Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• the present invention is directed to DNA molecules encoding imidazoline receptive polypeptides, preferably encoding human imidazoline receptive polypeptides, that can be used as an imidazoline receptor (abbreviated IR) .
• IR imidazoline receptor
• transcript (s) and protein sequences are predicted from the DNA clones.
• JEP-1A genomic DNA clone designated as JEP-1A.
• the cDNA clones according to the invention comprise cDNA homologous to portion (s) of this genomic clone; including 5A-1 cDNA, cloned by the inventors that established the open-reading frame for translation of mRNA from the gene, and established the immunoreactive properties of its polypeptide sequence in an expression systems. Also, the invention relates to cDNA clone EST04033, which is another clone identified to contain cDNA sequences from the JEP-1A gene, and of which the 5A-1 is a part, that encodes an active fragment of the IR polypeptide in transfection assays, and the protein sequences thereof.
• the invention also relates to methods for producing such genomic and cDNA clones, methods for expressing the IR protein and fragments, and uses thereof.
• brainstem imidazoline receptors possess binding site(s) for therapeutically relevant imidazoline compounds, such as clonidine and idazoxan.
• These drugs represent the first generation of ligands discovered for the binding site(s) of imidazoline receptors.
• clonidine and idazoxan were developed based on their high affinity for ⁇ 2 -adrenergic receptors.
• Second generation ligands, such as moxonidine possess somewhat improved selectivity for IR over ⁇ 2 -adrenergic receptors, but more selective compounds for IR are needed.
• imidazoline receptor clone is of particular interest because of its potential utility in identifying novel pharmaceutical agents having greater potency and/or more selectivity than currently available ligands have for imidazoline receptors.
• Recent technological advances permit pharmaceutical companies to use combinatorial chemistry techniques to rapidly screen a cloned receptor for ligands (drugs) binding thereto.
• a cloned imidazoline receptor would be of significant value to a drug discovery program.
• the molecular nature of imidazoline receptors remains unknown. For instance, no amino acid sequence data for a novel IR, e.g., by N-terminal sequencing, has been reported.
• imidazoline receptor candidates Three different techniques have been described in the literature by three different laboratories to visualize imidazoline-selective binding proteins (imidazoline receptor candidates) using gel electrophoresis. Some important consistencies have emerged from these results despite the diversity of the techniques employed. On the other hand, multiple protein bands have been identified, which suggests heterogeneity amongst imidazoline receptors. These reports are discussed below.
• I-site Any imidazoline-receptive binding site (e.g., encoded on IR)
• RVLM MW molecular weight NRL European abbreviation for RVLM (see below)
• RVLM Rostral Ventrolateral Medulla in brainstem SDS sodium dodecyl sulfate gel electrophoresis
• I-site refers to the imidazoline binding site, presumably defined within the imidazoline receptor protein.
• Reis antiserum was prepared by injecting the purified protein into rabbits [Wang et al, 1992]. The first immunization was done subcutaneously with the protein antigen (10 ⁇ g) emulsified in an equal volume of complete Freund's adjuvant, and the next three booster shots were given at 15-day intervals with incomplete Freund's adjuvant.
• the polyclonal antiserum has been mostly characterized by immunoblotting, but radioimmunoassays (RIA) and/or conjugated assay procedures, i.e., ELISA assays, are also conceivable [see “Radioimmunoassay of Gut Regulatory Peptides: Methods in Laboratory Medicine,” Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982].
• RIA radioimmunoassays
• ELISA assays conjugated assay procedures
• the present inventors and others [Escriba et al., Neurosci. Lett. 178: 81-84 (1994)] have characterized the Reis antiserum in several respects. For instance, the present inventors have discovered that human platelet immunoreactivity with Reis antiserum is mainly confined to a single protein band of MW ⁇ 33 kDa, although a trace band at ⁇ 85 kDa was also observed. The « 33 and « 85 kDa bands were enriched in plasma membrane fractions as expected for an imidazoline receptor.
• the intensity of the « 33 kDa band was found to be positively correlated with non-adrenergic I 5 PIC Bmax values at platelet IR, sites in samples from the same subjects, with an almost one-to-one slope factor.
• the nonadrenergic I25 PIC binding sites on platelets were discovered by the present inventors to have the same rank order of affinities as IR, binding sites in brainstem [Piletz and Sletten, J. Pharm. & Exper. Therap.. 267: 1493-1502
• the platelet » 33 kDa band may also be a product of a larger protein, since in human megakaryoblastoma cells, which are capable of forming platelets in tissue cultures, an « 85 kDa immunoreactive band was found to predominate. Immunoreactivity with Reis antiserum does not appear to be directed against human 2 AR and/or MAO A/B. This is a significant point because ⁇ 2 AR and MAO A/B have previously been cloned and also bind to imidazolines. The present inventors have obtained selective antibodies and recombinant preparations for 2 AR and MAO A/B, and these proteins do not correspond to the « 33, 70, or 85 kDa putative IR, bands. Thus, there is substantial evidence that, at least in human platelets, the Reis antiserum is IR, selective.
• the bands of MW ⁇ 41 and 44 kDa detected by Dontenwill antiserum may be derived from an ⁇ 85 kDa precursor protein, similar to that occurring in platelet precursor cells.
• An 85 kDa im unoreactive protein is obtained in fresh rat brain membranes only when a cocktail of 11 protease inhibitors is used.
• Reis antiserum detects the ⁇ 41 and 44 kDa bands in human brain when fewer protease inhibitors are used.
• the Dontenwill antiserum weakly detects a platelet « 33 kDa band.
• the present inventors have hypothesized that the « 41 and 44 kDa immunoreactive proteins may be alternative breakdown products of an ⁇ 85 kDa protein, as opposed to the platelet « 33 kDa breakdown product.
