ALPHA2 BETA1 INTEGRIN (VLA2) LIGAND INTERACTION

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Integrin alpha2beta1 (VLA-2) is receptor for collagen, laminin, Echovirus 1 and integrin alpha3beta1 (VLA-3). The alpha2beta1-ligand interaction may be a potential therapeutic target for many diseases. We recently identified the I domain of the alpha2 subunit as the ligand binding site by mapping the epitopes of function-inhibiting anti-alpha2 monoclonal antibodies (mAbs) utilizing human/bovine alpha2 chimeras and site-directed mutagenesis. Also we found a recombinant I domain fragment has collagen binding function, suggesting the I domain of alpha2 may have all the necessary components for ligand binding. Also, we identified the activation-dependent epitope of the beta1 subunit, which is induced upon activation by activating anti beta1 mAb, or stimulation of cells (e.g., T cells) by phorbol ester. Also a beta1 mutant (D130A) was found to express constitutively the activated epitope, indicating that the mutant has a conformation of highly activated integrins, while the mutation inactivate the ligand binding function. To understand the alpha2beta1 integrin adhesive function at the molecular level based on these preliminary data, we propose to: 1) identify residues in the I domain that are involved in ligand-alpha2beta1 interaction using site-directed mutagenesis, synthetic peptides and recombinant alpha2 fragments (fusion protein with glutathione S-transferase, designated GST-alpha2I); 2) study the alpha2beta1/ligand interaction by measuring binding of the recombinant I domain fragment to ligand and by visualizing the collagen/recombinant I domain fragment interaction by electron microscopy; 3) study the role of the beta1 subunit in the regulation of alpha2beta1 activity using mutants of Asp13O or recombinant beta1 fragments. The proposed work will define the residues and regions of the I domain of the alpha2 subunit that recognize ligands and the residues and regions of collagen that interact with alpha2beta1. Furthermore, the role of the beta1 subunit in regulation of alpha2beta1 function will be studied. Also synthetic peptides or recombinant fragments of the I domain, or recognition sequences in ligands (e.g., collagen) might affect the interactions between alpha2beta1 and ligands, and thus may represent potential inhibitors for immune response, viral infection, thrombus formation, tumor progression or metastasis.