, combinations of 5- alpha-reductase inhibitory compounds and alphal -adrenergic receptor blockers (terazosin, doxazosin, prazosin, bunazosin, indoramin, alfuzosin) were disclosed. However, no information as to the αiA, iB, or αic subtype specificity of these compounds was provided as these refinements were not yet available. The instant invention changes this situation by providing a cloned human αic adrenergic receptor and a method for identifying compounds which bind the human αic receptor.

Typically, identification of active compounds is through use of animal tissues known to be enriched in adrenergic receptors. Thus, rat tissues have been used to screen for potential adrenergic receptor antagonists. However, because of species variability, compounds which appear active in animal tissue may not be active or sufficiently selective in humans. This results in substantial wastage of time and effort, particularly where high volume compound screening programs are employed. There is also the danger that compounds, which might be highly effective in humans, would be missed because of their absence of appreciable affinity for the heterologous animal receptors. In this regard, it has been noted that even single amino acid changes between the sequence of biologically active proteins in one species may give rise to substantial pharmacological differences. Thus, Fong et al., (J. Biol. Chem.. 267:25668-25671, 1992) showed that there are 22 divergent amino acid residues between the sequence of the human neurokinin-1 receptor and the homologous rat receptor. They further showed, in studies with mutant receptors, that substitution of only two amino acid residues was both necessary and sufficient to reproduce the rat receptor's antagonist binding affinity in the human receptor. Oksenberg et al., (Nature. 360:161-163, 1992) showed that a single amino-acid difference confers major pharmacological variation between the human and the rodent 5-hydroxytryptamine receptors. Likewise, Kuhse et al., (Neuron. 5:867-873, 1990) showed that a single amino-acid exchange alters the pharmacology of the neonatal rat glycine receptor subunit. This difficulty and unpredictability has resulted in a need for a compound screen which will identify compounds that will be active in humans.

The instant inventors have solved these problems by cloning a novel human adrenergic receptor of the αic subtype. Their efforts have led to the development of a novel screening assay which enables them to identify compounds which specifically interact with the human αlC adrenergic receptor. Marshall et al (Br. J. Pfiarm.. 107:327 (1992)) speculated that compounds which specifically interact with the αlC adrenergic receptor may be responsible for contraction of the human prostate. The instant invention provides a method for identifying compounds which bind the human αic receptor. In addition, if the compounds are further tested for binding to other human alpha 1 receptor subtypes, as well as counterscreened against other types of receptors, the specificity of the compounds for the human αic adrenergic receptor may be defined.

Compounds identified according to this invention may be used to reduce the acute symptoms of BPH. New agents identified in this manner, or already known agents showing activity in this assay, may now be employed in a novel way to help BPH sufferers contend with the acute symptoms of the syndrome. Thus, this invention is useful to identify compounds which may be used alone or in conjunction with a more long-term anti-BPH therapeutics, such as PROSCAR®. Other uses for the invention include identification of compounds which induce highly tissue-specific, localized αic adrenergic receptor blockade. Effects of this blockade include reduction of intra-ocular pressure, control of cardiac arrhythmias, and possibly a host of alpha- IC receptor mediated central nervous system events. In addition, the cloned αic receptor can be used for screening of tissue specific expression of αic adrenergic receptors. Effects such as these, induced by or available to analysis with the αic adrenergic receptor also form part of this invention.

SUMMARY OF THE INVENTION

The human adrenergic receptor of the alphalC subtype is cloned and used in an in vitro assay to screen for compounds that bind to the receptor, including compounds which specifically inhibit the activity of the receptor. The invention includes the assay, the cloned receptor used in the assay (cDNA), an isolated human alphalC adrenergic receptor, cells expressing the cloned receptor, and compounds identified through the use of this novel, cloned receptor, which selectively bind to the human alphalC adrenergic receptor, including specific antagonists of the receptor. One embodiment of this invention is a method of treating benign prostatic hypeplasia (BPH) employing compounds having an affinity for the human alpha IC receptor that is at least 12 fold greater than for either the human alpha 1A or the human alpha IB receptors.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 : Sequence of cDNA obtained by PCR of human heart mRNA, SEQ. ID:4:.

Fig. 2: Comparison of the open reading frame obtained from human heart, SEQ ID:5:, and the bovine alpha-lC adrenergic receptor sequence, SEQ. LD:6:.

Fig. 3: Sequence of cDNA obtained by screening a human hippocampus cDNA library using the heart mRNA derived sequence from figure 1, SEQ. LD:7:

Fig. 4: Sequence of 3' coding region of human alpha- IC gene, obtained by PCR amplification of a human genomic DNA library with oligonucleotides, SEQ. ID:10:.

Fig. 5: Sequence of the ligated portions of human alpha- IC DNA shown in figures 3 and 4, SEQ. ID: 11:.

Fig. 6: The amino acid sequence of the human alpha- IC adrenergic receptor, SEQ ID: 12:.

Fig. 7: The alignment of the nucleotide and amino acid sequence of the human alpha- IC adrenergic receptor, showing the 5'-untranslated region, SEQ. ID:11: and SEQ. ID: 12:. Fig. 8: Expression of the human alpha- IC adrenergic receptor in COS cells: Binding data using membranes from cells transfected with the expression vector alone and the expression vector containing the human alpha- IC adrenergic receptor coding sequences.

Fig. 9: Binding curves of compounds using membranes from COS cells transfected with the human alpha- IC adrenergic receptor containing expression vector.

Fig. 10: Nucleotide sequence of the human alphal A receptor, SEQ. ID:13:

Fig. 11: Amino acid sequence of the human alphal A adrenergic receptor, SEQ. ID: 14:

Fig. 12: Partial sequence of the human alpha IB adrenergic receptor, SEQ. ID:17:

Fig. 13: Partial sequence of the human alphal B adrenergic receptor, SEQ. ID:20:

Fig. 14: Partial sequence of the human alphalB adrenergic receptor, SEQ. ID:23:

Fig. 15: Composite human/rat alphalB adrenoreceptror, SEQ. ID:24:

Fig. 16: Amino acid sequence of the composite human/rat alphalB adrenergic receptor, SEQ. LD:25:

Fig. 17: Binding curves of compounds using membranes from COS cells transfected with the human alphalA, IB, and IC adrenergic receptor expression vectors. Fig. 18: Sequence of truncated human alphalC adrenergic receptor, SEQ. LD:26:.

Fig. 19: Nucleotide sequence of the human αlC adrenergic receptor having a Pstl site, SEQ.LD:27:.

Fig. 20: Amino acid sequence of the human αlC adrenergic receptor encoded by the Pstl site encoding allele, SEQ.ID:28:.

Fig. 21 : Alignment of the nucleotide and amino acid sequences of figures 19 and 20, SEQ.LD:27: and SEQ.ID:28:.

FIG. 22: Nucleotide sequence of the human αiA adrenergic receptor, Seq.ID:29:.

Fig. 23: Amino acid sequence of the human αi A adrenergic receptor, SEQ.ID:30:.

Fig. 24: Alignment of the nucleotide and amino acid sequences of figures 22 and 23, SEQ.LD:29: and SEQ.ID:30:.

DETAILED DESCRIPTION OF THE INVENTION

The human alpha adrenergic receptor of the 1-C subtype was identified, cloned and expressed by the instant inventors. A partial coding region for this receptor was generated by reverse transcriptase- polymerase chain reaction technology, RT-PCR. Accordingly, degenerate oligonucleotides encoding amino acids conserved in the fifth and sixth transmembrane domains of all three αl receptor subtypes (A, B, C) were used to prime RT-PCR reactions using human heart mRNA as template. The predicted sized products were cloned and sequenced. Translation of the amplified cDNA yielded an open reading frame encoding a protein 95% homologous to the bovine αlC receptor (Fig.2, SEQ. ID:5: and SEQ. LD:6:). This partial sequence was used to obtain a larger cDNA clone from a human hippocampus library (Fig. 3, SEQ. LD:6:). The remaining coding region was obtained by PCR amplification of human genomic DNA using primers based on the cDNA sequence and the last six amino acids of bovine αlC receptor (Fig. 4, SEQ.LD:10:). The complete receptor was then assembled using the partial sequences shown in Fig. 3, SEQ._D:6: and Fig. 4, SEQ. ID:10:, to generate the sequence shown in Fig. 5, SEQ. ID: 11:. The translation of this sequence is shown in Fig. 6, SEQ. ID: 12:, and the alignment of the nucleotide and amino acid sequences, and the 5'- untranslated sequences, is shown in Fig. 7, SEQ. LD:11: and SEQ. ID:12:.

The 3 '-terminal six amino acids of the human αic adrenergic receptor were confirmed by screening a human genomic library with the radiolabeled 3'-terminal 512 nucleotides of the SEQ. ID: 10: clone previously obtained. A complete human exon 2 was generated in this manner and sequenced. The nucleotide sequence of this gene is provided in figure 19, SEQ. ID:27: and the amino acid sequence is provided in figure 20, SEQ. LD:28:. We discovered that this clone was identical to the original 3'-terminal portion of the gene, except that:

1) There are five silent nucleotide changes between the new clone and the previously obtained clone (the last five codons, including the stop codon, each have a silent change in the third nucleotide); and

2) At nucleotide position 1636 (amino acid 347), there is a cytosine to thymine base change resulting in the formation of a Pstl site at that location and a concommitant single amino acid change of Arg to Cys. Thus, we have confirmed and localized the site of the two-allele Pstl restriction fragment polymoφhism (RFLP) noted by Hoehe et al., fHuman Mol. Genetics.1(5):349 (8/92)]. Through pharmacological studies using clones of both alleles, we have confirmed that the Arg to Cys change appear to be pharmacologically indistinguishable (see Table II, Example 11, below).

The cloned human αlC receptor, when expressed in mammalian cell lines (see Fig. 8), is used to discover ligands that bind to the receptor and alter its function. In addition, the cloned αlC receptor enables quanititation of mRNA levels in human tissues, including the aorta and prostate, by RNase protection assays. For these puφoses, a complete coding sequence of the receptor is provided. . However, as long as the ligand binding and signal transduction segments of the receptor (G-protein interaction) are intact, truncation at the 3' end of the sequence does not affect the functioning of the receptor. Thus, in addition to the sequence provided in SEQ. ID: 11:, a sequence, truncated at the 3' end, SEQ. ED:26: is disclosed, which consists entirely of human alphalC sequence.

The specificty of binding of compounds showing affinity for the αlC receptor is shown by comparing affinity to membranes obtained from COS cells tranfected with the cloned αlC receptor and membranes from tissues known to express other types of alpha or beta adrenergic receptors. In addition, the cloned human αiA and a hybrid human/rat αiB (with only the cytoplasmic, carboxy terminal region being rat sequence) could be used for this pmpose, along with the human αlC receptor expressed in COS cells . Expression of the cloned human αiA, αiB, and αlC receptors and comparison of their binding properties with known selective antagonists provides a rational way for selection of compounds and discovery of new compounds with predictable pharmacological activities.

Once the human receptor is cloned and expressed in a cell such as COS cells or CHO cells, the receptor is free of other human proteins. The membranes from cells expressing different human alpha adrenergic receptor subtypes are then isolated according to methods well known in the art for membrane associated receptor binding assays. For example, the method of Schwinn, et al., (J. Biol. Chem.. 265:8183- 8189, 1990) may be used. A compound of interest is used to compete with the binding of a known, quantifiable alpha receptor ligand. Thus, radiolabled prazosin, niguldipine, 5-methyl urapidil, terazosin, dozazosin, phenoxybenzamine, WB4101, benoxathian, HEAT (2-[β-(4- hydroxy-3-iodophenyl)ethylaminomethyl]tetralone, or phentolamine may be used for this puφose (see, for example, Robert R. Ruffolo, Jr., q-Adrenoreceptors: Molecular Biology. Biochemistry and Pharmacology. (Progress in Basic and Clinical Pharmacology series, Karger, 1991), page 29). Because of the ease of 125lodine detection, 125I-HEAT may be preferred for this puφose. By increasing the amount of unlabeled, test compound, the labeled compound is competed off the receptor. From these experiments, IC50 values for each test compound and receptor subtype is determined.

Thus, according to this invention, a method is provided for identifying compounds specific for the human alphalC receptor comprising the following steps:

a. Cloning the human alphalC adrenergic receptor; b. Splicing the the cloned alphalC adrenergic receptor into an expression vector to produce a construct such that the alphalC receptor is operably linked to transcription and translation signals sufficient to induce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell; c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a human alphalC adrenergic receptor in the absence of said introduced construct; d. Incubating cells or membranes isolated from cells produced in step c. with a quantifiable compound known to bind to human alpha adrenergic receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the receptor; e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned human alpha adrenregic receptor of a subtype other than the human alphalC receptor under identical conditions to the incubation conducted in step d, and obtaining the IC50 of the test compound for the non-alphalC receptor; and f. Comparing the IC50 for the test compound for the alphalC receptor and for the alpha adrenergic receptor of a subtype other than the alphalC to identify compounds having a lower IC50 for the alphalC receptor.

