Before the enormous therapeutic potential of integrins can be fully exploited, the structural basis for their adhesion functions must be determined. Prominent unanswered questions are: What is the biochemical basis for an integrin having multiple functional forms? What are the functional consequences of an integrin having multiple structural forms? What domains are most important for ligand binding? What cell-cell adhesion ligands remain undiscovered? We propose to address these questions by analyzing three prototype integrins (VLA-2, VLA-3, and VLA-4), with the following specific aims: [1] Monoclonal antibodies will e generated which discriminate between i) VLA-2 that binds collagen and laminin, ii) VLA-2 that binds only collagen, and iii) VLA-2 that binds to neither. These """"""""function specific"""""""" antibodies will then be characterized using flow cytometry, adhesion blocking/stimulation assays, and rodent- human chimeras for epitope mapping. [2] We will use site-directed mutagenesis to prevent the conversion of VLA-4 (beta1alpha4/150) into either of two unique biochemical forms (beta1alpha4/80,70 and beta1alpha4/180), not seen for other integrin alpha subunits. Then, VLA-4 containing either alpha4/150, alpha4/80,70, or alpha4/180) will be expressed, and all known VLA-4 adhesion functions will be compared. Using monoclonal antibodies which can discriminate between the structural forms, their distribution, and regulation of interconversion will be analyzed. Also, to learn why alpha4/180 differs from alpha4/150, N-terminal amino acid sequencing will be done on purified alpha4/180. [3] We will use rodent/human alpha chain chimeras to map functional MAb binding sites within the VLA-2, VLA-3 and VLA-4 heterodimers. Then, key regions (presumably divalent cation sites and the alpha2 I-domain) will be altered by mutagenesis. Conserved ASP and ASN residues with putative cation sites will be mutated, and the functional consequences for metal-dependent adhesion will be determined. Also, peptides corresponding to the I-domain of alpha2 and cation sites of alpha2, alpha3, and alpha4 will be made, and tested for inhibition of adhesion. [4] We will search for new VLA-2, VLA- 3, and VLA-4 ligands, using cell-cell adhesion assays, involving cells transfected with alpha2, alpha3, or alpha4 cDNA. If we observe adhesion not explainable by currently known ligands, monoclonal antibodies will be generated and screened for blocking of these potentially novel cell adhesion pathways. Any newly discovered integrin ligands will then be characterized with respect to cell distribution and biochemistry.
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