• the present invention involves various cDNA clones (ie., 5A-1 and EST04033) and a genomic clone (JEP-1A) which are directed to an isolated polypeptide (s) that is receptive to (bind to) imidazoline compound (s) , and can be used to identify other compounds of interest.
• imidazoline compounds in this context are p-iodoclonidine and moxonidine.
• the inventors detected a polypeptide expressed by their cDNA clone (5A-1 isolated from a human hippocampus cDNA library) that immunoreacted with Reis antiserum and/or Dontenwill antiserum.
• a polypeptide includes a 651 amino acid sequence as shown in SEQ ID No. 5. This polypeptide is predicted from non-plasmid cDNA in EST04033; a clone which the inventors showed possesses sequences inclusive of 5A-1. Furthermore, transfection of EST04033 into COS cells yielded imidazoline receptivity by radioligand binding assays (described in detail later) . Other imidazoline receptive proteins homologous to this polypeptide are also contemplated.
• Such polypeptide (s) generally have a molecular weight of about 50 to 80 kDa. More particularly, one can have a molecular weight of about 70 kDa.
• a polypeptide in another aspect of this invention, includes a 390 amino acid sequence as shown in SEQ ID No. 6. This represents the polypeptide predicted from the non-plasmid DNA of the original 5A-1 clone.
• Such a polypeptide generally has a molecular weight of about 35 to 50 kDa. More particularly, it can have a molecular weight of about 43 kDa.
• DNA molecules encoding aforementioned imidazoline- receptive polypeptide are also contemplated.
• a DNA molecule e.g., a cDNA derived from mRNA
• a DNA molecule containing the 1954 base pairs (b.p.) (1954 b.p. encodes 651 amino acids) nucleotide sequence shown in SEQ ID No. 2 is contemplated. This represents the coding sequence for the polypeptide predicted by EST04033 transfections .
• a DNA molecule includes the longer nucleotide sequence shown in SEQ ID No. 3. This represents the cDNA predicted to have been translated + not predicted to have been translated in transfections experiments of EST04033.
• a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 6. In another aspect, it can include the
• the 1171 b.p. nucleic acid sequence shown in SEQ ID No. 4 is the 5A-1 non-plasmid DNA.
• the nucleic acid sequence of the genomic clone encoding the imidazoline receptor is further shown in SEQ ID No. 21.
• the nucleic acid and amino acid sequence of the predicted transcript (ie., cDNA) can be predicted from the description hereinbelow.
• the polypeptide encoded by the genomic DNA is shown in SEQ ID No. 22.
• Sequence similarity with the sequences indicated in SEQ ID protocols of the attached Sequence Listing is defined in connection with the present invention as a very close structural relationship of the relevant sequences with the sequences indicated in the respective SEQ ID protocols.
• sequence similarity in each case the structurally mutually corresponding sections of the sequence of the SEQ ID protocol and of the sequence to be compared therewith are superimposed in such a way that the structural correspondence between the sequences is a maximum, account being taken of differences caused by deletion or insertion of individual sequence members (DNA-codon or amino acid respectively) , and being compensated by appropriate shifts in sections of the sequences.
• sequence similarity in % results from the number of sequence members which now correspond to one another in the sequences ("homologous positions") relative to the total number of members contained in the sequences of the SEQ ID protocols. Differences in the sequences may be caused by variation, insertion or deletion of sequence members. Additionally in DNA sequences, different DNA-codons encoding for the same amino acid are considered identical in the context of the present invention. For amino acid sequences, conservative amino acid substitutions encoded by their corresponding DNA-codons, as well as naturally occurring homologs of the sequences, are considered within the context of sequence similarity.
• DNA molecules of substantial homology are an implicit aspect of this sort of invention.
• the inventors have already identified two possible splice variants in the amino acid coding sequence.
• artificially mutated receptor cDNA molecules can be routinely constructed by methods such as site-directed polymerase chain reaction-mediated mutagenesis [Nelson and Long, Anal. Biochem. 180: 147-151 (1989)]. It is commonly appreciated that highly homologous mutants frequently mimic their natural receptor.
• Kjelsberg et al. J. Biol. Chem. 267: 1430-1433 (1992)] showed that all 20 amino acid substitutions produce an active receptor at a single site in the ⁇ lb -adrenergic receptor.
• RNA molecules of > 75 % complementarity to an instant DNA molecule e.g., an mRNA molecule (sense) or a complementary cRNA molecule (antisense)
• RNA molecules of > 75 % complementarity to an instant DNA molecule e.g., an
• a further aspect of the invention is for a recombinant vector, as well as a host cell transfected with the recombinant vector, wherein the recombinant vector contains at least one of the nucleotide sequences shown in SEQ ID Nos. 1- 4, or sequences predicted by the genomic clone, or nucleotide sequences > 75 % homologous thereto.
• a method of producing an imidazoline receptor protein is another aspect of the invention. Such a method entails transfecting a host cell with an aforementioned vector, and culturing the transfected host cell in a culture medium to generate the imidazoline receptor.
• a method for producing homologous imidazoline receptor proteins, and the proteins produced thereby, are also considered an aspect of this invention.
• a significant further aspect of the invention is a method of screening for a ligand that binds to an imidazoline receptor.
• Such a method can comprise culturing an above- mentioned transfected cell in a culture medium to express imidazoline receptor proteins, followed by contacting the proteins with a labelled ligand for the imidazoline receptor under conditions effective to bind the labelled ligand thereto.
• the imidazoline receptor proteins can then be contacted with a candidate ligand, and any displacement of the labelled ligand from the proteins can be detected.
• Displacement of labelled ligand signifies that the candidate ligand is a ligand for the imidazoline receptor.
• Fig. 1 depicts a comparison of Reis antiserum (lane 1, 1:2000 dilution) and Dontenwill antiserum (lane 2, 1:5000 dilution) immunoreactivities for human NRL (same as RVLM) and hippocampus, as discussed in Example 1.
