The overall objective of the proposed research is to establish the mechanism of action of intracellular second messengers at the molecular level. The primary model will be how Ca++ acts to alter cell function via interaction with its ubiquitous intracellular receptor, calmodulin. We will focus on three areas. First it will be determined whether Ca++ plays a role in maintaining the intracellular levels of calmodulin. This will be accomplished by manipulating intracellular Ca++ levels and measuring calmodulin, calmodulin mRNA and rate of transcription of the calmodulin gene. We will evaluate the portion of the calmodulin gene necessary for Ca++ regulation by gene transfer methods. Finally we will use DNA/protein binding methods to determine if calmodulin interacts specifically with regions of the calmodulin gene.
The second aim i s to evaluate the consequences of altered calmodulin levels on cell functions such as growth, proliferation, and cell cycle. Eukaryotic expression vectors will be used to introduce calmodulin genes into cells under control of regulatable promoters. The constructions will allow production of both the sense and antisense strands of cDNA. Third we will evaluate the specific interaction of calmodulin with a model target enzyme, myosin light chain kinase (MLCK). The remainder of the MLCK cDNA will be cloned and the calmodulin binding fragment identified by the use of bacterial expression vectors and calmodulin binding assays of the peptides produced. The precise molecular interaction of MLCK and calmodulin will be solved by crystallographic procedures. Finally we will determine whether differences exist in the calmodulin binding domains of a phosphatase (calcineurin) and a multisubstrate kinase, calmodulin kinase II. These studies will provide the first details of interaction of an intracellular receptor with its acceptor proteins in chemically precise terms.
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