In addition to providing a sequence for the human αlC adrenergic receptor, the instant inventors have also discovered a different sequence than that reported by Bruno et al., rBBRC 179:1485- 1490 (1991)] for the human αiA adrenergic receptor. The new sequence is more homologous to the rat αiA adrenergic receptor sequence. Disclosed and claimed herein is the sequence for this new human αiA adrenergic receptor (see Example 12 and figures 22, 23, and 24, SEQ. 1D:29: and SEQ. 1D:30:). While no difference in ligand binding has thus far been observed based on the different amino terminal amino acid sequences between these two receptors, such differences cannot be ruled out except by screening compounds against both clones. Since a new human sequence is provided herein, compounds identified according to the method of this invention using the earlier reported human αiA adrenergic receptor sequence can now be confirmed against this clone.

As a result of the cloning, sequencing, expression, and screening efforts described above and further exemplified below, numerous compounds have been tested for their ability to specifically bind to the cloned human αlC receptor with high affinity. Compounds specific for the human alpha IC adrenergic receptor, that is compounds having an affinity for the human alpha IC receptor that is at least 12 fold greater than for either the human alpha 1 A or the human alpha IB receptors, are identified by this method. Thus, the compounds S(+) niguldipine, (S(+)- 1 ,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3 ,5- pyridinedicarboxylic acid 3-(4,4-diphenyl-l-piperidinyl)-propyl methyl ester hydrochloride), and 5-methyl urapidil (5-methyl-6[[3-[4-(2- methoxypheny 1)- 1 -piperazinyl]propyl] amino] -1,3 -dimethyluracil) have been discovered to selectively bind to the human αlC adrenergic receptor. These compounds may be administered in dosages effective to antagonize the alphalC receptor where such treatment is needed, as in BPH. Compounds identified according to the method of this invention as being selective human αlC adrenergic receptor antagonists may further be defined by counterscreening. This is accomplished, according to methods known in the art using other receptors responsible for mediating diverse biological functions Compounds which are both selective amongst the various human alphal adrenergic receptor subtypes and which have low affinity for other receptors, such as the alpha2 adrenergic receptors, the β-adrenergic receptors, the muscarinic receptors, the serotonin receptors, and others are particularly preferred. The absence of these non-specific activities may be confirmed by using cloned and expressed receptors in an analogous fashion to the method disclosed herein for identifying compounds which have high affinity for the various huaman alphal adrenergic receptors. Furthermore, functional biological tests are used to confirm the effects of identified compounds as alphalC adrenergic receptor antagonists.

Compounds identified according to this patent disclosure may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the human αlC adrenergic receptor while minimizing any potential toxicity. In addition, co- administration or sequential administration of other agents which alleviate the effects of BPH is desirable. Thus, in one embodiment, this includes administration of compounds identified according to this disclosure and a human testosterone 5-α reductase inhibitor. Many such compounds are now well known in the art and include such compounds as PROSCAR®, (also known as finasteride, a 4-Aza-steroid; see US Patents 4,377,584 and 4,760,071, for example, hereby incoφorated by reference). In addition to PROSCAR®, which is principally active in prostatic tissue due to its selectivity for human 5-α reductase isozyme 2, combinations of compounds which are specifically active in inhibiting isozyme 1 (found particularly in skin) and compounds which act at both of these isozymes, are useful in combination with compounds identified according to this invention.

In the treatment of hyperandrogenic disease conditions, e.g. benign prostatic hypeφlasia (BPH) and/or the prevention and treatment of prostatic cancer, and the treatment of prostatitis, it would be desirable to have one drug entity which is active against both isozymes to significantly inhibit dihydrotesterone production. It would also be desirable to have one drug entity that is active as a dual inhibitor of both isozymes for the treatment of conditions of the skin and scalp, e.g. acne vulgaris, seborrhea, female hirsutism, and androgenic alopecia. Additionally, such a dual inhibitor of 5α-reductase 1 and 2 could be used in combination with a 5α-reductase 1 inhibitor or with a 5α- reductase 2 inhibitor, e.g. finasteride (PROSCAR®), for combination therapy in the treatment of hyperandrogenic conditions, in combination with compounds identified according to this inventionas being selective human alphalC adrenergic receptor antagonists. The dual 5α-reductase isozyme inhibitor could also be used in combination with a potassium channel opener, e.g. minoxidil, for the treatment of male pattern baldness, and such combinations in combination with selective human alphalC adrenergic receptor antagonists also form part of the instant invention . Compounds that are active as dual 5a-reductase 1 and 2 inhibitors have been described in WO93/23420, EP 0572166; WO 93/23050; WO93/23038, ; WO93/23048; WO93/23041; WO93/23040; WO93/23039; W093/23376; W093/23419, EP 0572165; WO93/23051, each of which is hereby incoφorated by reference.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient for use in the specific antagonism of human alphalC adrenergic receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for systemic administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as an alphalC antagonistic agent.

The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0002 mg./kg to about 50 mg./kg. of body weight per day. The range is more particularly from about 0.001 mg./kg to 7 mg./kg. of body weight per day. The dosages of the alphalC adrenergic receptor and testosterone 5-alpha reductase inhibitors are adjusted when combined to achieve desired effects. As those skilled in the art will appreciate, less 5-alpha reductase inhibitor may be required when the acute symptoms of BPH are alleviated by treatment with alphalC adrenergic receptor inhibitors has been initiated. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. For the treatment of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, female hirsutism, benign prostatic hypeφlasia, prostatitis and the prevention and/or treatment of prostatic cancer, compounds exhibiting at least 12 fold selectivity for inhibition of the alphalC adrenergic receptor can be combined with a therapeutically effective amount of a 5α-reductase 2 inhibitor, such as finasteride, in addition to a 5α-reductase 1 inhibitor, such as 4,7β-dimethyl-4-aza-5α-cholestan-3-one, in a single oral, systemic, or parenteral pharmaceutical dosage formulation. Alternatively, a combined therapy can be employed wherein the alphalC adrenergic receptor antagonist and the 5α-reductase 1 or 2 inhibitor are administered in separate oral, systemic, or parenteral dosage formulations. Also, for the skin and scalp related disorders of acne vulgaris, androgenic alopecia including male pattern baldness, seborrhea, and female hirsutism, the compounds of the instant invention and dual inhibitors of 5α-reductase 1 and 2 could be formulated for topical administration. For example, niguldipine or 5-methyl urapidil and finasteride can be administered in a single oral or topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate oral dosage formulations, or an oral dosage formulation of finasteride in combination with a topical dosage formulation of a compound exhibiting dual inhibiton of both isozymes of 5α-reductase. See, e.g., U.S. Patent No.'s 4,377,584 and 4,760,071 which describe dosages and formulations for 5α-reductase inhibitors.

Furthermore, since administration of 5α-reductase inhibitors have been found to be useful in combination with a therapeutically effective amount of a potassium channel opener, such as minoxidil, cromakalin, pinacidil, a compound selected from the classes of S-triazine, thiane-1 -oxide, benzopyran, and pyridinopyran derivatives or a pharmaceutically acceptable salt thereof, compounds of this invention may also be used in combination therapy for the treatment of androgenic alopecia including male pattern baldness. The active agents can be administered in a single topical dosage formulation, or each active agent can be administered in a separate dosage formulation, e.g., in separate topical dosage formulations, or an oral dosage formulation of a compound of formula I in combination with a topical dosage formulation of, e.g., minoxidil. See, e.g., U.S. Patent No.'s 4,596,812, 4,139,619 and WO 92/02225, published 20 February 1992, for dosages and formulations of calcium channel openers.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incoφorated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

The liquid forms in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotomc preparations which generally contain suitable preservatives are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. See, e.g., EP 0 285 382.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl- pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl- amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl- eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

The following examples are provided to further define the invention without, however, limiting the invention to the particulars of these examples.

EXAMPLE 1

PCR amplification, cloning and sequencing of phαlX:

Based on the amino acid homologies of human αlA, rat αlB and bovine αlC receptors, degenerate oligonucleotides were designed to amplify cDNAs encoding all three receptor subtypes. These oligonucleotides are:

WL^* (SEQ.LD:1) TTTTCTAGATTRTTNARRTANCCNAGCC 28

MYC (SEQ.ID:2) TTTACTAGTATCSTNGTNATGTAYTG 16

WC^*(SEQ.ID:3) TTTTCTAGAGAARAANGGNARCCARC 26 Oligonucleotides MYC and WL' were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the RNA PCR kit from Perkin Elmer Cetus. Briefly, 0.5 ug of mRNA was reverse transcribed in a volume of 20 ul using either random oligonucleotide primers (reaction 1) or oligo dT primer (reaction 2). Reactions 1 and 2 were pooled and served as template for PCR amplification as follows:

PCR Reactions:

Primary reaction (50 ul)

5 ul 10X buffer from Perkin Elmer Cetus Gene Amp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 3ul first strand cDNA 1 ul 25 pMoles oligo MYC

1 ul 25 pMoles oligo WL'

0.25 ul 1.25 units Amplitaq DNA polymersase 31.75 ul water

Reaction conditions; 40 cycles of 94⁰C 1'; 45°C 2'; 72<>C 2'

Secondary reaction (100 ul)

9.5 ul 10X buffer from Perkin Elmer Cetus Gene Amp Kit 16ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 5ul first strand cDNA

2 ul 50 pMoles oligo MYC 2 ul 50 pMoles oligo WC

0.5 ul 2.5 units Amplitaq DNA polymersase 65 ul water

Reaction conditions; 40 cycles of 94<>C 1^*; 45°C 2'; 72°C 2'

Prep scale tertiary reaction 3 X 200 ul: 19.5 ul 10X buffer 32 ul 1.25 mM each stock of dATP, dCTP, dGTP, and dTTP 5 ul secondary PCR reaction 4 ul 100 pMoles oligo MYC 4 ul 100 pMoles oligo WC 1 ul 5 units Amplitaq DNA polymerase 134.5 ul water

Reaction conditions; 30 cycles of 94°C 1'; 5Q^OC 2'; 12^~C 2'

The PCR product was purified by Qiagen spin columns and digested with restriction endonucleases Spel and Xbal. The fragment was then ligated into Spel/Xbal cut pGEM9Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig. 1, SEQ. ID:4:.

EXAMPLE 2

Isolation of partial alphalC cDNA Clone:

A cDNA library prepared from mRNA isolated from human hippocampus (Stratagene) was screened by plaque hybridization using phαlX as a probe. Hybridization conditions were as follows:

5XSSC ( 1XSSC is 0.15M sodium chloride, 0.015M sodium citrate,

50% Formamide

5X Denhardt's Solution ( 1% Ficoll, 1 % polyvinylpyrrolidone, 1% bovine serum albumin)

0.15 mg/ml salmon sperm DNA hybridize overnight at 42° C. Filters were washed 3 times in 2XSSC, 0.1% SDS at room temperature for 5', then 1 time in IXSSC, 0.1% SDS at 50C for 30'. Positive clones were identified by autoradiography. Phagemid DNA was rescued from the positive plaques and sequenced by the dideoxy chain termination method. The base sequence obtained is shown in Fig. 3, SEQ. 1D:7:.

EXAMPLE 3

PCR amplification, cloning and sequencing of 3'CG of alphalC:

The 3' end of the coding region of human alphalC adrenergic receptor was amplified from human genomic DNA using two oligonucleotides:

S3C (SEQ ID:8:)

5' TTTGAATTCT GATTTCAAGC CCTCTG 3'

and

3'C (SEQ ID:9:)

5' TTTGAATTCT TANACYTCYT CNCCRTTYTC 3'

as follows:

10 ul 10X buffer from Perkin Elmer Cetus Gene Amp Kit 16 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 6 ul 1 ug human genomic DNA (Promega) 2 ul 50 pMoles oligo S3C 2 ul 50 pMoles oligo 3'C 0.5 ul 2.5 units Amplitaq DNA polymersase 63.5 ul water Reaction conditions; 40 cycles of 94< C 1'; 50^OC 2'; 72⁰C 2'

The PCR product was purified by Qiagen spin columns and digested with restriction endonuclease EcoRI. The fragment was then ligated into EcoRI cut pGEM3Zf(-). The ligation mix was used to transform E. coli XL-1 blue. Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 4, SEQ. 1D:10:.

ιi»XAM"Lιi, 4

Assembly of complete coding region of human alpha lc adrenergic receptor:

The complete coding region of human alphal c adrenergic receptor was assembled by ligating the cDNA clone (see Example 2, figure 3, SEQ ID:7:) and 3'CG (see Example 3, figure 4, SEQ 1D:10: ) at their common PvuII site ( 1552-1557 of figure 3, SEQ ID:7: and 59-64 of figure 4, SEQ ID: 10:). The complete nucleotide sequence is shown in figure 5, SEQ ID:11:. The amino acid sequence is shown in figure 6, SEQ. ID: 12:. Figure 7 shows the structure of the cDNA, including the 5'-untranslated sequences. The very 3' twenty seven nucleotides (6 amino acids) shown is the sequence of the PCR primer used to generate the sequence. However, the function of the receptor, both for ligand binding and signal transduction depends on sequences far removed from the carboxy terminus of the receptor. A completely human sequence is shown in figure 18, SEQ. ID:26: which is truncated at the 3' terimuns.

EXAMPLE 5

Expression of the cloned alphalC adrenergic receptor: The complete sequence (SEQ ID:11 :) of the human alphalC adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS -7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183- 8189, 1990. Membranes (5-25 ug, see figure 8) prepared from the COS-7 cells transfected with the vector containing the alphalC receptor gene specifically bound the alpha 1 antagonist [125 I] -HEAT; membranes prepared from the COS -7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I] -HEAT (figure 8), proving the expression of the alphalC adrenergic receptor. Binding reactions (total volume = 200 ul) contained 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 j-HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions were incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.

