Modulating robo: ligand interactions

Abstract

Disclosed are methods and compositions for identifying agents which modulate the interaction of Robo and a Robo ligand and for modulating the interaction of Robo and a Robo ligand. The methods for identifying Robo:ligand modulators find particular application in commercial drug screens. These methods generally comprise (1) combining a Robo polypeptide, a Slit polypeptide and a candidate agent under conditions whereby, but for the presence of the agent, the Robo and Slit polypeptides engage in a first interaction, and (2) determining a second interaction of the Robo and Slit polypeptides in the presence of the agent, wherein a difference between the first and second interactions indicates that the aget modulates the interaction of the Robo and Slit polypeptides. The subject methods of modulating the interaction of Robo and a Robo ligand involve combining a Robo polypeptide, a Slit polypeptide and a modulator under conditions whereby, but for the presence of the modulator, the Robo and Slit polypeptides engage in a first interaction, whereby the Robo and Slit polypeptides engage in a second interaction different from the first interaction. In a particular embodiment, the modulator is dominant negative form of the Robo or Slit polypeptide.

1 . An isolated Slit polypeptide comprising a vertebrate species-specific Slit fragment. 
     
     
         2 . A method of modulating the interaction of Robo and a Robo ligand, or said method comprising a step of
 combining a Robo polypeptide, a Slit polypeptide according to  claim 1 , and a modulator under conditions whereby, but for the presence of the modulator, the Robo and Slit polypeptide engage in a first interaction, wherein the Slit polypeptide specifically binds, activates or inhibits the activation of the Robo polypeptide and   whereby the Robo and Slit polypeptide engage in a second interaction different from the first interaction.   
     
     
         3 . A method of identifying agents which modulate the interaction of Robo and a Robo ligand, said method comprising the method of  claim 2 , wherein the steps are:
 combining a Robo polypeptide, a Slit polypeptide and a candidate agent under conditions whereby, but for the presence of the agent, the Robo and Slit polypeptides engage in a first interaction, wherein the Slit polypeptide specifically binds, activates or inhibits the activation of the Robo polypeptide and   determining a second interaction of the Robo and Slit polypeptides in the presence of the agent,   wherein a difference between the first and second interactions indicates that the agent modulates the interaction of the Robo and Slit polypeptides   
     
     
         4 . A method according to  claim 3 , wherein the modulator is a dominant negative form of the Robo or Slit polypeptide. 
     
     
         5 . An isolated vertebrate Slit polypeptide according to  claim 1 , wherein said vertebrate is human, mouse or rat. 
     
     
         6 . A recombinant nucleic acid encoding a vertebrate Slit polypeptide according to  claim 5 . 
     
     
         7 . A recombinant nucleic acid according to  claim 6  and comprising a strand of SEQ ID NO:01, or a fragment thereof having at least 24 consecutive nucleotides thereof, and sufficient to specifically hybridize with a polynucleotide having the sequence defined by the corresponding opposite strand of SEQ ID No:01, and is other than a natural drosophila Slit sequence. 
     
     
         8 . A method for specifically detecting a vertebrate Slit protein, comprising the steps of:
 specifically binding an antibody according to  claim 8  to the Slit protein; and   specifically detecting a resultant specific binding as an indication of the presence of the Slit protein.