• Fig. 2 depicts a comparison of Reis antiserum (1:15,000 dilution) and Dontenwill antiserum (1:20,000 dilution) immunoreactivities for plaques isolated from the human hippocampal cDNA library used in cloning as discussed in Example 2.
• the plaques contain the initial clone, designated herein as 5A-1, in a third stage of purification.
• Fig. 3 depicts the restriction map of the EST04033 cDNA clone.
• Fig. 4 depicts a competitive binding assay between 15 I- labelled p-iodoclonidine (PIC) and various ligands for the imidazoline receptor on membranes expressed in COS cells transfected with the EST04033 cDNA clone, as discussed in Example 4.
• PIC p-iodoclonidine
• Fig. 5 depicts the prediction of introns and exons of the genomic clone (as analyzed by the GENESCAN program and verified by the available CDNAS) .
• Fig. 6 depicts the distribution of MRNA homologous to our CDNA in human adult tissues (bar graph) and the two species of MRNA (6 and 9.5 kb) .
• DETAILED DESCRIPTION OF THE INVENTION The present invention is concerned with multiple aspects of an imidazoline receptor protein, and DNA molecules encoding the same, and fragments thereof, which have now been discovered.
• polypeptide having imidazoline binding activity contains the putative active site for binding, as discussed hereinafter.
• polypeptide (s) described herein has a binding affinity for an imidazoline compound, it may also have an enzymatic activity, such as do catalytic antibodies and ribozymes. In fact, one such domain within our protein predicts a cytochrome p450 activity (described later) .
• binding polypeptides are those containing either of the amino acid sequences shown in SEQ ID Nos. 5 or 6 (with the amino acid sequence predicted by EST04033 given in SEQ ID No. 5) .
• Functionally equivalent polypeptides are also contemplated, such as those having a high degree of homology with such aforementioned polypeptides, particularly when they contain the Glu-Asp-rich region described hereinafter which we believe may define an active imidazoline binding site.
• a polypeptide of the invention can be formed by direct chemical synthesis on a solid support using the carbodiimide method [R. Merrifield, JACS, 85: 2143 (1963)].
• an instant polypeptide can be produced by a recombinant DNA technique as described herein and elsewhere [e.g., U.S. Patent No. 4,740,470 (issued to Cohen and Boyer) , the disclosure of which is incorporated herein by reference] , followed by culturing transformants in a nutrient broth.
• a DNA molecule of the present invention encodes aforementioned polypeptide.
• a particularly preferred coding sequence is the 1954 b.p. sequence set forth in SEQ ID No. 2, which has now been discovered to be a nucleotide sequence that encodes a polypeptide capable of binding imidazoline compound (s) .
• a DNA molecule includes the 3318 b.p. nucleotide sequence shown in SEQ ID No. 3. This latter sequence is the entire EST04033 insert. It includes the nucleotide sequence of SEQ ID No. 2 which was predicted to have been translated into protein in the transfection experiments.
• a DNA molecule contains a nucleic acid sequence encoding the amino acid sequence (390 residues) shown in SEQ ID No. 6. This amino acid sequence corresponds to that derived from direct sequencing of the 5A-1 clone and represents a fragment of the native protein.
• the 5A-1 DNA molecule is defined by the 1171 b.p. nucleic acid sequence shown in SEQ ID No. 4.
• a DNA molecule of the present invention can be synthesized according to the phosphotriester method [Matteucci et al., JACS, 103: 3185 (1988)]. This method is particularly suitable when it is desired to effect site-directed mutagenesis of an instant DNA sequence, whereby a desired nucleotide substitution can be readily made.
• Another method for making an instant DNA molecule is by simply growing cells transformed with plasmids containing the DNA sequence, lysing the cells, and isolating the plasmid DNA molecules.
• an isolated DNA molecule of the invention is made by employing the polymerase chain reaction (PCR) [e.g., U.S. Patent No.
• a further aspect of the invention is for a vector, e.g., a plasmid, that contains at least one of the nucleotide sequences shown in SEQ ID Nos. 1-4 or those predicted by the genomic clone in SEQ ID No. 21.
• the vector encodes an IR polypeptide of the invention.
• fragments of the native IR protein are contemplated; as well as fusion proteins that incorporate an amino acid sequence as described herein.
• a recombinant vector of the invention can be formed by ligating an afore-mentioned DNA molecule to a preselected expression plasmid, e.g., with T4 DNA ligase.
• the plasmid and DNA molecule are provided with cohesive (overlapping) terminii, with the plasmid and DNA molecule operatively linked (i.e., in the correct reading frame).
• Another aspect of the invention is a host cell transfected with a vector of the invention.
• a protein expressed by a host cell transfected with such a vector is contemplated, which protein may be bound to the cell membrane.
• Such a protein can be identical with an aforementioned polypeptide, or it can be a fragment thereof, such as when the polypeptide has been partially digested by a protease in the cell.
• the expressed protein can differ from an aforementioned polypeptide, as whenever it has been subjected to one or more post-translational modifications.
• it should exhibit imidazoline binding capacity.
• a method of producing an imidazoline receptor protein is another aspect of the invention, which entails transfecting a host cell with an aforementioned vector, and culturing the transfected host cell in a culture medium to generate the imidazoline receptor.
• the receptor molecule can undergo any post-translational modification (s) , including proteolytic decomposition, whereby its structure is altered from the basic amino acid residue sequence encoded by the vector.
• a suitable transfection method is electroporation, and the like.
• a vector encoding an instant polypeptide can be transfected directly in animals.
• embryonic stem cells can be transfected, and the cells can be manipulated in embryos to produce transgenic animals. Methods for performing such an operation have been previously described [Bond et al., Nature , 374:272-276 (1995) ] . These methods for expressing an instant CDNA molecule in either tissue culture cells or in animals can be especially useful for drug discovery.