EXAMPLE 6

Screening assav: Alpha 1 C Adrenergic Receptor Binding ^■

Membranes prepared from the transfected COS-7 cells may also be used to identify compounds that bind to the human alphalC adrenergic receptor. These competition binding reactions (total volume = 200 ul) contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I] -HEAT, membranes prepared from COS-7 cells transfected with the alphalC expression plasmid and increasing amounts of unlabeled ligand. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and IC50s determined by an iterative curve fitting program. Results are shown in Figure 9.

EXAMPLE 7

Expression of human alphal A adrenergic receptor:

The complete coding region for the human alphal A adrenergic receptor (Bruno, et al., BBRC. 179:1485-1490, (1991); see figure 10, SEQ. ID:13: and figure 11, SEQ. ID:14: herein) was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183-8189, 1990. Membranes prepared from the COS-7 cells transfected with the vector containing the alphalA receptor gene specifically bound the alpha 1 antagonist [125 I] -HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I]-HEAT. Binding reactions (total volume = 200 ul) contained 50 mM Tris- HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [ 25 I]-HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non¬ specific binding was determined in the presence of 10 uM prazosin.

EXAMPLE 8

Expression of human alphalB adrenergic receptor:

1. PCR amplification of partial cDNA for human alphalB adrenergic receptor:

Amplification of 5XB clones

5XB, SEQ. ID:15: 5' TCT AGA CCA TGA AYC CNG AYC TGG 3^*

A1B, SEQ. ID:16: 5' TTT GAA TTC ACA TWC CGA CYA CAA TGC CC 3'

Oligonucleotides 5XB and A1B were used as primers in a reverse transcription PCR amplification of human heart mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligonuleotide WC as primer.

Primary reaction (50 ul)

5 ul 10X buffer from Perkin Elmer Cetus GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 2.5 ul first strand cDNA 1 ul 25 pMoles oligo 5XB 1 ul 25 pMoles oligo A1B 0.25 ul 1.25 units Amplitaq DNA polymersase 32.75 ul water

Reaction conditions; 40 cycles of 94<>C 1'; 58<>C 2'; 72<>C 2'

The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVαF' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 12, SEQ. ID:17:

2. Amplification of EFK clones

EFK, SEQ. ID:18: 5'GAAGGCGCGCTTGAACTC 3'

5B1,SEQ.ID:19: 5'AGAGAACCACCAAGAACC3'

Oligonucleotides EFK and 5B1 were used as primers in a reverse transcription PCR amplification of human aorta mRNA (Clontech) using the Invitrogen Copy Kit. Briefly, 1.0 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer.

Primary reaction (50 ul)

5 ul 10X buffer from Perkin Elmer Cetus

GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 2.0 ul first strand cDNA 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water

Reaction conditions; 40 cycles of 94< C 1'; 58°C 2'; 72⁰C 2^*

The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVaF (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 13, SEQ. ID:20:.

3. Assembly of partial cDNA for human alphalB adrenergic receptor

A partial cDNA clone encoding the human alphalB adrenergic receptor was assembled by joining the 5XB sequence (SEQ. ID: 17:) and the EFK sequence (SEQ. ID:20:) at their common BamHI site.

4. Amplification of the 3' end of rat alphalB adrenergic receptor

S4B, SEQ. ID:21: 5' TTT GAA TTC ATG TTC AAG GTG GTG TTC

3^*

3^*B2, SEQ. ID:22: 5^* TTT GAA TTC TAA AASTGN CCN GGN SCC AGN GGC AT 3^*

Oligonucleotides S4B and 3'B2 were used as primers in a reverse transcription PCR amplification of rat heart mRNA using the Invitrogen Copy Kit. Briefly, 0.6 ug of mRNA was reverse transcribed in a volume of 20 ul using oligo dT as primer. Primary reaction (50 ul)

5 ul 10X buffer from Perkin Elmer Cetus

GeneAmp Kit 8 ul 1.25 mM each stock of dATP,dCTP,dGTP, and dTTP 2.0 ul first strand cDNA 1 ul 25 pMoles oligo EFK 1 ul 25 pMoles oligo 5B1 0.25 ul 1.25 units Amplitaq DNA polymersase 33.25 ul water

Reaction conditions; 40 cycles of 94<>C 1'; 58°C 2'; 12-C 2'

The PCR product was directly ligated into pCR vector (Invitrogen) and used to transform E. coli INVαF' (Invitrogen). Plasmid DNA was isolated from white transformants and sequenced by the dideoxy chain termination method. The base sequence is shown in Fig. 14, SEQ. ID:23:.

5. Assembly and expression of a functional human/rat hybrid alphalB adrenergic receptor

The partial human alphalB adrenergic receptor cDNA was joined to the 3' end of the rat alphalB adrenergic receptor cDNA at their common BssHII restriction endonuclease site. This composite sequence is shown in figure 15, SEQ. ID:24:, and the amino acid sequence is shown in Fig. 16, SEQ. ID:25:

The complete coding region for the human/rat alphalB adrenergic receptor was subcloned into the eukaryotic expression vector pcDNAI-neo (Invitrogen). The resulting plasmid was transfected into COS-7 cells by electroporation. Cells were harvested after 72 hours and the membranes containing the expressed receptor protein were prepared as described in Schwinn, et al.. J. Biol. Chem.. 265:8183- 8189, 1990. Membranes prepared from the COS-7 cells transfected with the vector containing the alphalB receptor gene specifically bound the alpha 1 antagonist [125 I] -HEAT; membranes prepared from the COS-7 cells transfected with the vector alone did not bind the alpha 1 antagonist [125 I] -HEAT. Binding reactions (total volume = 200 ul) contained 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 I] -HEAT, and membranes prepared from COS-7 cells transfected with expression plasmids. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Non specific binding was determined in the presence of 10 uM prazosin.

EXAMPLE 9

Selective Binding assays

Membranes prepared from COS-7 cells transfected with the human alpha 1 receptor subtype expression vectors may also be used to identify compounds that selectively bind to the human alphalC adrenergic receptor. These competition binding reactions (total volume = 200 ul) contain 50 mM Tris-HCl pH. 7.4, 5 mM EDTA, 150 mM NaCl, 100 pM [125 i]_ HEAT, membranes prepared from COS-7 cells transfected with the respective alpha 1 subtype expression plasmid and increasing amounts of unlabeled ligand. Reactions are incubated at room temperature for one hour with shaking. Reactions were filtered onto Whatman GF/C glass fiber filters with a Brandel cell harvester. Filters were washed three times with ice cold buffer and bound radioactivity was determined. Binding data were analyzed and IC50s determined by an iterative curve fitting program. Table I shows the results from such an analysis. Compound

prazosm

terazosin

doxazosin

phenoxybenzamine

WB4101

benoxathian

phentolamine

5-methyl urapidil

S(+) niguldipine

EXAMPLE 10

IDENTIFICATION AND CLONING OF A NEW ALLELE FOR THE HUMAN ALPHA1-C ADRENERGIC RECEPTOR

Probes:

3'CG: A 525 bps fragment, specific to complete exon.2 of human alphal c AR, was PCR amplified from human genomic DNA using a sense primer based on the isolated cDNA clone and an antisense primer based on the last six amino acids of bovine alphal c cDNA. This PCR product was subcloned and confirmed by sequencing (see Example 3, SEQ.ID:10:). Genomic Library Screening:

Human W138 Fibroblast genomic library synthesized in the Lambda Fix II vector (2 x 10^ recombinants; Stratagene,La Jolla, CA) was screened with (3'CG). This probe was labelled with 32p_dCTP (Amersham) by random-primed labelling kit (Boehringer Mannheim,Indianapolis,IN). A Total 800,000 plaques were screened, using duplicate Hybond-N nylone filters (Amersham,UK). Prior to hybridization, filters were denatured (1.5M NaCl+0.5M NaOH), neutralized (1.5M NaCl+ IM Tris.Cl,pH 8.0) and washed (0.2M Tris.Cl pH 7.5 + 2 SSC), 5' for each. DNA was cross-linked with UV cross- linker (Stratagene,La Jolla,CA). The filters were, then, hybridized in 50% formamide, 5 x SSC(lxSSC= 0.15M NaCl, 0.015M Na citrate, pH7.0), 0.02% polyvinylpyrophosphate, 0.2% Ficoll, 0.2% bovine serum albumin, 150μg of sheared & boiled Salmon sperm DNA, 10^ cpm of 32p_iabelled probe at 42°C for 40hrs. Filters were washed in O.lx SSC +1% SDS solution at 60°C for 20'. Two more rounds of screening for 20 "positive" plaques/clones with 3'CG probe confirmed two clones for the alphalC adrenergic receptor, which were named 48.1C and 53.1C. Clone 53.1C was subjected to further analysis/investigation.

Sub-clonning of Exon.2 :

53. IC lambda DNA was amplified by plate lysis method and purified with Qiagen midi-lambda kit (Qiagen,Chatsworth,CA). A 2.6Kb band excised with EcoRI restriction enzyme was identified by Southern analysis using 3'CG probe. This fragment was then subcloned into pGEM3Zf(+) vector.

P A s-gqweπcing;

Nucleotide sequence analysis of DNA in both direction was performed by Sanger chain termination method. Result and Discussion:

Sequencing analysis of this genomic clone confirmed that clone 53.1c contains sequences for complete exon.2 flanked by an intron at 5'-end. It also revels that there is a nucleotide change from cytosine (C) to thymine(T) at nucleotide position 1636, amino acid position 347. This change creates a Pstl site and changes the codon for arginine (Arg) to cystine (Cys). This data differs from the known/published cDNA sequence of the gene. Southern analysis of human genomic DNA confirms the Pstl site in the gene/exon.2.

EXAMPLE 11

COMPARATIVE PHARMACOLOGY OF ALPHA1-C ALLELES

We have cloned two genes for the human alpha- lc receptor. The coding regions differ by a single nucleotide. The genes encode either Cys or Arg at amino acid 347 near the C terminus of the receptor. The nucleotide difference lies within a Pstl restriction enzyme recognition site thus creating a Restriction Fragment Length Polymoφhism (RFLP). The frequency of allele 1 (LRR) is 0.34; allele 2 (LCR) is 0.66 in 83 unrelated individuals (Hoehe et al "A two-allele Pstl RFLP for the alpha- IC adrenergic receptor gene" Human Molecular Genetics 1: 349, 1992; Allele I is defined by a 2.1 kb Pstl fragment; allele 2 yields two bands of 1.6 and 0.5 kb). Since the amino acid difference occurs within the intracellular tail of the receptor we would not expect any pharmacological differences between the expressed receptors. To investigate the pharmacological profiles of the two allelic forms of the human alpha- lc adrenergic receptor we ligated the genomic exon 2 fragment of allele 2 to a cDNA clone of allele 1 at a common PvuII restriction site. The two allelic forms were transiently expressed in COS-7 cells using pcDNAI/NEO (Invitrogen) expression vector. Competitive inhibition studies performed in the presence of 125τ_HEAT with various antagonists showed no significant difference in their pharmacological profiles (Table II): Table II COMPARATIVE PHARMACOLOGY OF ALPHA 1-C ALLELES

IC50 nM} LRR LCR

CLONING OF A NOVEL ALPHA1-A ADRENERGIC RECEPTOR

A cosmid library containing FG293 cell line genomic DNA in the double-cos vector sCos-1 was screened as follows: The published human αi_a receptor cDNA clone (Bruno et al., BBRC. 179:1485-1490 (1991), and see Fig. 10, SEQ.ID:13:) was cloned into the vector pcDNAl neo to generate the clone pEXαla. Filters containing approximately 200,000 clones were screened by colony hybridization ([Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, 1989 ]) using a mixed exon 1 probe generated by PCR corresponding to αi_a (TMD1-3) , αlb (TMD1-5) and αic (TMD1-5): 25 cycles of 95° C 1'; 52 °C 30 sec; 72 °C 1.5' using 10 ng of pEX αlb, pEX αic or pEX αla and 10 pmoles each of primers 5' MET (5' GAATCCCGACCTGGAC ), SEQ.ID:31:, and 3' BAM (5'GGATCCTCAGGGTC ), SEQ.ID:32:, for αlb, 5' 597 (5' CCATGGTGTTTCTCTCGGG), SEQ.ID:33: and 3' 1219 (5' GACGCGGCAGTACATGAC ), SEQ.ID:34: for αic or 5^* 76 (5^* GTCATGATGGCTGGGTACTTG ), SEQ.ID:35:, for αla in a 12 μl reaction containing 1.5 μM each unlabelled dNTP and 50 μCi 3000 Ci/mmol α-[³²P] dCTP. The filters were incubated with 1 x 10⁶ cpm/ml of probe in 5X SSC, 35% Formamide, 0.02% SDS, 0.1 % lauroyl sarcosine, 2% blocking buffer (Bohrenger Mannheim), at 42 °C for 18 hours. The filters were washed with 2 liters of 0.5X SSC, 0.1% SDS, 55 °C and exposed to Kodak XAR-5 film. Twelve primary positives were picked from master plates and re-screened using the αia- specific probe. Cosmid DNA was prepared from second round positive clones, digested with endonucleases Eco RI or Hind HI and subjected to Southern blot analysis: Fragments were resolved by electrophoresis, and transferred to a nitrocellulose membrane (Bohrenger Mannheim) with 20X SSC (IX SSC = 0.15M Sodium chloride, 0.015M Sodium citrate, pH 7.0) according to the method of Southern ([Southern, 1975 #14]) . The membrane was hybridized, washed and analyzed as described above. Alpha- la, αlb, and αic receptor clones were identified by comparison of restriction patterns with genomic southern blots performed with αla, <*lb, or αic specific probes. A Cosmid containing αla receptor exon 1 DNA was subjected to restriction digestion by endonuclease Pst I and subjected to southern blot analysis as above using the αla -specific probe. Two fragments of 2.3 and 1.6 kb were detected and subcloned into the Pst I site of PGEM 3ZF . The presence of the correct 5' terminal sequences in the 2.3 kb fragment was confirmed by sequencing across the junction between inverted repeat and non-repeat sequences. The 5' end of the αia receptor gene was ligated to the cDNA clone at their common Pstl site, see figures 22-24, SEQ.ID:29:, and SEQ.ID:30:.

