CD31 is a recently described trans-membrane protein which belongs to the immunoglobulin gene family of cell surface adhesion molecules. CD31 expression is restricted to vascular endothelium, leukocytes and platelets. In vascular endothelial cells, CD31 is localized to intercellular junctions. We have obtained several lines of data to suggest that CD31 may play a role in the acute inflammatory response of endothelium and in initial adhesion interactions occurring in the immune response: 1. Stimulation of platelets or endothelial ells with acute inflammatory agonists such as thrombin of histamine or T cell activation with phytohemagglutinin results in immediate phosphorylation of CD31 by a protein kinase C-dependent mechanism. 2. After T lymphocyte activation, CD31 expression is down-regulated. 3. A CD31 monoclonal antibody, LYP21, inhibits T lymphocyte activation in the allogeneic mixed lymphocyte reaction. The goals of this project build upon our earlier work with CD31.
Aim #1 : Characterization and functional consequences of CD31 phosphorylation. The stoichiometry of CD31 phosphorylation after cell activation will be determined. The CD31 amino acids which are phosphorylated with cell activation will be determined by incorporation of radiolabeled phosphate during platelet activation, followed by acid hydrolysis and amino acid chromatography. The functional consequences of CD31 phosphorylation will be determined by utilizing a homotypic adhesion model. CD31 protein will be purified and coated onto plastic in phosphorylated and unphosphorylated forms. COS cells transfected with CD31 cDNA will be tested for binding to purified CD31 under resting and PKC- activated conditions to determine: a) whether CD31 binding is homotypic or heterotypic and b) the effects of phosphorylation upon CD31 binding affinity.
Aim #2 : Determination of CD31 adhesion domains. The CD31 monoclonal antibody LYP21 interferes with the allogeneic mixed lymphocyte reaction, suggesting that this epitope may be a functionally important domain on the CD31 molecule. Based on our cloning data, we have tentatively localized the probable LYP21 domain to a 23 amino acid region of the molecule. The localization of adhesion epitopes of the related molecule ICAM-1 to the two most distal domains makes it logical that CD31 adhesive function is also at least in part mediated by epitopes in domains 1 and 2. Synthetic CD31 peptides based on the LYP21 epitope and sequences from the first and second domains predicted to be hydrophilic, exposed and available for binding interactions will be synthesized and tested for effects on CD31 binding.
Aim #3 : Biological function of CD31. Knowledge and reagents gained from the above aims will be used in two model systems to test the biological function of CD31. Endothelial permeability, endothelial-leukocyte adhesion and leukocyte trans-migration will be studied using cultured EC grown in a two chamber system separated by a porous membrane. The role of CD31 in T cell adhesion will be studied using the allogeneic mixed lymphocyte reaction. It is likely that other function roles of CD31 will become apparent as more is learned about this molecule. The detailed elucidation of the biology of CD31, in terms of its structure-function relationship and regulation of expression may shed important insights in our understanding of the molecular events which occur in the initial stages of T cell activation and the host response to acute inflammatory stimuli. Beyond the inherent scientific interest of studying such cellular adhesion phenomena, a more detailed understanding of these fundamental processes has the potential for addressing problems of great clinical importance, including the therapy of viral diseases, acute and chronic inflammatory diseases, dissection of adhesion phenomena required for generation of the immune response and hematogenous metastasis of malignant cells.
