Method for assaying for a substance that affects an SH2 phosphorylated ligand regulatory system

A method for assaying a medium for the presence of an agonist or antagonist substance of a Src homology region 2-phosphorylated ligand regulatory system comprising the steps of:

• (a) providing a Src homology region 2, and a phosphorylated ligand, the Src homology region 2 and the phosphorylated ligand being selected so that they bind to form a Src homology region 2-phosphorylated ligand complex which is capable of activating the Src homology region 2-phosphorylated ligand regulatory system, the Src homology region 2 and/or the phosphorylated ligand being present in a known concentration;
• (b) incubating said src homology region 2 and said phosphorylated ligand with a suspected agonist or antagonist substance, under conditions which permit the formation of the Src homology region 2-phosphorylated ligand complex;
• and
• (c) assaying for the Src homology region 2-phosphorylated ligand complex, free Src homology region 2, or non-complexed phosphorylated ligand and comparing to a control to determine the effect of the substance.

A method for assaying a medium for the presence of a substance that affects a Src homology region 2-phosphorylated ligand regulatory system comprising the steps of:

• (a) providing a Src homology region 2, and a phosphorylated ligand which is a Src homolog region 2 binding site on a transmembrane receptor with inducible protein-tyrosine kinase activity or on a deregulated protein-tyrosine kinase, the Src homology region 2 and the phosphorylated ligand being selected so that they bind to form a Src homology region 2-phosphorylated ligand complex, the Src homology region 2 and/or the phosphorylated ligand being present in a known concentration;
• (b) incubating said Src homology region 2 and said phosphorylated ligand with a test substance, under conditions which permit the formation of the Src homology region 2-phosphorylated ligand complex;
• (c) assaying for the Src homology region 2-phosphorylated ligand complex, free Src homology region 2, or non-complexed phosphorylated ligand;
• and
• (d) comparing to a control to determine the effect of the substance.

A method for assaying a medium for the presence of an agonist or antagonist substance of a Src homology region 2-phosphorylated ligand regulatory system, comprising the steps of:

• (a) providing a Src homology region 2, and a phosphorylated ligand which is a Src homology region 2 binding site on a transmembrane receptor with inducible protein-tyrosine kinase activity or on a deregulated protein-tyrosine kinase, the Src homology region 2 and the phosphorylated ligand being selected so that they bind to form a Src homology region 2-phosphorylated ligand complex which is capable of activating the Src homology region 2phosphorylated ligand regulatory system, the Src homology region 2 and/or the phosphorylated ligand being present in a known concentration;
• (b) incubating said src homology region 2 and said phosphorylated ligand with a suspected agonist or antagonist substance, under conditions which permit the formation of the Src homology region 2-phosphorylated ligand complex;
• (c) assaying for the Src homology region 2-phosphorylated ligand complex, free Src homology region 2, or non-complexed phosphorylated ligand;
• and
• (d) comparing to a control to determine the effect of the substance.

A method for screening for a phosphorylated ligand which is active in an SH2-phosphorylated ligand regulatory system comprising the steps of:

• (a) selecting an SH2 domain which is active in the SH2-phosphorylated ligand regulatory system;
• (b) reacting the SH2 domain with a phosphorylated ligand which is suspected of being capable of binding to the SH2 domain thereby activating the SH2-phosphorylated ligand regulatory system, under conditions which permit the SH2 domain and phosphorylated ligand to bind to form an SH2-phosphorylated ligand complex;
• (c) determining the mount of SH2-phosphorylated ligand complex, free SH2 domain, or non-complexed phosphorylated ligand;
• and
• (d) comparing in a control to determine if the phosphorylated ligand binds to Src homology region 2 domains.