EXAMPLE 1?

EXEMPLARY COUNTERSCREENS

1. Assay Title: Dopamine D2,D4 in vitro screen

Objective of the Assav:

The objective of this assay is to eliminate agents which specifically affect binding of [3H] spiperone to cells expressing human dopamine receptors D2, D3 or D4. Method;

Modified from VanTol et al (1991); Nature (Vol 350) Pg

610-613.

Frozen pellets containing specific dopamine receptor subtypes stably expressed^' in clonal cell lines are lysed in 2 ml lysing buffer (lOmM Tris-HCl/5mM Mg, pH 7.4). Pellets obtained after centrifuging these membranes (15' at 24,450 φm) are resuspended in 50mM Tris-HCl pH 7.4 containing EDTA, MgCl[2], KCl, NaCl, CaCl[2] and ascorbate to give a 1 Mg/mL suspension. The assay is initiated by adding 50-75 μg membranes in a total volume of 500 μl containing 0.2 nM [3H]-spiperone. Non-specific binding is defined using 10 μM apomoφhine. The assay is terminated after a 2 hour incubation at room temperature by rapid filtration over GF/B filters presoaked in 0.3% PEI, using 50mM Tris-HCl pH 7.4.

2. Assay Title: Serotonin 5HTla

Objective of the Assay

The objective of this assay is to eliminate agents which specifically affect binding to cloned human 5HTla receptor

Method:

Modified from Schelegel and Peroutka Biochemical Pharmacology 35: 1943-1949 (1986).

Mammalian cells expressing cloned human 5HTla receptors are lysed in ice-cold 5 mM Tris-HCl , 2 mM EDTA (pH 7.4) and homogenized with a polytron homogenizer. The homogenate is centrifuged at lOOOXg for 30', and then the supernatant is centrifuged again at 38,OOOXg for 30'. The binding assay contains 0.25 nM [3H]8- OH-DPAT in 50 mM Tris-HCl, 4 mM CaC12 and lmg/ml ascorbate. Non-specific binding is defined using 10 μM propranolol. The assay is terminated after a 1 hour incubation at room temperature by rapid filtration over GF/Cfilters EXAMPLE 14

EXEMPLARY FUNCTIONAL ASSAYS

In order to confirm the specificity of compounds for the human alphalC adrenergic receptor and to define the biological activity of the compounds, the following functional tests may be performed:

1. IN VITRO RAT, DOG AND HUMAN PROSTATE AND DOG URETHRA

Taconic Farms Sprague-Dawley male rats, weighing 250- 400 grams are sacrificed by cervical dislocation under anesthesia (methohexital; 50 mg/kg, i.p.). An incision is made into the lower abdomen to remove the ventral lobes of the prostate. Each prostate removed from a mongrel dog is cut into 6-8 pieces longitudinally along the urethra opening and stored in ice-cold oxygenated Krebs solution overnight before use if necessary. Dog urethra proximal to prostate is cut into approximately 5 mm rings, the rings are then cut open for contractile measurement of circular muscles. Human prostate chips from transurethral surgery of benign prostate hypeφlasia are also stored overnight in ice-cold Krebs solution if needed.

The tissue is placed in a Petri dish containing oxygenated Krebs solution [NaCl, 118 mM; KCl, 4.7 mM; CaCl2, 2.5 mM; KH2PO4, 1.2 mM; MgS04, 1.2 mM; NaHCθ3, 2.0 mM; dextrose, 11 mM] warmed to 37°C. Excess lipid material and connective tissue are carefully removed. Tissue segments are attached to glass tissue holders with 4-0 surgical silk and placed in a 5 ml jacketed tissue bath containing Krebs buffer at 37°C, bubbled with 5% Cθ2/95% 02- The tissues are connected to a Statham-Gould force transducer; 1 gram (rat, human) or 1.5 gram (dog) of tension is applied and the tissues are allowed to equilibrate for one hour. Contractions are recorded on a Hewlett-Packard 7700 series strip chart recorder.

After a single priming dose of 3 μM (for rat), 10 μM (for dog) and 20 μM (for human) of phenylephrine, a cumulative concentration response curve to an agonist is generated; the tissues are washed every 10 minutes for one hour. Vehicle or antagonist is added to the bath and allowed to incubate for one hour, then another cumulative concentration response curve to the agonist is generated.

EC50 values are calculated for each group using GraphPad Inplot software. pA2 (-log Kb) values were obtained from Schild plot when three or more concentrations were tested. When less than three concentrations of antagonist are tested, Kb values are calculated according to the following formula Kb = TB1. x-1 where x is the ratio of EC50 of agonist in the presence and absence of antagonist and [B] is the antagonist concentration.

2. MEASUREMENT OE TNT A-TTRETHRAL PRESSURE TN

ANESTHETIZED OGS

PURPOSE: Benign prostatic hypeφlasia causes decreased urine flow rate that may be produced by both passive physical obstruction of the prostatic urethra from increased prostate mass as well as active obstruction due to prostatic contraction. Alpha adrenergic receptor antagonists such as prazosin and terazosin prevent active prostatic contraction, thus improve urine flow rate and provide symptomatic relief in man. However, these are non-selective alpha- 1 receptor antagonists which also have pronounced vascular effects. Because we have identified the alpha- IC receptor subtype as the predominent subtype in the human prostate, it is now possible to specifically target this receptor to inhibit prostatic contraction without concomitant changes in the vasculature. The following model is used to measure adrenergically mediated changes in intra-urethral pressure and arterial pressure in anesthetized dogs in order to evaluate the efficacy and potency of selective alpha adrenergic receptor antagonists. The goals are to: 1) identify the alpha- 1 receptor subtypes responsible for prostatic/urethral contraction and vascular responses, and 2) use this model to evaluate novel selective alpha adrenergic antagonists. Novel and standard alpha adrenergic antagonists may be evaluated in this manner.

METHODS: Male mongrel dogs (7-12 kg) are used in this study.

The dogs are anesthetized with pentobarbital sodium (35 mg/kg, i.v. plus 4 mg/kg/hr iv infusion). An endotracheal tube is inserted and the animal ventilated with room air using a Harvard instruments positive displacement large animal ventilator. Catheters (PE 240 or 260) are placed in the aorta via the femoral artery and vena cava via the femoral veins (2 catheters, one in each vein) for the measurement of arterial pressure and the administration of drugs, respectively. A supra-pubic incision ~l/2 inch lateral to the penis is made to expose the urethers, bladder and urethra. The urethers are ligated and cannulated so that urine flows freely into beakers. The dome of the bladder is retracted to facilitate dissection of the proximal and distal urethra. Umbilical tape is passed beneath the urethra at the bladder neck and another piece of umbilical tape is placed under the distal urethra approximately 1-2 cm distal to the prostate. The bladder is incised and a Millar micro-tip pressure transducer is advanced into the urethra. The bladder incision is sutured with 2-0 or 3-0 silk (purse-string suture) to hold the transducer. The tip of the transducer is placed in the prostatic urethra and the position of the Millar catheter is verified by gently squeezing the prostate and noting the large change in urethral pressure. Phenylephrine, an alpha- 1 adrenergic agonist, is administered (0.1-100 ug/kg, iv; 0.05 ml/kg volume) in order to construct dose response curves for changes in intra-urethral and arterial pressure. Following administration of increasing doses of an alpha adrenergic antagonist (or vehicle), the effects of phenylephrine on arterial pressure and intra-urethral pressure are re-evaluated. Four or five phenylephrine dose-response curves are generated in each animal (one control, three or four doses of antagonist or vehicle). The relative antagonist potency on phenylephrine induced changes in arterial and intra-urethral pressure are determined by Schild analysis. The family of averaged curves are fit simultaneously (using ALLFIT software package) with a four paramenter logistic equation constraining the slope, minimum response, and maximum response to be constant among curves. The dose ratios for the antagonist doses (rightward shift in the dose-response curves from control) are calculated as the ratio of the ED50's for the respective curves. These dose-ratios are then used to construct a Schild plot and the Kb (expressed as ug/kg, iv) determined. The Kb (dose of antagonist causing a 2-fold rightward shift of the phenylephrine dose-response curve) is used to compare the relative potency of the antagonists on inhibiting phenylephrine responses for intra-urethral and arterial pressure. The relative selectivity is calculated as the ratio of arterial pressure and intra-urethral pressure Kb's. Effects of the alpha- 1 antagonists on baseline arterial pressure are also monitored. Comparison of the relative antagonist potency on changes in arterial pressure and intra-urethral pressure provide insight as to whether the alpha receptor subtype responsible for increasing intra-urethral pressure is also present in the systemic vasculature. According to this method, one is able to confirm the selectivity of alphalC adrenergic receptor antagonists that prevent the increase in intra-urethral pressure to phenylephrine without any activity at the vasculature.

At the end of the experiment, the dogs are killed via an overdose of intravenously administered pentobarbital or saturated KCl.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bayne, Marvin L

Clineschmidt, Bradley V Strader, Catherine D

(ii) TITLE OF INλENTION: CLONED HUMAN ALPHAIC ADRENERGIC RECEPTOR

(iii) NUMBER OF SEQUENCES: 35

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Merck & Co., Inc.

(B) STREET: 126 Lincoln Avenue (C) CITY: Rahway

(D) STATE: New Jersey

(E) COUNTRY: United States of America

(F) ZIP: 07065

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOΞ/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/032,849 (B) FILING DATE: 15-MAR-1993

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bencen, Gerard H

(B) REGISTRATION NUMBER: 35,746

(C) REFERENCE/DOCKET NUMBER: 189 3IA

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (908)594-3901 (B) TELEFAX: (908)594-4720

(C) TELEX: 138825

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTTTCTAGAT TRTTNARRTA NCCNAGCC 28

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TTTACTAGTA TCSTNGTNAT GTAYTG 26 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TTTTCTAGAG AARAANGGNA RCCARC 26

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 235 base pairs

(B) TYPE: nucleic acid.

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC CTCAAGλCCG 60

ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC CCGGCAGGAG 120

GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC CTCAAGTTCT 180

CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGCTGCTTC GTCCT 235 (2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly 1 5 10 15

Leu Lys Thr Asp Lys Ser Asp Ser Glu Gin Val Thr Leu Arg lie His 20 25 30

Arg Lys Asn Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr 35 40 45

Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys 50 55 60

Ala Ala Lys Thr Leu Gly lie Val Val Gly Cys Phe Val Leu 65 70 75

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal ( i) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Leu Val Met Tyr Cys Arg Val Tyr Val Val Ala Lys Arg Glu Ser Arg 1 5 10 15

Gly Leu Lys Ser Gly Leu Lys Thr Asp Lys Ser Asp Ser Glu Gin Val 20 25 30

Thr Leu Arg lie His Arg Lys Asn Ala Gin Val Gly Gly Ser Gly Val 35 40 45

Thr Ser Ala Lys Asn Lys Thr His Phe Ser Val Arg Leu Leu Lys Phe 50 55 60

Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Gly He Val Val Gly Cys 65 70 75 80

Phe Val Leu Cys Trp Leu Pro Phe Phe Leu Val Met Pro 85 90

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1601 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60

GTAACTGCGG GCCAGGAGCA GCGCCCAGAT GCCATCGGTC CCTGCCTTTG AGCGTCGACG 120

GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG AATCATGTGC 180

AGAATCGTGA ATCTTCCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240

TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG CTTCGGGTAC 300

GGGAGGGAGT CCGGGTCCCG GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360

CGCGCGCCCC TGGCCATGTC TTTAATGCCC TGCCCCTTCA TGTGGCCTTC TGAGGGTTCC 420

CAGGGCTGGC CAGGGTTGTC TCCCACCCGC GCGCGCCGTC TCACCCCCAG CCAAACCCAC 480

CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG CTCCCGCGCT CCCGCCTCCG 540

CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC CGGCCCCGGC TGGGACCATG 600

