LOCALIZATION OF MULTIPLE FUNCTIONAL DOMAINS ON HUMAN PECAM-1 (CD31) BY MONOCLONAL-ANTIBODY EPITOPE MAPPING

PECAM-1, a cell adhesion molecule of the immunoglobulin gene (Ig) supe rfamily, has been implicated in white cell transmigration, integrin ac tivation on lymphocytes, and cell-cell adhesion. The purpose of this i nvestigation was to identify specific regions of the PECAM-1 extracell ular domain mediating these functions by identifying the location of e pitopes of bioactive anti-PECAM-1 monoclonal antibodies. The binding r egions of mAbs important in PECAM-1-mediated leukocyte transmigration (Hec 7.2 and 3D2) were mapped to N-terminal Ig-like domains. The epito pes of monoclonal antibodies that activated integrin function on lymph ocytes were dispersed over the entire extracellular region, but those that had the strongest activating effect were preferentially localized to the N-terminus of the molecule. The binding regions of mAbs that b locked PECAM-1-mediated heterophilic L-cell aggregation were located e ither in Ig-like domain 2 (NIH31.4) or Ig-like domain 6 (4G6 and 1.2). Site-directed mutagenesis further pinpointed the epitope of the 4G6 m Ab to a hexapeptide, CAVNEG, within Ig-like domain 6. These results de monstrate that PECAM-1 contains multiple functional domains. Regions w ithin N-terminal Ig-like domains appear to be required for transmigrat ion. In contrast, two distinct regions were implicated in L-cell media ted heterophilic aggregation.