EndoCAM/PECAM (CD31) is a newly described member of the immunoglobulin superfamily that is expressed at the cell-cell borders of endothelial cells where it appears to function in the initiation of cell-cell contact. The molecule has been cloned and we have recently expressed the protein in nonendothelial mammalian cells. Because it is also present on the surface of platelets and some leukocytes, endoCAM/PECAM may also be a more general vascular cell adhesion molecule involved in inflammation, thrombosis and wound healing. A more complete understanding of endoCAM/PECAM could thus lead to novel approaches to the treatment of inflammatory and vascular disease. The long term goal of this proposal Is to more fully characterize the structure and functions of endoCAM/PECAM. To accomplish this goal, we will use a molecular biologic approach by expressing and analyzing native and mutated endoCAM/PECAM cDNA.
The specific aims of this proposal are: 1) To determine If endoCAM/PECAM binds homophilically or heterophilically and to study the characteristics of this binding. The approach will include aggregation assays using transfected cells, binding studies using purified endoCAM/PECAM protein incorporated into covaspheres and liposomes, analysis of the binding characteristics of soluble endoCAM/PECAM protein to the surface of cells, and analysis of other cellular proteins that may bind to immobilized endoCAM/PECAM proteins. 2) To identify regions of the extracellular and cytoplasmic domains of endoCAM/PECAM that are Important In ligand Interactions and cellular targeting. The structure/function relationships of the extracellular domain will be evaluated by creating a series of deletion mutants and soluble fragments to study binding and/or inhibition of ligand binding. Point mutations in areas of interest will follow. In order to preserve tertiary structure, chimeric receptors will be prepared by exchanging domains of endoCAM/PECAM with portions of other members of the immunoglobulin superfamily with similar structure but different functions. The role of the cytoplasmic domain in membrane targeting and overall function will be studied by the creation and analysis of a series of deletion mutants, point mutations, and chimeric receptors containing cytoplasmic regions of other adhesion molecules. These studies will result in an improved understanding of the characteristics of this novel vascular cell adhesion molecule and may ultimately lead to the development of novel therapeutic agents that may be of use in the treatment of inflammatory or thrombotic disorders.