GTGTTTCTCT CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660

AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720 CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC CACTACTACA TCGTCAACCT GGCGGTGGCC GACCTCCTGC TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC TGGGCCTTCG TGGGCGGCAG TGGATGTGCT GTGCTGCACC GCGTCCATCA ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC AGGGGTCTCA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC CTCTTCGGCT GGAGGCAGCC GGCCCCCGAG GACGAGACCA CCGGGCTACG TGCTCTTCTC GGCTCTGGGC TCCTTCTACC GTCATGTACT GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA CTCAAGACCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC CCGGCAGGAG GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GTCCTCTGCT GGCTGCCTTT TTTCTTAGTC ATGCCCATTG AAGCCCTCTG AAACAGTTTT TAAAATAGTA TTTTGGCTCG AACCCCATCA TATACCCATG CTCCAGCCAA GAGGGAATTC (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTTGAATTCT GATTTCAAGC CCTCTG 26 (2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTTGAATTCT TANACYTCYT CNCCRTTYTC 30 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 512 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CTGATTTCAA GCCCTCTGAA ACAGTTTTTA AAATAGTATT GCTGCATCAA CCCCATCATA TACCCATGCT CCAGCCAAGA ATGTCTTGAG AATCCAGTGT CTCCGCAGAA AGCAGTCTTC CCCTGCACCC GCCCAGCCAG GCCGTGGAAG GGCAACACAA TGGGATCAAG AGAGACCTTC TACAGGATCT CCAAGACGGA TTTTCTCTTC CATGCCCCGT GGATCTGCCA GGATTACAGT GTACCACAGC CCGGGTGAGA AGTAAAAGCT TTTTGCAGGT CAACCCCCAG CCTTGACAAG AACCATCAAG TTCCAACCAT TCAGTGAGAA CGGCGAAGAG GTTTAAGAAT TC (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2004 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60

GTAACTGCGG GCCAGGAGCA GCGCCCAGAT GCCATCGGTC CCTGCCTTTG AGCGTCGACG 120

GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG AATCATGTGC 180

AGAATCGTGA ATCTTCCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240

TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG CTTCGGGTAC 300

GGGAGGGAGT CCGGGTCCCG GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360

CGCGCGCCCC TGGCCATGTC TTTAATGCCC TGCCCCTTCA TGTGGCCTTC TGAGGGTTCC 420

CAGGGCTGGC CAGGGTTGTC TCCCACCCGC GCGCGCCGTC TCACCCCCAG CCAAACCCAC 480

CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG CTCCCGCGCT CCCGCCTCCG 540

CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC CGGCCCCGGC TGGGACCATG 600

GTGTTTCTCT CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660

AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720

CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC GACACCTGCA CTCAGTCACG 780

CACTACTACA TCGTCAACCT GGCGGTGGCC GACCTCCTGC TCACCTCCAC GGTGCTGCCC 840

TTCTCCGCCA TCTTCGAGGT CCTAGGCTAC TGGGCCTTCG GCAGGGTCTT CTGCAACATC 900

TGGGCGGCAG TGGATGTGCT GTGCTGCACC GCGTCCATCA TGGGCCTCTG CATCATCTCC 960

ATCGACCGCT ACATCGGCGT GAGCTACCCG CTGCGCTACC CAACCATCGT CACCCAGAGG 1020 AGGGGTCTCA TGGCTCTGCT CTGCGTCTGG GCACTCTCCC TGGTCATATC CATTGGACCC 1080

CTCTTCGGCT GGAGGCAGCC GGCCCCCGAG GACGAGACCA TCTGCCAGAT CAACGAGGAG 1140

CCGGGCTACG TGCTCTTCTC GGCTCTGGGC TCCTTCTACC TGCCTCTGGC CATCATCCTG 1200

GTCATGTACT GCCGCGTCTA CGTGGTGGCC AAGAGGGAGA GCCGGGGCCT CAAGTCTGGC 1260

CTCAAGACCG ACAAGTCGGA CTCGGAGCAA GTGACGCTCC GCATCCATCG GAAAAACGCC 1320

CCGGCAGGAG GCAGCGGGAT GGCCAGCGCC AAGACCAAGA CGCACTTCTC AGTGAGGCTC 1380

CTCAAGTTCT CCCGGGAGAA GAAAGCGGCC AAAACGCTGG GCATCGTGGT CGGCTGCTTC 1440

GTCCTCTGCT GGCTGCCTTT TTTCTTAGTC ATGCCCATTG GGTCTTTCTT CCCTGATTTC 1500

AAGCCCTCTG AAACAGTTTT TAAAATAGTA TTTTGGCTCG GATATCTAAA CAGCTGCATC 1560

AACCCCATCA TATACCCATG CTCCAGCCAA GAGTTCAAAA AGGCCTTTCA GAATGTCTTG 1620

AGAATCCAGT GTCTCCGCAG AAAGCAGTCT TCCAAACATG CCCTGGGCTA CACCCTGCAC 1680

CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACATGG TGCGCATCCC CGTGGGATCA 1740

AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GTGAATGGAA ATTTTTCTCT 1800

TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC CTGTACCACA 1860 GCCCGGGTGA GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC CTCAACCCCC 1920

AGCCTTGACA AGAACCATCA AGTTCCAACC ATTAAGGTCC ACACCATCTC CCTCAGTGAG 1980

AACGGCGAAG AGGTTTAAGA ATTC 2004 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 466 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gin 1 5 10 15

Pro Pro Ala Pro Val Asn He Ser Lys Ala He Leu Leu Gly Val He 20 25 30

Leu Gly Gly Leu He Leu Phe Gly Val Leu Gly Asn He Leu Val He 35 40 45

Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60

He Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80

Pro Phe Ser Ala He Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95

Val Phe Cys Asn He Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110

Ser He Met Gly Leu Cys He He Ser He Asp Arg Tyr He Gly Val 115 120 125

Ser Tyr Pro Leu Arg Tyr Pro Thr He Val Thr Gin Arg Arg Gly Leu 130 135 140

Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val He Ser He Gly 145 150 155 160

Pro Leu Phe Gly Trp Arg Gin Pro Ala Pro Glu Asp Glu Thr He Cys 165 170 175

Gin He Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180 185 190 Phe Tyr Leu Pro Leu Ala He He Leu Val Met Tyr Cys Arg Va] Tyr 195 200 205

Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215 220

Asp Lys Ser Asp Ser Glu Gin Val Thr Leu Arg He His Arg Lys Asn 225 230 235 240

Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His 245 250 255

Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270

Thr Leu Gly He Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 280 285

Phe Leu Val Met Pro He Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290 295 300

Glu Thr Val Phe Lys He Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320

He Asn Pro He He Tyr Pro Cys Ser Ser Gin Glu Phe Lys Lys Ala 325 330 335

Phe Gin Asn Val Leu Arg He Gin Cys Leu Arg Arg Lys Gin Ser Ser 340 345 350

Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gin Ala Val Glu 355 360 365

Gly Gin His Lys Asp Met Val Arg He Pro Val Gly Ser Arg Glu Thr ³⁷⁰ 375 380

Phe Tyr Arg He Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 395 400

Ser Ser Met Pro Arg Gly Ser Ala Arg He Thr Val Ser Lys Asp Gin 405 410 415

Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gin Val 420 425 430

Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gin 435 440 445

Val Pro Thr He Lys Val His Thr He Ser Leu Ser Glu Asn Gly Glu 450 455 460

Glu Val ⁴⁶⁵

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1621 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CCCGTGCAGG GGCCCTACGG ACACCACCAG GGCTACGACC CAGAGCAGGG CCAGGATGGC 60

GGCCGCCTTG CGCTCGGTCA TGATGGCTGG GTACTTGAGT GAGTGGCGCA CGCCCACGTA 120

CCGGTCCACG GAGATGGTGC AGAGGCTGAG GATGGAGGCC GTGCAGCACA GCACGTCCAC 180 GGCGGCCGTC GGGGGACTGG TGGTGAGCGC GCAGGGCGTG GGCGTGGGCG TCTTCCTGGC 240

AGCCTTCATC CTTATGGCCG TGGCAGGTAA CCTGCTTGTC ATCCTCTCAG TGGCCTGCAA 300

CCGCCACCTG CAGACCGTCA CCAACTATTT CATCGTGAAC CTGGCCGTGG CCGACCTGCT 360

GCTGAGCGCC ACCGTACTGC CCTTCTCGGC CACCATGGAG GTTCTGGGCT TCTGGGCCTT 420

TGGCCGCGCC TTCTGCGACG TATGGGCCGC CGTGGACGTG CTGTGCTGCA CGGCCTCCAT 480

CCTCAGCCTC TGCACCATCT CCGTGGACCG GTACGTGGGC GTGCGCCACT CACTCAAGTA 540

CCCAGCCATC ATGACCGAGC GCAAGGCGGC CGCCATCCTG GCCCTGCTCT GGGTCGTAGC 600

CCTGGTGGTG TCCGTAGGGC CCCTGCTGGG CTGGAAGGAG CCCGTGCCCC CTGACGAGCG 660

CTTCTGCGGT ATCACCGAGG AGGCGGGCTA CGCTGTCTTC TCCTCCGTGT GCTCCTTCTA 720 CCTGCCCATG GCGGTCATCG TGGTCATGTA CTGCCGCGTG TACGTGGTCG CGCGCAGCAC 780

CACGCGCAGC CTCGAGGCAG GCGTCAAGCG CGAGCGAGGC AAGGCCTCCG AGGTGGTGCT 840

GCGCATCCAC TGTCGCGGCG CGGCCACGGG CGCCGACGGG GCGCACGGCA TGCGCAGCGC 900

CAAGGGCCAC ACCTTCCGCA GCTCGCTCTC CGTGCGCCTG CTCAAGTTCT CCCGTGAGAA 960

GAAAGCGGCC AAGACTCTGG CCATCGTCGT GGGTGTCTTC GTGCTCTGCT GGTTCCCTTT 1020

CTTCTTTGTC CTGCCGCTCG GCTCCTTGTT CCCGCAGCTG AAGCCATCGG AGGGCGTCTT 1080

CAAGGTCATC TTCTGGCTCG GCTACTTCAA CAGCTGCGTG AACCCGCTCA TCTACCCCTG 1140

TTCCAGCCGC GAGTTCAAGC GCGCCTTCCT CCGTCTCCTG CGCTGCCAGT GCCGTCGTCG 1200

CCGGCGCCGC CGCCCTCTCT GGCGTGTCTA CGGCCACCAC TGGCGGGCCT CCACCAGCGG 1260 CCTGCGCCAG GACTGCGCCC CGAGTTCGGG CGACGCGCCC CCCGGAGCGC CGCTGGCCCT 1320

CACCGCGCTC CCCGACCCCG ACCCCGAACC CCCAGGCACG CCCGAGATGC AGGCTCCGGT 1380

CGCCAGCCGT CGAAGCCACC CAGCGCCTTC CGCGAGTGGA GGCTGCTGGG GCCGTTCCGG 1440

AGACCCACGA CCCAGCTGCG CGCCAAAGTC GCCAGCCTGT CGCACAAGAT CGCCGCCGGG 1500

GGCGCGCAGC GCGCAGAGGC AGCGTGCGCC CAGCGCTCAG AGGTGGAGGC TGTGTCCCTA 1560 GGCGTCCCAC ACGAGGTGGC CGAGGGCGCC ACCTGCCAGG CCTACGAATT GGCCGACTAC 1620

A 1621

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 501 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Ala Ala Ala Leu Arg Ser Val Met Met Ala Gly Tyr Leu Ser Glu 1 5 10 15

Trp Arg Thr Pro Thr Tyr Arg Ser Thr Glu Met Val Gin Arg Leu Arg 20 25 30

Met Glu Ala Val Gin His Ser Thr Ser Thr Ala Ala Val Gly Gly Leu 35 40 45

Val Val Ser Ala Gin Gly Val Gly Val Gly Val Phe Leu Ala Ala Phe ^{50 55} 60

He Leu Met Ala Val Ala Gly Asn Leu Leu Val He Leu Ser Val Ala 65 70 75 80

Cys Asn Arg His Leu Gin Thr Val Thr Asn Tyr Phe He Val Asn Leu 85 90 95

Ala Val Ala Asp Leu Leu Leu Ser Ala Thr Val Leu Pro Phe Ser Ala 100 105 110

Thr Met Glu Val Leu Gly Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp 115 120 125

Val Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala Ser He Leu Ser 130 135 140

Leu Cys Thr He Ser Val Asp Arg Tyr Val Gly Val Arg His Ser Leu 145 150 155 160

Lys Tyr Pro Ala He Met Thr Glu Arg Lys Ala Ala Ala He Leu Ala 165 170 175

Leu Leu Trp Val Val Ala Leu Val Val Ser Val Gly Pro Leu Leu Gly 180 185 190

Trp Lys Glu Pro Val Pro Pro Asp Glu Arg Phe Cys Gly He Thr Glu 195 200 205

Glu Ala Gly Tyr Ala Val Phe Ser Ser Val Cys Ser Phe Tyr Leu Pr- 210 215 220

Met Ala Val He Val Val Met Tyr Cys Arg Val Tyr Val Val Ala Arg 225 230 235 240 Ser Thr Thr Arg Ser Leu Glu Ala Gly Val Lys Arg Glu Arg Gly Lys

245 250 255

Ala Ser Glu Val Val Leu Arg He His Cys Arg Gly Ala Ala Thr Gly 260 265 270

Ala Asp Gly Ala His Gly Met Arg Ser Ala Lys Gly His Thr Phe Arg 275 280 285 Ser Ser Leu Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala 290 295 300

Ala Lys Thr Leu Ala He Val Val Gly Val Phe Val Leu Cys Trp Phe 305 310 315 320

Pro Phe Phe Phe Val Leu Pro Leu Gly Ser Leu Phe Pro Gin Leu Lys 325 330 335 Pro Ser Glu Gly Val Phe Lys Val He Phe Trp Leu Gly Tyr Phe Asn

