Cyclic AMP phosphodiesterase isoforms and methods of use

Abstract

Human and rat cAMP phosphodiesterase isoforms (denoted PDE4Ds), as well as the DNA (RNA) encoding such polypeptides, are disclosed. Also disclosed are methods for utilizing such polypeptides in diagnostic assays for identifying mutations in nucleic acid sequences encoding the polypeptides of the present invention, for detecting altered levels of the polypeptide of the present invention as a means of detecting diseases and methods of screening potential modulators, especially inhibitors, of the novel PDE4Ds (denoted PDE4D6) disclosed herein as a means of increasing cyclic AMP in neurons and thus treating neurological problems, such as long term memory loss, if not preventing such maladies entirely. Transgenic animals expressing polypeptides disclosed herein are also described.

What is claimed is:  
     
         1 . An isolated polypeptide comprising a segment having an amino acid sequence at least 65% identical to SEQ ID NO: 5.  
     
     
         2 . The isolated polypeptide of  claim 1  wherein the segment is at least 80% identical to SEQ ID NO: 5.  
     
     
         3 . The isolated polypeptide of  claim 1  wherein the segment is at least 95% identical to SEQ ID NO: 5.  
     
     
         4 . The isolated polypeptide of  claim 1  wherein the segment is the sequence of SEQ ID NO: 5.  
     
     
         5 . An isolated polypeptide comprising a segment of SEQ ID NO: 5 and containing at least 10 amino acids.  
     
     
         6 . The isolated polypeptide of  claim 5  wherein the segment contains at least 12 amino acids.  
     
     
         7 . The isolated polypeptide of  claim 6  wherein the segment contains at least 14 amino acids.  
     
     
         8 . The isolated polypeptide of  claim 7  wherein the segment contains at least 15 amino acids.  
     
     
         9 . A phosphodiesterase comprising a polypeptide selected from the group consisting of the polypeptides of claims  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , and  8 .  
     
     
         10 . An isolated polypeptide selected from the group consisting of the polypeptides of claims  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , and  8 .  
     
     
         11 . The isolated polypeptide of  claim 10  wherein said polypeptide is purified.  
     
     
         12 . An isolated 15-mer containing a segment of at least 12 amino acid residues of SEQ ID NO:5.  
     
     
         13 . The isolated 15-mer of  claim 12  wherein the segment contains at least 14 amino acid residues of SEQ ID NO:5.  
     
     
         14 . An isolated polynucleotide coding for a polypeptide selected from the group consisting of the polypeptides of claims  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , and  8 .  
     
     
         15 . The polynucleotide of  claim 14  wherein the polynucleotide is a DNA.  
     
     
         16 . The polynucleotide of  claim 15  wherein the DNA is a cDNA.  
     
     
         17 . The polynucleotide of  claim 14  wherein the polynucleotide is RNA.  
     
     
         18 . The complement of the polynucleotide of  claim 15 .  
     
     
         19 . An antibody specific for a polypeptide only when said polypeptide contains the segment of claims  1 ,  2 ,  3 ,  4 ,  5 ,  6 ,  7 , or  8 .  
     
     
         20 . The antibody of  claim 19  wherein said antibody is a monoclonal antibody.  
     
     
         21 . A method of making a recombinant vector comprising inserting the polynucleotide of  claim 15  into a vector.  
     
     
         22 . A recombinant vector comprising the polynucleotide of  claim 15 .  
     
     
         23 . A recombinant cell containing the polynucleotide of  claim 14 .  
     
     
         24 . A method for producing a polypeptide comprising expressing from the recombinant cell of  claim 23  the polypeptide encoded by said polynucleotide.  
     
     
         25 . A process for diagnosing a disease in an animal afflicted therewith, or diagnosing a susceptibility to a disease in an animal at risk thereof, wherein said disease is related to an over-expression of a phosphodiesterase of  claim 9  comprising determining the level of said phosphodiesterase activity in a cell from said animal.  
     
     
         26 . The process of  claim 25  wherein said animal is a human.  
     
     
         27 . The method of  claim 25  wherein the cell is a neuron.  
     
     
         28 . The method of  claim 25  wherein said disease involves the loss of memory.  
     
     
         29 . The method of  claim 28  wherein said loss of memory is a loss of long term memory.  
     
     
         30 . A process for diagnosing a disease in an animal afflicted therewith, or a susceptibility to a disease in an animal at risk thereof, comprising determining a mutation in the genome of a neuron from said animal and wherein the mutation occurs in a nucleotide sequence coding for the 15-mer of SEQ ID NO:5.  
     
     
         31 . The process of  claim 30  wherein said animal is a human.  
     
     
         32 . The process of  claim 30  wherein said disease involves a loss of memory.  
     
     
         33 . The process of  claim 32  wherein said loss of memory is a loss of long term memory.  
     
     
         34 . A process for screening chemical agents for phosphodiesterase modulating activity comprising: contacting a cell expressing a polypeptide coded for by the polynucleotide of  claim 10  with a chemical agent having potential phosphodiesterase inhibitory activity and measuring the level of total cyclic adenosine monophosphate (cAMP) in said cell following said exposure wherein an altered level of cAMP in said cell following said exposure is an indicator of such moderating activity.  
     
     
         35 . The method of  claim 34  wherein the modulating activity is inhibitory activity.  
     
     
         36 . A transgenic animal comprising within its genome one or more copies of the polynucleotide of  claim 15 .  
     
     
         37 . The transgenic animal of  claim 36  wherein said animal overproduces the expression product of said polynucleotide relative to a non-transgenic animal.  
     
     
         38 . The transgenic animal of  claim 37  wherein said animal is a mouse.  
     
     
         39 . A transgenic animal whose genome lacks a gene expressing a functional PDE4D6 isoform or functional analog thereof.  
     
     
         40 . The transgenic animal of  claim 39  wherein said animal is a mouse.  
     
     
         41 . A transgenic non-human animal whose genome comprises one or more genes coding for the human isoform of PDE4D6 in place of the mammalian gene otherwise coding for said isoform.  
     
     
         42 . The transgenic animal of  claim 41  wherein said animal is a mouse.