PECAM-1 (CD31, endoCAM) is a member of the immunoglobulin superfamily present on endothelial cells, leukocytes and platelets implicated in angiogenesis, leukocyte transmigration, and leukocyte activation. The goal of this proposal is to study the molecular basis of PECAM-1 function. Preliminary data indicate that PECAM-I functions as an adhesion molecule capable of mediating either heterophilic (PECAM-to unknown counter- receptor) or homophilic (PECAM to PECAM) binding depending on the structure of the cytoplasmic domain. Multiple alternatively spliced isoforms of murine PECAM-1 have recently been identified and it appears that presence of exon 14 within the cytoplasmic domain regulates ligand specificity. To understand the molecular basis of this unique property and to define the biological significance of alternatively spliced isoforms, the following specific aims are proposed: 1. To precisely localize the regions of the cytoplasmic domain that regulate the ability of PECAM-1 to bind in a homophilic or heterophilic fashion and to define the mechanisms by which the cytoplasmic domain regulates PECAM-1 function. This will be accomplished by construction and characterization of PECAM-1 mutants with deletions and point mutations based on the results of the naturally occurring isoforms that will allow more precise localization of the """"""""regulatory"""""""" region(s) with the cytoplasmic domain, exploration of mechanisms by which the cytoplasmic domain regulates PECAM-1 function, and use of chimeric constructs containing various isoforms of the cytoplasmic domain of PECAM-1 transferred by adenoviral vectors to endothelial cells. 2. To define the regions of the extracellular domain responsible for heterophilic or homophilic binding. This will be accomplished by mapping the epitopes of a series of bioactive murine specific anti -PECAM-1 antibodies, evaluation of PECAM-1 constructs containing specific point mutations in key regions identified by epitope mapping, and by testing the ability of peptides that mimic the putative functional regions of the extracellular domain to block homophilic and heterophilic PECAM-1- mediated aggregation. 3. To determine the biological significance of the multiple PECAM-1 isoforms. This will be accomplished by evaluating peptides that mimic the putative functional regions of the extracellular domain for their ability to block human leukocyte transmigration through an endothelial cell monolayer, testing the ability of peptides with defined in vitro functions to block leukocyte transmigration into thioglycollate stimulated peritoneum in an in vivo model of murine inflammation, and by determination of in vitro and in vivo expression status of muPECAM-l isoforms. These studies will provide both a better understanding of the basic mechanisms of cell-cell adhesion and of the structure/function relationships of PECAM-I and may thus ultimately be of value in designing therapies for inflammatory and malignant diseases.