340 345 350

Ser Cys Val Asn Pro Leu He Tyr Pro Cys Ser Ser Arg Glu Phe Lys 355 360 365

Arg Ala Phe Leu Arg Leu Leu Arg Cys Gin Cys Arg Arg Arg Arg Arg 370 375 380 ^Ar9 ^Ar9 ^{Pro Leu Tr}P ^Ar9 ^Val 1Y^{r G}ly His ^Hi^s Trp Arg Ala Ser Thr 385 390 395 400

Ser Gly Leu Arg Gin Asp Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro 405 410 415

Gly Ala Pro Leu Ala Leu Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro 420 425 430 Pro Gly Thr Pro Glu Met Gin Ala Pro Val Ala Ser Arg Arg Ser His 435 440 445

Pro Ala Pro Ser Ala Ser Gly Gly Cys Trp Gly Arg Ser Gly Asp Pro 450 455 460

Arg Pro Ser Cys Ala Pro Lys Ser Pro Ala Cys Arg Thr Arg Ser Pro 465 470 475 480 Pro Gly Ala Arg Ser Ala Gin Arg Gin Arg Ala Pro Ser Ala Gin Arg

485 490 495

Trp Arg Leu Cys Pro 500

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TCTAGACCAT GAAYCCNGAY CTGG 24 (2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TTTGAATTCA CATWCCGACY ACAATGCCC 29

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 921 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

( i) SEQUENCE DESCRIPTION: SEQ ID NO:17:

TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG 60

GAGAGTTGAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC 120

CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GCTGGGCGCC TTCATCCTCT 180

TTGCCATCGT GGGCAACATC CTAGTCATCT TGTCTGTGGC CTGCAACCGG CACCTGCGGA 240 CGCCCACCAA CTACTTCATT GTCAACCTGG CCATGGCCGA TCCTGCCCTT CTCAGCGGCC CTAGAGGTGC TCGGCTACTG GTGACATCTG GGCAGCCGTG GATGTCCTGT GCTGCACAGC CCATCTCCAT CGATCGCTAC ATCGGGGTGC GCTACTCTCT CCCGGAGGAA GGCCATCTTG GCCCTGCTCA GTGTCTGGGT TCGGGCCTCT CCTTGGGTGG AAGGAGCCGG CACCCAACGA CCGAAGAACC CTTCTATGCC CTCTTCTCCT CTCTGGGCTC TCATTCTAGT CATGTACTGC CGTGTCTATA TAGTGGCCAA AGGCAGGAGT CATGAAGGAG ATGTCCAACT CCAAGGAGCT AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA TTGTGGTCGG TATGTGAATT C (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAAGGCGCGC TTGAACTC (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGAGAACCAC CAAGAACC (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 389 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA ^' (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

AAGAGAACCA CCAAGAACCT AGAGGCAGGA GTCATGAAGG AGATGTCCAA CTCCAAGGAG 60

CTGACCCTGA GGATCCATTC CAAGAACTTT CACGAGGACA CCCTTAGCAG TACCAAGGCC 120

AAGGGCCACA ACCCCAGGAG TTCCATAGCT GTCAAACTTT TTAAGTTCTC CAGGGAAAAG 180

AAAGCAGCTA AGACGTTGGG CATTGTGGTC GGTATGTTCA TCTTGTGCTG GCTACCCTTC 240

TTCATCGCTC TACCGCTTGG CTCCTTGTTC TCCACCCTGA AGCCCCCCGA CGCCGTGTTC 300

AAGGTGGTGT TCTGGCTGGG CTACTTCAAC AGCTGCCTCA ACCCCATCAT CTACCCATGC 360

TCCAGCAAGG AGTTCAAGCG CGCTTTCGT 389 (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TTTGAATTCA TGTTCAAGGT GGTGTTC 27

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TTTGAATTCT AAAASTGNCC NGGNSCCAGN GGCAT 35

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 582 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GAATTCATGA TTCAAGGTGG TGTTCTGGCT GGGCTACTTC CATCTACCCG TGCTCCAGCA AGGAGTTCAA GCGCGCCTTC GTGCCGCGGT GGCCGCCGCC GCCGCCGCCG TCGCCGTCTA CCGGCCGTGG ACCCGCGGCG GCTCGCTGGA GAGATCACAG TGACAGCGGC AGCTGCATGA GCGGCCAGAA GAGGACCCTG GGGCTACCTG GGTCGAGGAA CGCAGCCACC CGTGGAGCTG ACCCGGGGCG CTGCTCAGCT TGCCAGAGCC TCCTGGCCGC GCCACTCTTC ACCTTCAAGC TCCTGGGCGA TCCTGAGAGC CAGCAACGGG GGCTGCGACA CCACGACCGA CCTGGCCAAC CAACATGCCC CTGGGCCCGG GCCACTTTTA AAAGCCGAAT (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1567 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

TCTAGACCAT GAATCCCGAC CTGGACACCG GCCACAACAC ATCAGCACCT GCCCACTGGG 60

GAGAGTTGAA AAATGCCAAC TTCACTGGCC CCAACCAGAC CTCGAGCAAC TCCACACTGC 120

CCCAGCTGGA CATCACCAGG GCCATCTCTG TGGGCCTGGT GCTGGGCGCC TTCATCCTCT 180

TTGCCATCGT GGGCAACATC CTAGTCATCT TGTCTGTGGC CTGCAACCGG CACCTGCGGA 240

CGCCCACCAA CTACTTCATT GTCAACCTGG CCATGGCCGA CCTGCTGTTG AGCTTCACCG 300

TCCTGCCCTT CTCAGCGGCC CTAGAGGTGC TCGGCTACTG GGTGCTGGGG CGGATCTTCT 360

GTGACATCTG GGCAGCCGTG GATGTCCTGT GCTGCACAGC GTCCATTCTG AGCCTGTGCG 420

CCATCTCCAT CGATCGCTAC ATCGGGGTGC GCTACTCTCT GCAGTATCCC ACGCTGGTCA 480

CCCGGAGGAA GGCCATCTTG GCCCTGCTCA GTGTCTGGGT CTTGTCCACC GTCATCTCCA 540

TCGGGCCTCT CCTTGGGTGG AAGGAGCCGG CACCCAACGA TGACAAGGAG TGCGGGGTCA 600

CCGAAGAACC CTTCTATGCC CTCTTCTCCT CTCTGGGCTC CTTCTACATC CCTCTGGCGG 660

TCATTCTAGT CATGTACTGC CGTGTCTATA TAGTGGCCAA GAGAACCACC AAGAACCTAG 720

AGGCAGGAGT CATGAAGGAG ATGTCCAACT CCAAGGAGCT GACCCTGAGG ATCCATTCCA 780

AGAACTTTCA CGAGGACACC CTTAGCAGTA CCAAGGCCAA GGGCCACAAC CCCAGGAGTT 840 CCATAGCTGT CAAACTTTTT AAGTTCTCCA GGGAAAAGAA AGCAGCTAAG ACGTTGGGCA 900

TTGTGGTCGG TATGTTCATC TTGTGCTGGC TACCCTTCTT CATCGCTCTA CCGCTTGGCT 960

CCTTGTTCTC CACCCTGAAG CCCCCCGACG CCGTGTTCAA GGTGGTGTTC TGGCTGGGCT 1020

ACTTCAACAG CTGCCTCAAC CCCATCATCT ACCCATGCTC CAGCAAGGAG TTCAAGCGCG 1080

CCTTCATGCG TATCCTTGGG TGCCAGTGCC GCGGTGGCCG CCGCCGCCGC CGCCGTCGCC 1140

GTCTAGGCGC GTGCGCTTAC ACCTACCGGC CGTGGACCCG CGGCGGCTCG CTGGAGAGAT 1200

CACAGTCGCG GAAGGACTCT CTGGATGACA GCGGCAGCTG CATGAGCGGC CAGAAGAGGA 1260

CCCTGCCCTC GGCGTCGCCC AGCCCGGGCT ACCTGGGTCG AGGAACGCAG CCACCCGTGG 1320

AGCTGTGCGC CTTCCCCGAG TGGAAACCCG GGGCGCTGCT CAGCTTGCCA GAGCCTCCTG 1380

GCCGCCGCGG CCGTCTCGAC TCTGGGCCAC TCTTCACCTT CAAGCTCCTG GGCGATCCTG 1440

AGAGCCCGGG AACCGAAGCG ACAGCCAGCA ACGGGGGCTG CGACACCACG ACCGACCTGG 1500

CCAACGGGCA GCCCGGCTTC AAGAGCAACA TGCCCCTGGG CCCGGGCCAC TTTTAAAAGC 1560

CGAATTC 1567 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 515 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Met Asn Pro Asp Leu Asp Thr Gly His Asn Thr Ser Ala Pro Ala His

1 5 10 15

Trp Gly Glu Leu Lys Asn Ala Asn Phe Thr Gly Pro Asn Gin Thr Ser 20 25 30

Ser Asn Ser Thr Leu Pro Gin Leu Asp He Thr Arg Ala He Ser Val 35 40 45

Gly Leu Val Leu Gly Ala Phe He Leu Phe Ala He Val Gly Asn He 50 55 60

Leu Val He Leu Ser Val Ala Cys Asn Arg His Leu Arg Thr Pro Thr 65 70 75 80

Asn Tyr Phe He Val Asn Leu Ala Met Ala Asp Leu Leu Leu Ser Phe ⁸⁵ 90 95

Thr Val Leu Pro Phe Ser Ala Ala Leu Glu Val Leu Gly Tyr Trp Val 100 105 110

Leu Gly Arg He Phe Cys Asp He Trp Ala Ala Val Asp Val Leu Cys 115 120 125

Cys Thr Ala Ser He Leu Ser Leu Cys Ala He Ser He Asp Arg Tyr 130 135 140

He Gly Val Arg Tyr Ser Leu Gin Tyr Pro Thr Leu Val Thr Arg Arg 145 150 155 160

Lys Ala He Leu Ala Leu Leu Ser Val Trp Val Leu Ser Thr Val He 165 170 175

Ser He Gly Pro Leu Leu Gly Trp Lys Glu Pro Ala Pro Asn Asp Asp 180 185 190

Lys Glu Cys Gly Val Thr Glu Glu Pro Phe Tyr Ala Leu Phe Ser Ser 195 200 205

Leu Gly Ser Phe Tyr He Pro Leu Ala Val He Leu Val Met Tyr Cys 210 215 220

Arg Val Tyr He Val Ala Lys Arg Thr Thr Lys Asn Leu Glu Ala Gly 225 230 235 240

Val Met Lys Glu Met Ser Asn Ser Lys Clu Leu Thr Leu Arg He His 245 250 255

Ser Lys Asn Phe His Glu Asp Thr Leu Ser Ser Thr Lys Ala Lys Gly 260 265 270 His Asn Pro Arg Ser Ser He Ala Val Lys Leu Phe Lys Phe Ser Arg

275 280 285

Glu Lys Lys Ala Ala Lys Thr Leu Gly He Val Val Gly Met Phe He 290 295 300

Leu Cys Trp Leu Pro Phe Phe He Ala Leu Pro Leu Gly Ser Leu Phe 305 310 315 320 Ser Thr Leu Lys Pro Pro Asp Ala Val Phe Lys Val Val Phe Trp Leu

325 330 335

Gly Tyr Phe Asn Ser Cys Leu Asn Pro He He Tyr Pro Cys Ser Ser 340 345 350

Lys Glu Phe Lys Arg Ala Phe Met Arg He Leu Gly Cys Gin Cys Arg 355 360 365 Gly Gly Arg Arg Arg Arg Arg Arg Arg Arg Leu Gly Ala Cys Ala Tyr 370 375 380

Thr Tyr Arg Pro Trp Thr Arg Gly Gly Ser Leu Glu Arg Ser Gin Ser 385 390 395 400

Arg Lys Asp Ser Leu Asp Asp Ser Gly Ser Cys Met Ser Gly Gin Lys 405 410 415 ^Ar9 ^{τhr Leu Pro Ser A}l^{a Ser Pro} Ser ^{Pro G} y Tyr Leu Gly Arg Gly

420 425 430

Thr Gin Pro Pro Val Glu Leu Cys Ala Phe Pro Glu Trp Lys Pro Gly 435 440 445

Ala Leu Leu Ser Leu Pro Glu Pro Pro Gly Arg Arg Gly Arg Leu Asp 450 455 460 Ser Gly Pro Leu Phe Thr Phe Lys Leu Leu Gly Asp Pro Glu Ser Pro 465 470 475 480

Gly Thr Glu Ala Thr Ala Ser Asn Gly Gly Cys Asp Thr Thr Thr Asp 485 490 495

Leu Ala Asn Gly Gin Pro Gly Phe Lys Ser Asn Met Pro Leu Gly Pro 500 505 510 Gly His Phe

515

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1987 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both (D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

