Functional analyses of two cellular binding domains of bovine lactadherin

The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises t wo EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD- dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadh erin and different mammalian cell types were investigated. Using recombinan t forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expr essing the alpha(v)beta(5) integrin receptor were shown to bind specificall y to RGD containing lactadherin but not to a mutated RGE lactadherin. A mon oclonal antibody against the alpha(v)beta(5) integrin receptor and a synthe tic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadh erin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lacta dherin via both integrins. To investigate the interaction of lipid with lac tadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentrat ion-dependent manner with an affinity similar to native lactadherin (K-d = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain in hibited the binding of lactadherin to phospholipid in a concentration-depen dent manner, and finally it was shown that lactadherin mediates binding bet ween artificial phosphatidyl serine membranes and MCF-7 cells. Taken togeth er these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipi ds through its C-terminus.