• a method of screening for a ligand (drug) that binds to an imidazoline receptor comprises culturing an above- mentioned host cell in a culture medium to express an instant imidazoline receptive polypeptide, then contacting the polypeptides with a labelled ligand, e.g., radiolabelled p- iodoclonidine, for the imidazoline receptor under conditions effective to bind the labelled ligand thereto.
• the polypeptides are further contacted with a candidate ligand, and any displacement of the labelled ligand from the polypeptides is detected.
• Displacement signifies that the candidate ligand actually binds to the imidazoline receptor.
• steps could be performed on intact host cells, or on proteins isolated from the cell membranes of the host cells.
• a suitable drug screening protocol involves preparing cells (or possibly tissues from transgenic animals) that express an instant imidazoline receptive polypeptide.
• categories of chemical structure are systematically screened for binding affinity or activation of the receptor molecule encoded by the transfected CDNA. This process is currently referred to as combinatorial chemistry.
• a number of commercially available radioligands e.g., 125 PIC, can be used for competitive drug binding affinity screening.
• An alternative approach is to screen for drugs that elicit or block a second messenger effect known to be coupled to activation of the imidazoline receptor, e.g., moxonidine- stimulated arachidonic acid release.
• a second messenger effect known to be coupled to activation of the imidazoline receptor, e.g., moxonidine- stimulated arachidonic acid release.
• a preferred compound drug
• Identification of this compound would lead to animal testing and upwards to human trials.
• the initial rationale for drug discovery becomes vastly improved with an instant cloned imidazoline receptor.
• a drug screening method is contemplated in which a host cell of the invention is cultured in a culture medium to express an instant imidazoline receptive polypeptide.
• Intact cells are then exposed to an identified agent (ie., agonist, inverse agonist, or antagonist) under conditions effective to elicit a second messenger or other detectable responses upon interacting with the receptor molecule.
• the imidazoline receptive polypeptides are then contacted with one or more candidate chemical compounds (drugs) , and any modification in a second messenger response is detected.
• candidate chemical compounds drug compounds
• Compounds that mimic an identified agonist would be agonist candidates, and those producing the opposite response would be inverse agonist candidates.
• Those compounds that block the effects of a known agonist would be antagonist candidates for an in vivo imidazoline receptor.
• the contacting step with a candidate compound is preferably conducted at a plurality of candidate compound concentrations.
• a method of probing for another gene encoding an imidazoline receptor or homologous protein is further contemplated.
• Such a method comprises providing a radiolabelled DNA molecule identical or complementary to one of the above-described CDNA molecules (probe) .
• the probe is then placed in contact with genetic material suspected of containing a gene encoding an imidazoline receptor or encoding a homologous protein, under stringent hybridization conditions (e.g., a high stringency wash condition is 0.1 x SSC, 0.5% SDS at 65 °C) , and identifying any portion of the genetic material that hybridizes to the DNA molecule.
• stringent hybridization conditions e.g., a high stringency wash condition is 0.1 x SSC, 0.5% SDS at 65 °C
• a method of selectively producing antibodies comprises injecting a mammal with an aforementioned polypeptide, and isolating the antibodies produced by the mammal. This aspect is discussed in more detail in an example presented hereinafter.
• the present inventors began their search for a human imidazoline receptor CDNA by screening a ⁇ gtll phage human hippocampus CDNA expression library. Their research had indicated that both of the known antisera (Reis and
• Example 1 Selectivity of the Antisera.
• the obtained Reis antiserum had been prepared against a purified imidazoline binding protein isolated from BAC cells, which protein runs in denaturing-SDS gels at 70 Kda [Wang et al. , 1992, 1993].
• the Dontenwill antiserum is anti-idiotypic, and thus is believed to detect the molecular configuration of an imidazoline binding site domain in any species. Prior to being used for screening plaques, both antisera were cleaned by stripping out possible antibacterial antibodies.
• both antisera have been tested to ensure that they are in fact selective for a human imidazoline receptor.
• both of these antisera detected identical bands in human platelets and hippocampus, and in brainstem RVLM (NRL) by Western blotting (see Fig. 1) .
• ECL Enhanced Chemiluminescence
• the linearity of response of the ECL system was demonstrated with a standard curve.
• ECL detection was demonstrated to be very quantifiable and about ten times more sensitive than other screening methods previously used with these antisera.
• Western blots with antiserum dilutions of 1:3000 revealed immunoreactivity with as little as 1 ng of protein from a human hippocampal homogenate by dot blot analysis.
• human hippocampal homogenate (30 ⁇ g) and NRL membrane proteins (lO ⁇ g) were electrophoresed through a 12.5% SDS-polyacrylamide gel, electrotransfered to nitrocellulose and sequentially incubated with (1) the Reis antibody (1:2000 dilution) and (2) the
• Dontenwill antibody (1:5000 dilution). Immunoreactive bands were visualized with an Enhanced Chemiluminescence (ECL) detection kit (Amersham) using anti-rabbit Ig-HRP conjugated antibody at a dilution of 1:3000 and the ECL detection reagents. Following detection with the antibody, blots were stripped and reprocessed omitting the primary antibody to check for complete removal of this antibody. In panels A and B, lane 1 shows the immunoreactive bands observed with the Reis antibody and lane 2 shows the bands detected with the Dontenwill antibody. Protein molecular weight standards are indicated to the left of each panel (in Kda) .
• both of these antisera detected a similar 85 Kda protein in human brain and other tissues.
• a 33 Kda band was found in human platelets.
• the 33 Kda band is of smaller size than that reported for other tissues [Wang et al., 1993; Escriba et al., 1994; Greney et al., 1994]
• the fact that both antisera detected it suggests that both the 85 Kda and 33 Kda bands may be imidazoline binding polypeptides.
