Inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) mediate the activation of numerous cell types by stimulating increases in cytoplasmic free Ca2+. IP3 stimulates Ca2+ release from internal stores, and IP4 (possibly in concert with IP3) stimulates Ca2+ influx through the plasma membrane. To examine how these two cell regulators function at the receptor level, two types of reagents are needed: 1) high specific activity radiolabeled IP3 and IP4 (for measuring the binding affinity of these agents and their analogs to specific receptors) and 2) photoaffinity labels (to covalently label the receptors and to act as non-competitive inhibitors). In Phase I, phosphatidyl inositol-4-phosphate (PIP) kinase was purified and used to label PIP to produce (32P)PIP2, which was then hydrolyzed to produce (32P)IP3. In Phase II, the efficiency of labeling (32P)PIP2 will be increased from the 5-6% obtained in Phase I to a goal of >50%, and the activity of (32P)IP3 will be assayed in receptor studies. An enzyme-linked system using IP3 3- kinase will be used to produce (32P)IP4, which will also be tested in receptor assays. Finally, photoaffinity derivatives of IP3 and (32P)IP3 will be synthesized and used both to inhibit IP3 action and to label IP3 receptors.
