Compositions and methods for identifying modulators of transducisomes, a new class of therapeutic targets

Abstract

The invention provides cells and methods for identifying modulators of signal transduction, based on transducisome proteins that coordinate and assemble many types of signal transduction proteins. A transducisome is a PDZ domain containing protein that binds at least one signal transduction protein or a PDZ domain containing protein with at least one signal transduction protein bound. Examples of transducisome proteins include INAD, GRIP and other recently identified multi-PDZ domain proteins. Examples of signal transduction proteins include GPCRs, tyrosine kinase receptors, tyrosine phosphatase receptors, ion channels, phospholipases, adenylate cyclases, kinases and G-proteins. Also provided are methods for identifying modulators of signal transduction, proteins (and polynucleotides encoding the same) corresponding to transducisomes, modified transducisomes or defective transducisomes to use in assays of signal transduction, and a screening assay system for detecting protein-protein interactions.

1 . A fly comprising an amino acid mutation in a transducisome protein that prevents functional binding of a signal transduction protein, wherein said amino acid mutation is not a naturally occurring mutation of inaD.  
     
     
         2 . The fly of  claim 1 , wherein said amino acid mutation results from a mutation selected from the group consisting of inaD 2  and inaD 1 .  
     
     
         3 . An isolated cell comprising a polynucleotide encoding a transducisome protein with an amino acid mutation that prevents functional binding of a signal transduction protein, wherein said amino acid mutation is a naturally occurring mutation of inaD.  
     
     
         4 . The cell of  claim 3 , wherein said cell is a fly cell.  
     
     
         5 . The cell of  claim 4 , wherein said amino acid mutation results from a mutation selected from the group consisting of inaD 2  and inaD 1 .  
     
     
         6 . An isolated polynucleotide comprising a coding region for a transducisome protein with an amino acid mutation in a PDZ domain that prevents functional binding of a signal transduction protein, wherein said amino acid mutation is not a naturally occurring mutation inaD 215 .  
     
     
         7 . The isolated polynucleotide of  claim 6 , wherein said transducisome protein is INAD.  
     
     
         8 . The isolated polynucleotide of  claim 7 , wherein said amino acid mutation results from a mutation selected from the group consisting of inaD 2  and inaD 1 .  
     
     
         9 . An isolated protein comprising a polypeptide of SEQ ID NO.:1 with an amino acid mutation in a PDZ domain that prevents functional binding of a signal transduction protein, wherein said amino acid mutation is not a naturally occurring mutation inaD 215 .  
     
     
         10 . The isolated protein of  claim 9 , wherein said amino acid mutation results from a mutation selected from the group consisting of inaD 2  and inaD 1 .  
     
     
         11 . A chimeric transducisome protein comprising at least one first PDZ domain that binds a first signal transduction protein and at least one second PDZ domain binds a second signal transduction protein, wherein said chimeric transducisome protein is not a naturally occurring protein.  
     
     
         12 . The chimeric transducisome protein of  claim 11 , wherein said first signal transduction protein is selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein.  
     
     
         13 . The chimeric transducisome protein of  claim 11 , wherein said second signal transduction protein is selected from the groups PKC, TRP, and PLCβ.  
     
     
         14 - 29 . (canceled)  
     
     
         30 . An isolated, non-naturally occurring cell, comprising: 
 a) a heterologously expressed transducisome protein comprising one or more PDZ domains and    b) an expressed protein comprising a signal transduction protein that binds to one or more said PDZ domains.    
     
     
         31 . An isolated, non-naturally occurring cell, comprising: 
 a) a cell capable of expressing: 
 i) a non-naturally occurring polynucleotide comprising a coding region for a transducisome protein comprising one or more PDZ domains and  
 ii) a non-naturally occurring polynucleotide comprising a coding region for a heterologous protein comprising a signal transduction protein.  
   
     
     
         32 . The isolated, non-naturally occurring cell of  claim 31 , wherein said signal transduction protein is selected from the group of a kinase, a phosphatase, a GPCR, a tyrosine kinase receptor, a tyrosine phosphatase receptor, an ion channel, a G-protein, a phospholipase and a calcium binding protein.  
     
     
         33 . The isolated, non-naturally occurring cell of  claim 32 , wherein further comprising a test chemical.  
     
     
         34 . The isolated, non-naturally occurring cell of  claim 33 , wherein further comprising a signal transduction detection system.  
     
     
         35 - 40 . (canceled)