• the 85 and 33 Kda bands were enriched in plasma membrane fractions, as is known to be the case for IR, binding, but not I 2 binding [Piletz and Sletten, 1993].
• a commercially available human hippocampal cDNA ⁇ gtll expression library (Clontech Inc. , Palo Alto, CA) was screened for immunoreactivity sequentially using both the anti- idiotypic Dontenwill antiserum and the Reis antiserum.
• Positive plaques were pulled and rescreened until tertiary screenings yielded only positive plaques.
• Clone 5A-1 has been deposited under the Budapest Treaty with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD, USA, 20852, on August 28, 1997 and has been assigned deposit accession no. ATCC 209217. Tertiary-screened plaques of 5A-1 were all immuno-positive with either of the two known anti-imidazoline receptor antisera, but not with either preimmune antisera. These results suggested that clone 5A-1 encoded a fusion peptide similar to or identical with one of the predominant bands detected in human Western blots by both the Dontenwill and Reis antisera.
• DNA sequencing was performed using T7 DNA polymerase and the dideoxy nucleotide termination reaction.
• DNA sequencing was performed by the fluorescent dye terminator labelling method using AmpliTaq DNA polymerase (Applied Biosystems Inc. , Prizm DNA Sequencing Kit, Perkin- Elmer Corp. , Foster City, CA) .
• the primer walking method was used.
• the primers actually used were a subset of those shown in SEQ ID Nos. 7- 20.
• BLASTN is a program used to compare known DNA sequences from international databases, regardless of whether they encode a polypeptide. Neither of the two EST cDNA sequences having high homology to 5A-1, to our knowledge have been reported anywhere else except on the Internet. Both were derived as Expressed Sequence Tags (ESTs) in random attempts to sequence the human cDNA repertoire [as described in Adams et al., Science, 252: 1651-1656 (1991)]. As far as can be determined, the people who generated these ESTs lack any knowledge of what protein (s) they encode.
• One cDNA, designated HSA09H122 contained 250 b.p. with 7 unknown/ incorrect base pairs (97% homology) versus 5A-1 over the same region.
• HSA09H122 was generated in France (Genethon, B.P. 60, 91002 Evry Cedex France) from a human lymphoblast cDNA library.
• the other EST designated EST04033, contained 155 b.p. with 12 unknown/ incorrect base pairs (92% homology) versus 5A-1 over the same region.
• EST04033 was generated at the Institute for Genomic Research (Gaithersburg, MD) from a human fetal brain cDNA clone (HFBDP28) . Thus, both of these ESTs are short DNA sequences and contain a number of errors
• HSA09H122 Based on the BLASTN search, the owner of HSA09H122 was contacted in an effort to obtain that clone.
• the current owner of the clone appears to be Dr. Charles Auffret (Paul Brousse Hospital, Genetique, B.P. 8, 94801 Villejuif Cedex, France) .
• Dr. Auffret indicated by telephone that his clone came from a lot of clones believed to be contaminated with yeast DNA, and he did not trust it for release. Contamination with yeast DNA of that clone was later confirmed to have been reported within an Internet database. Thus, HSA09H122 was not reliable.
• the other partial clone (EST04033) was purchased from American Type Culture Collection in Rockville, MD (ATCC Catalog no. 82815) .
• EST04033 was 3389 b.p. (SEQ ID No. 1) , with a 3,318 b.p. nonplasmid insert (see SEQ ID No. 3).
• the nucleotide sequence of the entire clone is shown in SEQ ID No. 1. In this sequence, an identical overlap was observed for the sequence obtained previously for the 5A-1 clone and the sequence obtained for EST04033. The 5A- 1 overlap began at EST04033 b.p. 2,181 (SEQ. No.l) and continued to the end of the molecule (b.p. 3,351).
• cDNA of the present invention encode a protein that is immunoreactive with both of the known selective antisera for an imidazoline receptor, i.e., Reis antiserum and Dontenwill antiserum.
• an instant cDNA molecule produces a protein immunologically related to a purified imidazoline receptor and has the antigenic specificity expected for an imidazoline binding site.
• These antisera have been documented in the scientific literature as being selective for an "imidazoline receptor" , which provides strong evidence that such an imidazoline receptor has indeed been cloned.
• our instant cDNA sequence contains open reading frame distinct from any previously described proteins. Therefore, the encoded protein is novel, and it is unrelated to ⁇ 2 -adrenoceptors or monoamine oxidases. Small hydrophobic domains in the predicted amino acid sequence suggest that the protein is probably membrane bound, as expected for an imidazoline receptor.
• Example 3 Cloning of a Human Gene
• a pre-made genomic library of human placental DNA was purchased from Stratagene (La Jolla, CA) to screen for an IR gene by hybridization.
• the genomic library was constructed in Stratagene's vector ⁇ FIX® II (catalog # 946206), and it was grown in XLl-Blue MRA (P2) host bacteria. It was titered to yield approximately 50,000 plaques per 137 mm plate. Lifts from six such plates were screened in duplicate by hybridization.
• the DNA probe used for screening was a 1.85 kb EcoRI fragment from EST 04033 cDNA (uniquely related to our sequences based on the BLASTN) .
• the 1.85 kb fragment was extracted from an agarose electrophoresis gel, cleaned according to the GENECLEAN® III kit manual (BIO 101, Inc., P.O. Box 2284, La Jolla, CA) , and radiolabeled with [ ⁇ - 32 P] d-CTP according to Stratagene's Prime-It® II Random Primer Labeling Kit manual. Plaques were lifted onto 137 mm Duralon-UVTM membranes
• This hybridization procedure is essentially described in Stratagene's vector ⁇ FIX® II instruction manual. Positive plaques were localized by developing Kodak BioMax films. Two positive genomic clones of identical size were retained through three rounds of screening
• JEP 1-A One of the positive genomic clones (designated JEP 1-A) was selected for complete characterization. It was found to contain an « 17 kb insert. Large-scale preparations of this genomic clone DNA were performed using the ⁇ QUICK! SPIN kit (BIO101, La Jolla, CA) . To verify that we had cloned a gene corresponding to 5A-1 and EST04033 cDNA, some restriction site positions in the genomic clone were determined using the FLASH Nonradioactive Gene Mapping Kit (Stratagene) and compared to Southern blots of human DNA.