GAATTCCCTC CTAGAAGCTG GAGAGAGCAG GAGCCTTCGG TGGGGCAGCT CAAAATGTAG 60

GTAACTGCGG GCCAGGAGCA GCGCCCAGAT GCCATCGGTC CCTGCCTTTG AGCGTCGACG 120

GCTGATCTTT TGGTTTGAGG GAGAGACTGG CGCTGGAGTT TTGAATTCCG AATCATGTGC 180

AGAATCGTGA ATCTTCCCCC AGCCAGGACG AATAAGACAG CGCGGAAAAG CAGATTCTCG 240

TAATTCTGGA ATTGCATGTT GCAAGGAGTC TCCTGGATCT TCGCACCCAG CTTCGGGTAC 300

GGGAGGGAGT CCGGGTCCCG GCTAGGCCAG CCCGCAGGTG GAGAGGGTCC CCGGCAGCCC 360

CGCGCGCCCC TGGCCATGTC TTTAATGCCC TGCCCCTTCA TGTGGCCTTC TGAGGGTTCC 420 CAGGGCTGGC CAGGGTTGTC TCCCACCCGC GCGCGCCGTC TCACCCCCAG CCAAACCCAC 480

CTGGCAGGGC TCCCTCCAGA AGAGACCTTT TGATTCCCGG CTCCCGCGCT CCCGCCTCCG 540

CGCCAGCCCG GGAGGTGGCC CTGGACAGCC GGACCTCGCC CGGCCCCGGC TGGGACCATG 600

GTGTTTCTCT CGGGAAATGC TTCCGACAGC TCCAACTGCA CCCAACCGCC GGCACCGGTG 660

AACATTTCCA AGGCCATTCT GCTCGGGGTG ATCTTGGGGG GCCTCATTCT TTTCGGGGTG 720

CTGGGTAACA TCCTAGTGAT CCTCTCCGTA GCCTGTCACC GACACCTGCA CTCAGTCACG 780

CACTACTACA TCGTCAACCG CTAGTGGCGG TGGCCGACCT CCTGCTCACC TCCACGGTGC 840

TGCCCTTCTC CGCCATCTTC GAGGTCCTAG GCTACTGGGC CTTCGGCAGG GTCTTCTGCA 900

ACATCTGGGC GGCAGTGGAT GTGCTGTGCT GCACCGCGTC CATCATGGGC CTCTGCATCA 960 TCTCCATCGA CCGCTACATC GGCGTGAGCT ACCCGCTGCG CTACCCAACC ATCGTCACCC 1020

AGAGGAGGGG TCTCATGGCT CTGCTCTGCG TCTGGGCACT CTCCCTGGTC ATATCCATTG 1080

GACCCCTCTT CGGCTGGAGG CAGCCGGCCC CCGAGGACGA GACCATCTGC CAGATCAACG 1140

AGGAGCCGGG CTACGTGCTC TTCTCGGCTC TGGGCTCCTT CTACCTGCCT CTGGCCATCA 1200

TCCTGGTCAT GTACTGCCGC GTCTACGTGG TGGCCAAGAG GGAGAGCCGG GGCCTCAAGT 1260

CTGGCCTCAA GACCGACAAG TCGGACTCGG AGCAAGTGAC GCTCCGCATC CATCGGAAAA 1320

ACGCCCCGGC AGGAGGCAGC GGGATGGCCA GCGCCAAGAC CAAGACGCAC TTCTCAGTGA 1380

GGCTCCTCAA GTTCTCCCGG GAGAAGAAAG CGGCCAAAAC GCTGGGCATC GTGGTCGGCT 1440

GCTTCGTCCT CTGCTGGCTG CCTTTTTTCT TAGTCATGCC CATTGGGTCT TTCTTCCCTG 1500 ATTTCAAGCC CTCTGAAACA GTTTTTAAAA TAGTATTTTG GCTCGGATAT CTAAACAGCT 1560

GCATCAACCC CATCATATAC CCATGCTCCA GCCAAGAGTT CAAAAAGGCC TTTCAGAATG 1620

TCTTGAGAAT CCAGTGTCTC CGCAGAAAGC AGTCGCTAGT TCCAAACATG CCCTGGGCTA 1680

CACCCTGCAC CCGCCCAGCC AGGCCGTGGA AGGGCAACAC AAGGACATGG TGCGCATCCC 1740

CGTGGGATCA AGAGAGACCT TCTACAGGAT CTCCAAGACG GATGGCGTTT GTGAATGGAA 1800

ATTTTTCTCT TCCATGCCCC GTGGATCTGC CAGGATTACA GTGTCCAAAG ACCAATCCTC 1860

CTGTACCACA GCCCGGGTGA GAAGTAAAAG CTTTTTGCAG GTCTGCTGCT GTGTAGGGCC 1920

CTCAACCCCC AGCCTTGACA AGAACCATCA AGTTCCAACC ATTAAGGTCC ACACCATCTC 1980

CCTCAGT 1987 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1997 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

AATTCCCTCC TAGAAGCTGG AGAGAGCAGG AGCCTTCGGT GGGGCAGCTC AAAATGTAGG 60

TAACTGCGGG CCAGGAGCAG CGCCCAGATG CCATCGGTCC CTGCCTTTGA GCGTCGACGG 120

CTGATCTTTT GGTTTGAGGG AGAGACTGGC GCTGGAGTTT TGAATTCCGA ATCATGTGCA 180

GAATCGTGAA TCTTCCCCCA GCCAGGACGA ATAAGACAGC GCGGAAAAGC AGATTCTCGT 240

AATTCTGGAA TTGCATGTTG CAAGGAGTCT CCTGGATCTT CGCACCCAGC TTCGGGTACG 300

GGAGGGAGTC CGGGTCCCGG CTAGGCCAGC CCGCAGGTGG AGAGGGTCCC CGGCAGCCCC 360

GCGCGCCCCT GGCCATGTCT TTAATGCCCT GCCCCTTCAT GTGGCCTTCT GAGGGTTCCC 420

AGGGCTGGCC AGGGTTGTCT CCCACCCGCG CGCGCCGTCT CACCCCCAGC CAAACCCACC 480

TGGCAGGGCT CCCTCCAGAA GAGACCTTTT GATTCCCGGC TCCCGCGCTC CCGCCTCCGC 540

GCCAGCCCGG GAGGTGGCCC TGGACAGCCG GACCTCGCCC GGCCCCGGCT GGGACCATGG 600

TGTTTCTCTC GGGAAATGCT TCCGACAGCT CCAACTGCAC CCAACCGCCG GCACCGGTGA 660

ACATTTCCAA GGCCATTCTG CTCGGGGTGA TCTTGGGGGG CCTCATTCTT TTCGGGGTGC 720

TGGGTAACAT CCTAGTGATC CTCTCCGTAG CCTGTCACCG ACACCTGCAC TCAGTCACGC 780 ACTACTACAT CGTCAACCTG GCGGTGGCCG ACCTCCTGCT CACCTCCACG GTGCTGCCCT 840

TCTCCGCCAT CTTCGAGGTC CTAGGCTACT GGGCCTTCGG CAGGGTCTTC TGCAACATCT 900

GGGCGGCAGT GGATGTGCTG TGCTGCACCG CGTCCATCAT GGGCCTCTGC ATCATCTCCA 960

TCGACCGCTA CATCGGCGTG AGCTACCCGC TGCGCTACCC AACCATCGTC ACCCAGAGGA 1020

GGGGTCTCAT GGCTCTGCTC TGCGTCTGGG CACTCTCCCT GGTCATATCC ATTGGACCCC 1080

TCTTCGGCTG GAGGCAGCCG GCCCCCGAGG ACGAGACCAT CTGCCAGATC AACGAGGAGC 1140

CGGGCTACGT GCTCTTCTCG GCTCTGGGCT CCTTCTACCT GCCTCTGGCC ATCATCCTGG 1200

TCATGTACTG CCGCGTCTAC GTGGTGGCCA AGAGGGAGAG CCGGGGCCTC AAGTCTGGCC 1260

TCAAGACCGA CAAGTCGGAC TCGGAGCAAG TGACGCTCCG CATCCATCGG AAAAACGCCC 1320

CGGCAGGAGG CAGCGGGATG GCCAGCGCCA AGACCAAGAC GCACTTCTCA GTGAGGCTCC 1380

TCAAGTTCTC CCGGGAGAAG AAAGCGGCCA AAACGCTGGG CATCGTGGTC GGCTGCTTCG 1440

TCCTCTGCTG GCTGCCTTTT TTCTTAGTCA TGCCCATTGG GTCTTTCTTC CCTGATTTCA 1500

AGCCCTCTGA AACAGTTTTT AAAATAGTAT TTTGGCTCGG ATATCTAAAC AGCTGCATCA 1560 ACCCCATCAT ATACCCATGC TCCAGCCAAG AGTTCAAAAA GGCCTTTCAG AATGTCTTGA 1620

GAATCCAGTG TCTCTGCAGA AAGCAGTCTT CCAAACATGC CCTGGGCTAC ACCCTGCACC 1680

CGCCCAGCCA GGCCGTGGAA GGGCAACACA AGGACATGGT GCGCATCCCC GTGGGATCAA 1740

GAGAGACCTT CTACAGGATC TCCAAGACGG ATGGCGTTTG TGAATGGAAA TTTTTCTCTT 1800

CCATGCCCCG TGGATCTGCC AGGATTACAG TGTCCAAAGA CCAATCCTCC TGTACCACAG 1860

CCCGGGTGAG AAGTAAAAGC TTTTTGCAGG TCTGCTGCTG TGTAGGGCCC TCAACCCCCA 1920

GCCTTGACAA GAACCATCAA GTTCCAACCA TTAAGGTCCA CACCATCTCC CTCAGTGAGA 1980

ACGGGGAGGA AGTCTAG 1997 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 466 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: N-terminal

( i) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Met Val Phe Leu Ser Gly Asn Ala Ser Asp Ser Ser Asn Cys Thr Gin 1 5 10 15 Pro Pro Ala Pro Val Asn He Ser Lys Ala He Leu Leu Gly Val He 20 25 30

Leu Gly Gly Leu He Leu Phe Gly Val Leu Gly Asn He Leu Val He 35 40 45

Leu Ser Val Ala Cys His Arg His Leu His Ser Val Thr His Tyr Tyr 50 55 60

He Val Asn Leu Ala Val Ala Asp Leu Leu Leu Thr Ser Thr Val Leu 65 70 75 80

Pro Phe Ser Ala He Phe Glu Val Leu Gly Tyr Trp Ala Phe Gly Arg 85 90 95

Val Phe Cys Asn He Trp Ala Ala Val Asp Val Leu Cys Cys Thr Ala 100 105 110

Ser He Met Gly Leu Cys He He Ser He Asp Arg Tyr He Gly Val 115 120 125

Ser Tyr Pro Leu Arg Tyr Pro Thr He Val Thr Gin Arg Arg Gly Leu 130 135 140

Met Ala Leu Leu Cys Val Trp Ala Leu Ser Leu Val He Ser He Gly 145 150 155 160

Pro Leu Phe Gly Trp Arg Gin Pro Ala Pro Glu Asp Glu Thr He Cys 165 170 175

Gin He Asn Glu Glu Pro Gly Tyr Val Leu Phe Ser Ala Leu Gly Ser 180 185 190

Phe Tyr Leu Pro Leu Ala He He Leu Val Met Tyr Cys Arg Val Tyr 195 200 205

Val Val Ala Lys Arg Glu Ser Arg Gly Leu Lys Ser Gly Leu Lys Thr 210 215 220

Asp Lys Ser Asp Ser Glu Gin Val Thr Leu Arg He His Arg Lys Asn 225 230 235 240

Ala Pro Ala Gly Gly Ser Gly Met Ala Ser Ala Lys Thr Lys Thr His 245 250 255

Phe Ser Val Arg Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys 260 265 270

Thr Leu Gly He Val Val Gly Cys Phe Val Leu Cys Trp Leu Pro Phe 275 .280 285

Phe Leu Val Met Pro He Gly Ser Phe Phe Pro Asp Phe Lys Pro Ser 290 295 300

Glu Thr Val Phe Lys He Val Phe Trp Leu Gly Tyr Leu Asn Ser Cys 305 310 315 320

He Asn Pro He He Tyr Pro Cys Ser Ser Gin Glu Phe Lys Lys Ala 325 330 335

Phe Gin Asn Val Leu Arg He Gin Cys Leu Cys Arg Lys Gin Ser Ser 340 345 350

Lys His Ala Leu Gly Tyr Thr Leu His Pro Pro Ser Gin Ala Val Glu 355 360 365

Gly Gin His Lys Asp Met Val Arg He Pro Val Gly Ser Arg Glu Thr 370 375 380 Phe Tyr Arg He Ser Lys Thr Asp Gly Val Cys Glu Trp Lys Phe Phe 385 390 395 400

Ser Ser Met Pro Arg Gly Ser Ala Arg He Thr Val Ser Lys Asp Gin 405 410 415

Ser Ser Cys Thr Thr Ala Arg Val Arg Ser Lys Ser Phe Leu Gin Val 420 425 430 Cys Cys Cys Val Gly Pro Ser Thr Pro Ser Leu Asp Lys Asn His Gin 435 440 445

Val Pro Thr He Lys Val His Thr He Ser Leu Ser Glu Asn Gly Glu 450 455 460

Glu Val 465 (2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1776 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

