Tissue protective cytokine receptor complex, assays for identifying tissue protective compounds and uses thereof

A method for identifying a compound that modulates a tissue protective activity, comprising:

• (a) contacting a test compound with a tissue protective cytokine receptor complex-expressing cell;
• and
• (b) measuring the level of tissue protective cytokine receptor complex activity in the cell;
• (c) identifying a test compound which increases or decreases the tissue protective cytokine receptor complex activity as compared to the level of tissue protective cytokine receptor complex activity measured in the absence of the test compound;
• and
• (d) assaying for the identified test compound for a tissue protective activity.

A method for identifying a compound that modulates a tissue protective activity, comprising:

• (a) contacting a test compound with a cell which is recombinantly engineered to express an EPO receptor, wherein said cell is transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a β common receptor polypeptide;
• (b) measuring the level of tissue protective cytokine receptor complex activity in the cell;
• (c) identifying a test compound that increases or decreases the level of tissue protective cytokine receptor complex activity in the cell relative to the level of tissue protective cytokine receptor complex activity measured in a control cell, wherein the control cell is of the same cell type as the cell of step (a) and is not transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a β common receptor polypeptide;
• and
• (d) assaying the identified test compound for a tissue protective activity.

A method for identifying a compound that modulates a tissue protective activity, comprising:

• (a) contacting a test compound with a recombinant cell that expresses a β common receptor, wherein said cell is transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a EPO receptor polypeptide;
• (b) measuring the level of tissue protective cytokine receptor complex activity in the cell;
• (c) identifying a test compound that increases or decreases the level of tissue protective cytokine receptor complex activity in the cell relative to the level of tissue protective cytokine receptor complex activity measured in a control cell, wherein the control cell is of the same cell type as the cell of step (a) and is not transformed with a nucleic acid comprising a nucleotide sequence that (i) is operably linked to a promoter, and (ii) encodes a β common receptor polypeptide;
• and
• (d) assaying the identified test compound for a tissue protective activity.

A method of identifying a compound that modulates a tissue protective activity, comprising:

• (a) contacting a test compound with a cell comprising:
• (i) a nucleic acid sequence comprising a reporter gene operably liked to a binding site specific for a DNA binding domain of a transcriptional activator;
• (ii) a first fusion protein comprising (A) the DNA binding domain of the transcriptional activator, and (B) a first tissue protective cytokine receptor polypeptide or a fragment thereof;
• and
• (iii) a second fusion protein comprising (A) an activation domain of the transcriptional activator and (B) a second tissue protective cytokine receptor,
• (b) detecting reporter gene expression,
• such that if the reporter gene expression in (b) differs relative to the reporter gene expression detected in the absence of the test compound, a compound that modulates a tissue protective activity is identified.

A method for identifying a compound that binds to a tissue protective cytokine receptor complex, comprising:

• (a) contacting a tissue protective cytokine receptor complex with (i) a tissue protective cytokine receptor complex ligand attached to a first label and (ii) an equivalent amount of a test compound attached to a second label under conditions conducive to binding;
• (b) removing unbound material from the tissue protective cytokine receptor complex;
• and
• (c) detecting the level of the first and second labels wherein if the second label is present the compound binds the complex and if the level of the first label decreases relative to the level of the first label where the labeled ligand is contacted with a tissue protective cytokine receptor complex under conditions conducive to binding in the absence of a test compound after removal of unbound material,
• then a compound that binds to a tissue protective cytokine receptor complex y is identified.

A method for identifying a compound that modulates the binding of a tissue protective cytokine receptor complex ligand to a tissue protective cytokine receptor complex, comprising:

• (a) contacting a tissue protective cytokine receptor complex ligand with a tissue protective cytokine receptor complex in the presence of one or more test compounds under conditions conducive to binding;
• and
• (b) measuring the amount of tissue protective cytokine receptor complex ligand bound to the tissue protective cytokine receptor complex;
• such that if the amount of bound tissue protective cytokine receptor complex ligand measured in (b) differs from the amount of bound tissue protective cytokine receptor complex ligand measured in the absence of the one or more test compounds, then a compound that modulates the binding of a tissue protective cytokine receptor complex ligand to the tissue protective cytokine receptor complex is identified.

A method for identifying a compound that modulates the interaction between a tissue protective cytokine receptor complex and a tissue protective cytokine receptor complex ligand, comprising:

• (a) contacting a tissue protective cytokine receptor complex with one or more test compounds;
• and
• (b) measuring the tissue protective cytokine receptor complex activity,
• such that if the activity measured in (b) differs from the tissue protective cytokine receptor complex activity in the absence of the one or more test compounds, then a compound that modulates the interaction between the tissue protective cytokine receptor complex and the tissue protective cytokine receptor complex ligand is identified.

A method for identifying a compound that binds a tissue protective cytokine receptor complex, comprising:

• (a) contacting a test compound with a ligand-binding tissue protective receptor complex fragment comprising at least one EPO receptor extracellular domain and at least one β common receptor extracellular domain fused to an Fc fragment attached to a solid support;
• and
• (b) removing unbound test compounds from the solid support;
• (c) identifying the compound attached to the tissue protective cytokine receptor complex fragment,
• such that a compound bound to the solid support is identified as a compound that binds to a tissue protective cytokine receptor complex.

A method for identifying a compound that modulates a tissue protective activity in a mammal, comprising:

• (a) administering the compound to a first animal immediately following infliction of an injury, wherein the first animal endogenously expresses a tissue protective cytokine receptor complex;
• and
• (b) administering the compound to a second animal immediately following infliction of the same injury as in step (a), wherein the second animal is deficient in expression of a tissue protective cytokine receptor complex;
• such that if recovery from the injury differs in the animal of step (a) as compared to the animal of step (b), a compound that modulates a tissue protective activity is identified.