DNA molecules encoding imidazoline receptive polypeptides and polypeptides encoded thereby

Abstract

A genomic DNA encoding a human imidazoline receptor is described. cDNAs encoding the receptor and fragments thereof are also provided. An amino acid sequence predicted to be 120,000 MW for nearly the entire protein is identified, as well as a middle fragment believed to contain the imidazoline binding site of the receptor. The protein is highly unique in its sequence and may represent the first in a novel family of receptor proteins. Methods of cloning the cDNA and expressing the imidazoline receptor in a host cell are described. Methods of preparing antibodies against the transfected protein are also described. Also, a screening method for identifying additional subtypes of this receptor are identified. Also, screening methods for identifying drugs that interact with the imidazoline receptor are described.

What is claimed is:  
     
         1 . A DNA molecule encoding for a polypeptide including an amino acid sequence which is receptive to imidazoline compounds, said DNA molecule containing a DNA sequence with at least 75% sequence similarity with the DNA sequence shown in SEQ ID No. 4.  
     
     
         2 . A DNA molecule according to  claim 1 , containing a DNA sequence with at least 75% sequence similarity with the DNA sequence shown in SEQ ID No. 2.  
     
     
         3 . A DNA molecule according to  claim 2 , containing a DNA sequence with at least 75% sequence similarity with the DNA sequence of SEQ ID No. 3.  
     
     
         4 . A DNA molecule according to  claim 3 , containing a DNA sequence with at least 75% sequence similarity with the DNA sequence of SEQ ID No. 1.  
     
     
         5 . A DNA molecule according to any one of  claims 1  to  4 , containing a DNA sequence with at least 80% sequence similarity with the sequence of said SEQ ID No.  
     
     
         6 . A DNA molecule according to any one of  claims 1  to  4 , containing a DNA sequence with at least 85% sequence similarity with the sequence of said SEQ ID No.  
     
     
         7 . A DNA molecule according to any one of  claims 1  to  4 , containing a DNA sequence with at least 90% sequence similarity with the sequence of said SEQ ID No.  
     
     
         8 . A DNA molecule according to any one of  claims 1  to  4 , containing a DNA sequence with at least 95% sequence similarity with the sequence of said SEQ ID No.  
     
     
         9 . A DNA molecule according to  claim 1 , which is deposited with the ATCC under deposit accession no. ATCC 209217.  
     
     
         10 . A genomic DNA molecule encoding for a polypeptide including an amino acid sequence which is receptive to imidazoline compounds, and wherein exon portions of said genomic DNA molecule include the DNA sequence as defined in  claim 1 .  
     
     
         11 . A genomic DNA molecule according to  claim 10 , which is deposited with the ATCC under deposit accession no. ATCC 209216.  
     
     
         12 . A 1110 bp ApaI-EcoRI restriction fragment of the DNA molecule according to  claim 1 .  
     
     
         13 . A 1.85 kb EcoRI restriction fragment of the DNA molecule according to  claim 4 .  
     
     
         14 . A vector containing a DNA sequence as defined in any one of claims  1 - 13 .  
     
     
         15 . A host cell transfected with a vector as defined in  claim 14 .  
     
     
         16 . An isolated polypeptide including a site which is receptive to imidazoline compounds, said polypeptide containing an amino acid sequence with at least 80% sequence similarity with the amino acid sequence shown in SEQ ID No. 6.  
     
     
         17 . A polypeptide as defined in  claim 16 , having a molecular weight of about 35 to 45 kDa.  
     
     
         18 . A polypeptide as defined in  claim 17 , having a molecular weight of about 37 kDa.  
     
     
         19 . An isolated polypeptide including a site which is receptive to imidazoline compounds, said polypeptide containing an amino acid sequence with at least 80% sequence similarity with the amino acid sequence shown in SEQ ID No. 5.  
     
     
         20 . A polypeptide as defined in  claim 19 , having a molecular weight of about 60 to 85 kDa.  
     
     
         21 . A polypeptide as defined in  claim 20 , having a molecular weight of about 70 kDa.  
     
     
         22 . A fragment of the amino acid sequence shown in SEQ ID No. 5 or 6, which fragment is receptive to imidazoline compounds.  
     
     
         23 . A polypeptide according to any one of  claims 16  to  22 , which is immunoreactive with at least one of Reis antiserum and Dontenwill antiserum.  
     
     
         24 . A polypeptide according to any one of  claims 16  to  23 , which is a human polypeptide.  
     
     
         25 . A method of producing an isolated polypeptide including an amino acid sequence which is receptive to imidazoline compounds, said method comprising: 
 transfecting a host cell with a vector as defined in  claim 14;  and    culturing the transfected host cell in a culture medium to express the polypeptide.    
     
     
         26 . An isolated polypeptide including an amino acid sequence which is receptive to imidazoline compounds, which polypeptide is expressed by the method of  claim 25 .  
     
     
         27 . A method of screening for a ligand of an imidazoline receptor, which method comprises: 
 culturing a host cell as defined in  claim 15  in a culture medium to express a polypeptide including an amino acid sequence which is receptive to imidazoline compounds;    contacting said polypeptide with a labelled ligand for the imidazoline receptor under conditions effective to bind the labelled ligand thereto;    contacting said polypeptide with a candidate ligand; and    detecting any displacement of the labelled ligand from said polypeptide, wherein displacement signifies that the candidate ligand is a ligand for the imidazoline receptor.    
     
     
         28 . The method of  claim 27 , wherein said contacting steps are performed in an intact cultured host cell.  
     
     
         29 . The method of  claim 27 , further comprising isolating the cell membrane of said cultured host cell prior to performing said contacting steps.  
     
     
         30 . The method of  claim 27 , wherein said contacting of said imidazoline receptive polypeptide with said candidate ligand is conducted at a plurality of candidate ligand concentrations.  
     
     
         31 . The method of  claim 27 , wherein the labelled ligand is radiolabelled.  
     
     
         32 . A method of obtaining a DNA material encoding a polypeptide which is receptive to imidazoline compounds, said method comprising: 
 providing a labelled DNA probe by labelling a DNA molecule identical or complementary to a DNA molecule as defined in any one of  claims 1  to  9  or a restriction fragment thereof;    contacting said DNA probe with genetic material suspected of encoding said imidazoline receptive polypeptide;    hybridizing said DNA probe and said genetic material under stringent hybridization conditions;    identifying any portion of the genetic material which hybridizes to said DNA probe; and    isolating said identified material.    
     
     
         33 . A method according to  claim 32 , wherein the genetic material is derived from a library selected from the group consisting of RNA library, cDNA library and genomic DNA library.  
     
     
         34 . A method according to  claim 33 , wherein said library is a human library.  
     
     
         35 . A method according to  claim 32 , wherein the labelled DNA probe is provided by labelling a restriction fragment according to  claim 12  or  13 .  
     
     
         36 . A method of raising antibodies immunoreactive with a polypeptide which is receptive to an imidazoline compound, which method comprises: 
 injecting an animal with a polypeptide as defined in any one of  claims 16  to  24  and  26 ; and    isolating antibodies produced by the animal.