CTCCCTGCCG GCCGCTCGTT CTGTGCCCCG GCCCGGCCAC CGACGGCCGG CGTTGAGATG 60

ACTTTCCGCG ATCTCCTGAG CGTCAGTTTC GAGGGACCCC GCCCGGACAG CAGCGCAGGG 120

GGCTCCAGCG CGGGCGGCGG CGGGGGCGGC GCGGGCGGCG CGGCCCCCTC GGAGGGCCCG 180

GCGGTGGGCG GCGTGCCGGG GGGCGCGGGC GGCGGCGGCG GCGTGGTGGG CGCAGGCAGC 240

GGCGAGGACA ACCGGAGCTC CGCGGGGGAG CCGGGGAGCG CGGGCGCGGG CGGCGACGTG 300

AATGGCACGG CGGCCGTCGG GGGACTGGTG GTGAGCGCGC AGGGCGTGGG CGTGGGCGTC 360

TTCCTGGCAG CCTTCATCCT TATGGCCGTG GCAGGTAACC TGCTTGTCAT CCTCTCAGTG 420

GCCTGCAACC GCCACCTGCA GACCGTCACC AACTATTTCA TCGTGAACCT GGCCGTGGCC 480

GACCTGCTGC TGAGCGCCAC CGTACTGCCC TTCTCGGCCA CCATGGAGGT TCTGGGCTTC 540 TGGGCCTTTG GCCGCGCCTT CTGCGACGTA TGGGCCGCCG TGGACGTGCT GTGCTGCACG 600

GCCTCCATCC TCAGCCTCTG CACCATCTCC GTGGACCGGT ACGTGGGCGT GCGCCACTCA 660

CTCAAGTACC CAGCCATCAT GACCGAGCGC AAGGCGGCCG CCATCCTGGC CCTGCTCTGG 720

GTCGTAGCCC TGGTGGTGTC CGTAGGGCCC CTGCTGGGCT GGAAGGAGCC CGTGCCCCCT 780

GACGAGCGCT TCTGCGGTAT CACCGAGGAG GCGGGCTACG CTGTCTTCTC CTCCGTGTGC 840

TCCTTCTACC TGCCCATGGC GGTCATCGTG GTCATGTACT GCCGCGTGTA CGTGGTCGCG 900

CGCAGCACCA CGCGCAGCCT CGAGGCGGGC GTCAAGCGCG AGCGAGGCAA GGCCTCCGAG 960

GTGGTGCTGC GCATCCACTG TCGCGGCGCG GCCACGGGCG CCGACGGGGC GCACGGCATG 1020

CGCAGCGCCA AGGGCCACAC CTTCCGCAGC TCGCTCTCCG TGCGCCTGCT CAAGTTCTCC 1080

CGTGAGAAGA AAGCGGCCAA GACTCTGGCC ATCGTCGTGG GTGTCTTCGT GCTCTGCTGG 1140

TTCCCTTTCT TCTTTGTCCT GCCGCTCGGC TCCTTGTTCC CGCAGCTGAA GCCATCGGAG 1200

GGCGTCTTCA AGGTCATCTT CTGGCTCGGC TACTTCAACA GCTGCGTGAA CCCGCTCATC 1260

TACCCCTGTT CCAGCCGCGA GTTCAAGCGC GCCTTCCTCC GTCTCCTGCG CTGCCAGTGC 1320

CGTCGTCGCC GGCGCCGCCG CCCTCTCTGG CGTGTCTACG GCCACCACTG GCGGGCCTCC 1380

ACCAGCGGCC TGCGCCAGGA CTGCGCCCCG AGTTCGGGCG ACGCGCCCCC CGGAGCGCCG 1440

CTGGCCCTCA CCGCGCTCCC CGACCCCGAC CCCGAACCCC CAGGCACGCC CGAGATGCAG 1500

GCTCCGGTCG CCAGCCGTCG AAAGCCACCC AGCGCCTTCC GCGAGTGGAG GCTGCTGGGG 1560

CCGTTCCGGA GACCCACGAC CCAGCTGCGC GCCAAAGTCT CCAGCCTGTC GCACAAGATC 1620

CGCGCCGGGG GCGCGCAGCG CGCAGAGGCA GCGTGCGCCC AGCGCTCAGA GGTGGAGGCT 1680

GTGTCCCTAG GCGTCCCACA CGAGGTGGCC GAGGGCGCCA CCTGCCAGGC CTACGAATTG 1740

GCCGACTACA GCAACCTACG GGAGACCGAT ATTTAA 1776 (2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 572 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: Met Thr Phe Arg Asp Leu Leu Ser Val Ser Phe Glu Gly Pro Arg Pro 1 5 10 15

Asp Ser Ser Ala Gly Gly Ser Ser Ala Gly Gly Gly Gly Gly Gly Ala 20 25 30

Gly Gly Ala Ala Pro Ser Glu Gly Pro Ala Val Gly Gly Val Pro Gly 35 40 45

Gly Ala Gly Gly Gly Gly Gly Val Val Gly Ala Gly Ser Gly Glu Asp 50 55 60

Asn Arg Ser Ser Ala Gly Glu Pro Gly Ser Ala Gly Ala Gly Gly Asp 65 70 75 80

Val Asn Gly Thr Ala Ala Val Gly Gly Leu Val Val Ser Ala Gin Gly 85 90 95

Val Gly Val Gly Val Phe Leu Ala Ala Phe He Leu Met Ala Val Ala 100 105 110

Gly Asn Leu Leu Val He Leu Ser Val Ala Cys Asn Arg His Leu Gin 115 120 125

Thr Val Thr Asn Tyr Phe He Val Asn Leu Ala Val Ala Asp Leu Leu 130 135 140

Leu Ser Ala Thr Val Leu Pro Phe Ser Ala Thr Met Glu Val Leu Gly 145 150 155 160

Phe Trp Ala Phe Gly Arg Ala Phe Cys Asp Val Trp Ala Ala Val Asp 165 170 175

Val Leu Cys Cys Thr Ala Ser He Leu Ser Leu Cys Thr He Ser Val 180 185 190

Asp Arg Tyr Val Gly Val Arg His Ser Leu Lys Tyr Pro Ala He Met 195 200 205

Thr Glu Arg Lys Ala Ala Ala He Leu Ala Leu Leu Trp Val Val Ala 210 215 220

Leu Val Val Ser Val Gly Pro Leu Leu Gly Trp Lys Glu Pro Val Pro 225 230 235 240

Pro Asp Glu Arg Phe Cys Gly He Thr Glu Glu Ala Gly Tyr Ala Val 245 250 255

Phe Ser Ser Val Cys Ser Phe Tyr Leu Pro Met Ala Val He Val Val 260 265 270

Met Tyr Cys Arg Val Tyr Val Val Ala Arg Ser Thr Thr Arg Ser Leu 275 280 285

Glu Ala Gly Val Lys Arg Glu Arg Gly Lys Ala Ser Glu Val Val Leu 290 295 300

Arg He His Cys Arg Gly Ala Ala Thr Gly Ala Asp Gly Ala His Gly 305 310 315 320

Met Arg Ser Ala Lys Gly His Thr Phe Arg Ser Ser Leu Ser Val Arg 325 330 335 Leu Leu Lys Phe Ser Arg Glu Lys Lys Ala Ala Lys Thr Leu Ala He 340 345 350

Val Val Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe Val Leu 355 360 365

Pro Leu Gly Ser Leu Phe Pro Gin Leu Lys Pro Ser Glu Gly Val Phe 370 375 380

Lys Val He Phe Trp Leu Gly Tyr Phe Asn Ser Cys Val Asn Pro Leu 385 390 395 400

He Tyr Pro Cys Ser Ser Arg Glu Phe Lys Arg Ala Phe Leu Arg Leu 405 410 415

Leu Arg Cys Gin Cys Arg Arg Arg Arg Arg Arg Arg Pro Leu Trp Arg 420 425 430

Val Tyr Gly His His Trp Arg Ala Ser Thr Ser Gly Leu Arg Gin Asp 435 440 445

Cys Ala Pro Ser Ser Gly Asp Ala Pro Pro Gly Ala Pro Leu Ala Leu 450 455 460

Thr Ala Leu Pro Asp Pro Asp Pro Glu Pro Pro Gly Thr Pro Glu Met 465 470 475 480

Gin Ala Pro Val Ala Ser Arg Arg Lys Pro Pro Ser Ala Phe Arg Glu 485 490 495

Trp Arg Leu Leu Gly Pro Phe Arg Arg Pro Thr Thr Gin Leu Arg Ala 500 505 510

Lys Val Ser Ser Leu Ser His Lys He Arg Ala Gly Gly Ala Gin Arg 515 520 525

Ala Glu Ala Ala Cys Ala Gin Arg Ser Glu Val Glu Ala Val Ser Leu 530 535 540

Gly Val Pro His Glu Val Ala Glu Gly Ala Thr Cys Gin Ala Tyr Glu 545 550 555 560

Leu Ala Asp Tyr Ser Asn Leu Arg Glu Thr Asp He 565 570

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GAATCCCGAC CTGGAC 16

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGATCCTCAG GGTC 14

(2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CCATGGTGTT TCTCTCGGG 19 (2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GACGCGGCAG TACATGAC 18

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: GTCATGATGG CTGGGTACTT G 21

Claims (21)

WHAT IS CLAIMED IS:

1. A DNA which consists essentially of a DNA which codes for a human adrenergic receptor of the alpha 1-C subtype.

2. The DNA of Claim 1, wherein the DNA coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell.

3. The DNA of Claim 2, wherein the receptor has the nucleic acid sequence SEQ ID: 11:, figure 5, a variant thereof which, through redundancy of the genetic code, encodes a human alphalC receptor, or a truncation thereof, which continues to encode a functional human alphalC adrenergic receptor with intact G-protein and ligand binding sites.

4. The DNA of Claim 3 having the sequence, SEQ. ID:11 :, figure 5; SEQ. ID:26:; figure 18; or SEQ.ID:27, figure 19.

5. A human alphalC adrenergic receptor expressed by a cell which contains a cloned nucleic acid construct encoding said receptor.

A cell expressing a cloned human alphalC adrenergic receptor.

7. A method for identifying compounds which specifically bind to the human alphalC adrenergic receptor which comprises the steps of: a. Cloning the human alphalC adrenergic receptor; b. Splicing the the cloned alphalC adrenergic receptor into an expression vector to produce a construct such that the alphalC receptor is operably linked to transcription and translation signals sufficient to induce expression of said receptor upon introduction of said construct into a prokaryotic or eukaryotic cell; c. Introducing said construct into a prokaryotic or eukaryotic cell which does not express a human alphalC adrenergic receptor in the absence of said introduced construct; d. Incubating cells or membranes isolated from cells produced in step c. with a quantifiable compound known to bind to human alpha adrenergic receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable compound becomes displaced from the receptor; e. Incubating cells or membranes of cells which naturally express or have an introduced, cloned human alpha adrenergic receptor of a subtype other than the human alphalC receptor under identical conditions to the incubation conducted in step d, and obtaining the IC50 of the test compound for the non-alpha IC receptor; and f. Comparing the IC50 for the test compound for the alphalC receptor and for the alpha adrenergic receptor of a subtype other than the alphalC to identify compounds having a lower IC50 for the alphalC receptor.

8. A method of alleviating the effects of BPH which comprises administering a pharmaceutically effective amount of a compound which specifically binds to the human alphalC adrenergic receptor.

9. The method of Claim 8 wherein the compound is S(+)-niguldipine, (S(+)-l,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)- 3 ,5-pyridinedicarboxylic acid 3-(4,4-diphenyl- 1 -piperidinyl)-propyl methyl ester hydrochloride), or 5-methyl urapidil, 5-methyl-6[[3-[4-(2- methoxyphenyl)- 1 -piperaziny l]propy 1] amino] - 1 ,3 -dimethy luracil .

10. The method of Claim 8 wherein said compound is administered in conjunction with a compound effective to inhibit human testosterone 5-alpha reductase.

11. The method of Claim 10 wherein the compound effective to inhibit human testosterone 5-alpha reductase is a 5-alpha reductase isozyme 1 inhibitor.

12. The method fo Claim 11 wherein the 5-alpha reductase inhibitor is finasteride.

13. The method of Claim 10 wherein the compound effective to inhibit human testosterone 5-alpha reductase is a dual 5- alpha reductase isozyme 1 and an isozyme 2 inhibitor.

14. A method of alleviating the effects of BPH which comprises administering a pharmaceutically effective amount of finasteride and S(+)-niguldipine or 5-methyl urapidil.

15. A DNA which consists essentially of a DNA which codes for an entirely human adrenergic receptor of the alpha 1-A subtype.

16. The DNA of Claim 15, wherein the DNA coding for the receptor is operably linked to regulatory sequences such that the receptor may be expressed upon introduction into a prokaryotic or eukaryotic cell.

17. The DNA of Claim 16, wherein the receptor has the nucleic acid sequence SEQ ID:29:, figure 22, a variant thereof which, through redundancy of the genetic code, encodes a human alphal A adrenergic receptor, or a truncation thereof, which continues to encode a functional human alphal A adrenergic receptor with intact G-protein and ligand binding sites.

18. A human alphal A adrenergic receptor expressed by a cell which contains a cloned nucleic acid construct encoding said receptor.

19. A cell expressing a cloned, human alphal A adrenergic receptor having a sequence greater than 95% homologous with the sequence of SEQ.ID:29:.

20. A method treating BPH which comprises administering to a patient in need of such treatment an inhibitorily effective amount of compound having at least 12 fold specificity for the human alphalC adrenergic receptor as compared with the human alphal A and alphalB receptor.

21. A composition comprising an inhibitorily effective amount of compound having at least 12 fold specificity for the human alphalC adrenergic receptor as compared with the human alphal A and alphalB receptor and an inhibitorily effective amount of a human testosterone 5 -alpha reductase inhibitor.