• genomic sequences highly related to (or identical to) those of our cDNA clones was determined by high stringency hybridization (as above) with the following 2 P-labeled probe: a 1110 bp Apal-EcoRI fragment from the cDNA clone 5A-1. This fragment was chosen as the probe because it lacks the GAG repeat (encoding glutamic acids) , which might have complicated matters if it were found to be repeated elsewhere in the genome.
• genomic clone JEPl-A we detected a 14.1 kb .EcoRI fragment and a 7.7 kb SacI fragment that hybridized with this probe.
• the procedure consisted of sequencing all these subclones and parent clones with vector forward and reverse primers. Subsequently, this initial round of sequencing was supplemented with primer walking using custom oligonucleotides.
• the Sac I fragments were joined together by primer walking using the 2 Xba I fragments of 3 and 10 Kb. Then, the largest Sac I fragment (8 kb) and the 10 kb Xba I fragment were used as templates for a transposon sequencing method.
• the method used was the Primer Island Transposition Kit (Perkin-Elmer Corp. , Norwalk, CT; Applied Biosystems) (ABI) .
• the kit consists of a synthetic transposon (Tyl) containing forward and reverse primers and the integrase enzyme which inserts the transposon randomly into the target plasmid DNA.
• Transposon insertion is an alternative to subcloning or primer walking when sequencing a large region of DNA (Devine and Boeke, Nucleic Acids Res. 22: 3765-3772 (1994); Devine et al., Genome Res., in press, (1997); Kimmel et al., In Genome Analysis, a Laboratory Manual, Cold Spring Harbor Press, NY, NY, in press (1997). A total of over 250 individual sequencing reactions were performed. Sequencing was done on ABI model 373 and 377 automated sequencers using ABI dye-terminator sequencing kits.
• Primers were designed using Gene Runner software (Hastings Software, Hastings On Hudson, NY) . Oligonucleotides were purchased from Gibco-BRL (Gaithersburg, MD) . Sequence assembly was performed using Sequencer Software (Gene Codes Corp. , Ann Arbor, MI) from 4- fold redundancy of sequences.
• the entire sequence of our JEP-1A genomic clone is shown in SEQ. 21.
• the computer program, GENSCAN 1.0 was able to identify splice sites of known topology. As expected, this gene contained a number of introns. See Table 1 hereinbelow. Only one continuous open reading frame was identified within our genomic clone. This open reading frame was interrupted by a number of introns (which is typical of eukaryotic transcripts) as shown in Fig. 5.
• the predicted polypeptide is encoded by the genomic DNA beginning at b.p. # 971 of SEQ ID No. 21.
• the predicted amino acid sequence of the polypeptide encoded thereby is shown in SEQ ID No. 22.
• JEP-1A has more nearly defined the full- length transcript (by at least 102 more coding nucleotides than the cDNAs alone) .
• This region of the IR protein delineates a highly unique span of 59 amino acids, 36 of which are Glu or Asp residues (61%) .
• This region was largely discovered within clone 5A-1 and it is present within all discovered and predicted transcripts from the gene (EST04033 included) .
• This sequence lies between two potential transmembrane loops (hydrophobic domains) .
• the identification of this unique Glu/Asp-rich domain within our clones is consistent with an expected negatively charged pocket capable of binding clonidine and agmatine, both of which are highly positively charged ligands.
• clone 5A-1 might encode an imidazoline binding site.
• this glu/asp-rich sequence is located within the longest stretch of homology that the clone has with any known protein, i.e., the ryanodine receptor (as determined by on BLASTN) .
• the ryanodine receptor as determined by on BLASTN
• the total nucleic acid homology is 67% with the ryanodine receptor DNA over the stretches encompassing this region. However, this is not sufficient to indicate that the imidazoline receptor is a subtype of the ryanodine receptor, because this homologous stretch is still a minor portion of the overall transcript (s) identified in the gene. Instead, this significant homology may reflect a commonality in function between this region of the IR and the ryanodine receptor.
• the Glu/Asp-rich region within the ryanodine receptor has also been reported to define a calcium and ruthenium red dye binding domain that modulates the ryanodine receptor/Ca ++ release channel located within the sarcoplasmic reticulum.
• the only other charged amino acids within the Glu/Asp-rich region of our clones are two arginines (the ryanodine receptor has uncharged amino acids at the corresponding positions) .
• IL-2R/3 interleukin-2R receptor
• IL-2R/5 possesses the following regions over a span of 286 amino acids: ser-rich region, followed by glu/asp-rich region, followed by proline-rich region.
• our predicted protein has the same three regions, in the same order, over a span of about 625 amino acids. This suggests that our protein might function similarly as cytokine receptors.
• GTPase activator protein (2) There appear to be four small hydrophobic domains indicative of transmembrane domain receptors. (3) A number of potential protein kinase C (PKC) phosphorylation sites appear near to the carboxy side of the protein, and we have previously found that treatment of membranes with PKC leads to an enhancement of native IR binding. Thus, these observations are all consistent with other observations expected for native IR.
• PKC protein kinase C
• the 6 kb band is weakly detectable in some non-CNS tissues, it is enriched in brain. An enrichment of the 6 kb mRNA is observed in brainstem, although not exclusively. The regional distribution of the mRNA is somewhat in keeping with the reported distribution of IR binding sites, when extrapolated across species (Fig. 6). Thus, the rank order of Bmax values for IR in rat brain has been reported to be frontal cortex > hippocampus > medulla oblongata > cerebellum [Kamisaki et al., Brain Res. , 514: 15- 21 (1990)]. Therefore, with the exception of human cerebellum, which showed two mRNA bands, the distribution of the mRNA for our the present cloned cDNA is consistent with it belonging to IR.
• IR binding sites are commonly considered to be low in cerebral cortex compared to brainstem, this is in fact a misinterpretation of the literature based only on comparisons to the alpha-2 adrenoceptor ' s Bmax, rather than on absolute values.
• IR Bmax values have actually been reported to be slightly higher in the cortex than the brainstem, but they only "appear” to be low in the cortex in comparison to the abundance of alpha-2 binding sites in cortex. Therefore, the distribution of the IR mRNA is reasonably in keeping with the actual Bmax values for radioligand binding to the receptor [Kamisaki et al., (1990)].
• the JEP-lA clone clearly contains most of the gene. Within it we have identified at least 3,776 nucleotides for transcript (s) (encoding 1,065 amino acids plus 587 b.p. of untranslated region down to the polyT + tail) . This has been lengthened by at least 66 coding nucleotides upstream (22 amino acids) in comparison to overlapping ESTs. In addition to this, we are quite confident of the splice site for the two observed mRNA sizes. Most of the functional sequences are predicted to be encoded within our genomic clone. A summary of the evidence that a gene encoding an imidazoline receptor protein has been cloned is summarized in Table 2 hereinbelow.
• COS-7 cells were transfected with a vector containing EST04033 cDNA, which was predicted based on sequence analysis to contain the glu/asp rich region thought to be important for ligand binding to the imidazoline receptor protein.
• the EST04033 cDNA was subcloned into pSVK3 (Pharmacia LKB Biotechnology, Piscataway, NJ) using standard techniques [Sambrook, supra] , and transfected via the DEAE-dextran technique as previously described [Choudhary et al., Mol. Pharmacol. , 42: 627-633 (1992); Choudhary et al., Mol. Pharmacol.
• a restriction map of the EST04033 cDNA is shown in Fig. 3.
• the restriction enzymes Sal I and Xba I were used for subcloning into pSVK3. Briefly stated, COS-7 cells were seeded at 3 x 10 6 cells/100 mm plate, grown overnight and exposed to 2 ml of DEAE-dextran/plasmid mixture. After a 10-15 min.
• Transfected samples were also analyzed by Western blots.
• the protocol used for Western blot assay of transfected cells is as follows.
• Cell membranes were prepared in a special cocktail of protease inhibitors (1 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethyl-sufonylfluoride, 10 M e-aminocaproic acid, 0.1 mM benzamide, 0.1 mM benzamide-HCl , 0.1 mM phenanthroline, 10 ⁇ g/ml pepstatin A, 5 mM iodoacetamide, 10 ⁇ g/ml antipain, 10 ⁇ g/ml trypsin-chymotrypsin inhibitor, 10 ⁇ g/ml leupeptin, and 1.67 ⁇ g/ml calpain inhibitor) in 0.25 M sucrose, 1 mM MgCl 2 , 5 mM Tris, pH 7.4.
• the protocol to fully characterize radioligand binding in the transfected cells entails the following. First, the presence of IR and/or I 2 binding sites are scanned over a range of protein concentrations using a single concentration of [ 125 I]-p-iodoclonidine (l.OnM) and H-idazoxan (8nM) , respectively. Then, rate of association binding experiments (under a 10 ⁇ M mask of NE to remove ⁇ 2 AR interference) are performed to determine if the kinetic parameters are similar to those reported for native imidazoline receptors [Ernsberger et al. Annals NY Acad. Sci.. 763: 163-168 (1995)].
• Stable transfections can be obtained by subcloning the imidazoline receptor cDNA into a suitable expression vector, e.g., pRc/CMV (Invitrogen, San Diego, CA) , which can then be used to transform host cells, e.g. CHO and HEK-293 cells, using the Lipofectin reagent (Gibco/BRL, Gaithersburg, MD) according to the manufacturer's instructions. These two host cell lines can be used to increase the permanence of expression of an instant clone. The inventors have previously ascertained that parent CHO cells lack both alpha 2 -adrenoceptor and IR binding sites [Piletz et al., J. Pharm. & Exper. Ther..
• Direct probing of other human genomic and cDNA libraries can be performed by preparing labelled cDNA probes from different subcloned regions of our clone.
• Commercially available human DNA libraries can be used.
• another genomic library is EMBL (Clontech) , which integrates genomic fragments up to 22 kbp long. It is reasonable to expect that introns may exist within other human IR genes so that only by obtaining overlapping clones can the full-length genes be sequenced.
• a probe encompassing the 5' end of an instant cDNA is generally useful to obtain the gene promoter region.
• Clontech's Human PromoterFinder DNA Walking procedure provides a method for "walking" upstream or downstream from cloned sequences such as cDNAs into adjacent genomic DNA.
• Example 7 Methods for Preparing Antibodies to Imidazoline Receptive Proteins.
• An instant imidazoline receptive polypeptide can also be used to prepare antibodies immunoreactive therewith.
• synthetic peptides (based on deduced amino acid sequences from the DNA) can be generated and used as immunogens .
• transfected cell lines or other manipulations of the DNA sequence of an instant imidazoline receptor can provide a source of purified imidazoline receptor peptides in sufficient quantities for immunization, which can lead to a source of selective antibodies having potential commercial value.
• kits for assaying imidazoline receptors can be developed that include either such antibodies or the purified imidazoline receptor protein.
• a purification protocol has already been published for the bovine imidazoline receptor in BAC cells [Wang et al, 1992] and an immunization protocol has also been published [Wang et al., 1993]. These same protocols can be utilized with little if any modification to afford purified human IR protein from transfected cells and to yield selective antibodies thereto.
• the peptide may be linked to a suitable soluble carrier to which antibodies are unlikely to be encountered in human serum.
• Illustrative carriers include bovine serum albumin, keyhole limpet hemocyanin, and the like.
• the conjugated peptide is injected into a mouse, or other suitable animal, where an immune response is elicited.
• Monoclonal antibodies can be obtained from hybridomas formed by fusing spleen cells harvested from the animal and myeloma cells [see, e.g., Kohler and Milstein, Nature, 256: 495-497 (1975)]. Once an antibody is prepared (either polyclonal or monoclonal) , procedures are well established in the literature, using other proteins, to develop either RIA or ELISA assays [see, e.g., "Radioimmunoassay of Gut Regulatory Peptides; Methods in Laboratory Medicine," Vol. 2, chapters 1 and 2, Praeger Scientific Press, 1982]. In the case of RIA, the purified protein can also be radiolabelled and used as a radioactive antigen tracer.
• Suitable assay techniques can employ polyclonal or monoclonal antibodies, as has been previously described [U.S. Patent No. 4,376,110 (issued to David et al.), the disclosure of which is incorporated herein by reference] . Summary
• the present inventors also demonstrated that an imidazoline receptive site can be expressed in cells transfected with the EST 04033 cDNA clone, and this site has the proper potencies of an IR. We have deduced most of the complete cDNA encoding this protein.
• ADDRESSEE WENDEROTH, LIND & PONACK STREET: 805 Fifteenth St. N.W., Suite 700 CITY: Washington STATE: District of Columbia COUNTRY: U.S.A. ZIP: 20005 COMPUTER READABLE FORM:
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• TELEPHONE (202) 371-8850
• TELEFAX (202) 371-8856 INFORMATION FOR SEQ ID NO: 1
• SEQUENCE CHARACTERISTICS :
• ORGANISM Homo sapiens IMMEDIATE SOURCE:
• GGT GGC AAA AGA AGC ATT GCA GGT CTG ACA CTT GTG AGG CCG CTC AGA 1478 Gly Gly Lys Arg Ser lie Ala Gly Leu Thr Leu Val Arg Pro Leu Arg 15 20 25
• GCC GCC ATC CCC TAC TGG CTG TTG CTC ACG CCC CAG CAC CTC AAC GTC 2918 Ala Ala lie Pro Tyr Trp Leu Leu Leu Thr Pro Gin His Leu Asn Val 495 500 505 ATC AAG GCC GAC TTC AAC CCC ATG CCC AAC CGT GGC ACC CAC AAC TGT 2966 lie Lys Ala Asp Phe Asn Pro Met Pro Asn Arg Gly Thr His Asn Cys 510 515 520
• TYPE nucleic acid
• TYPE nucleic acid
• TGTCCCATAC CAAGTCTCAT TGATATTTCT GCAGAATATC AGATGAAAAT CTATTTCTAA 660 AGACCATTGG GAGAATGGGT GGTGGAGAAG GAGTTGGAGT GGGGTTGGGGGGCAGTTAAA 720 AATGAATAAA AATCTCTCAG CTACAGAACC CAAACATCAC TTCCCTCCGC ATTCACAGCA 780 TTTCCCAGCA GTCCCCAGAT GGTTGTTTCC GTGGGGACAC AGCAGCTGCC TCATTTCCCT 840 TCAGGCCCCA TGGGCTGCTG GTCAACCTCA GGATCTACTA AAGATGACGC AAATGCCGAC 900 TGAACAATCT GAAACCCAAA GGACTCGAGG AGAGACATGT TCTGCTGAGG AGAGAAAGGT 960
• CTGAGGCCGT CAAGTCCGCC GCCATCCCCT ACTGGCTGTT GCTCACGCCC CAGCACCTCA 2880
• GCTGTACCCA GCCTCGGGGC GCCTTTGCTG ATGGCCACGT GCTAGAGCTG CTCGTGGGGT 3060
• TYPE nucleic acid
• CTGGGCGCGG ACGAGGACTT CCTGCTGGAG CACATCCGCA TCCTCAAGGT GCTGTGGTGC 120
• TYPE nucleic acid
• TYPE nucleic acid
• TYPE nucleic acid
• TYPE nucleic acid
• TYPE nucleic acid
• CTATTTGTGT CCCACCTCCC TCTTTGTGGC CGCAAGTGCC CCTTCCTCCA CACAGTCACA 360
• CTTCAAGTGG TCTAGAGGTG ATCTGAGGTG GAGTAACAGG TCCAGATAGG CTACGTTCAT 4740
• TTCATCTGGT TTTAATCTAT CCTGGTTTTT AAAAAATGTG TCTGTGGAAG TTTAATTTTT 5460
• ATGTAGTCAC ATCTCAGTTT TTTTCCATTG CATTTATTCT CAGAATGCTT CTCCCTGCCC 5520
• TTTCAGCCTC CCTCCTTCCC TTCCCCACCC ACCTTGGTTG ATGGGAACAG GCAGTTCTCT 6120
• CTGTTTTTAG TAGGGACAGG ATTTCGCCAT GTTGGACAGT TACATTCTTA AAGGGCTGCT 7860 GAAGATCGTA TGGACATGGT AGCCCATAAA TCCCAAAATG TGTACTCTGA CCCTTTACAG 7920
• CTGTGCCAGG CACCTATGGT CCAAGCCCCT AGAAGCATAG ACTCTGACCA AACTGGCGAC 8220
• GGCTGCCAAC CAGCGGGAGG AGGGCCAGGG TGAACAGGGC GAGGAGGAGG ATGAGGAGGA 8640
• GCACATCCGC ATCCTCAAGG TGCTGTGGTG CTTCCTGATC CATGTGCAGG GCAGTATCCG 8880

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