The overall goal of this research grant is to understand how fibronectin molecules assemble into covalently linked fibrils. Such fibrils are found in extracellular matrices of many tissues and represent a substratum on which cells adhere, migrate, and (de)differentiate. Assembly occurs at specialized sites on the surfaces of cultured fibroblasts. Dimeric (protameric) fibronectin binds reversibly to high affinity sites where thiol-disulfide exchange occurs to form disulfide-bonded multimers. In addition, a transglutaminase can catalyze the formation of intermolecular episolon-( -glutamyl)-lysyl crosslinks. A full understanding of the process of fibronectin assembly will include the characterization of molecule(s) at cell surface assembly sites, elucidation of fibronectin molecules after assembly. I will focus on 2 aims - to learn how the aminoterminal portion of fibronectin directs assembly and to learn how other domains of fibronectin are involved in assembly. The technique of oligonucleotide-directed mutagenesis will be used to make variants of the aminoterminal portion of rat fibronectin in a baculoviral expression system. I will be particularly interested in the minimal structure required for binding of recombinant proteins to assembly sites of human fibroblasts and whether binding requires just one or several of the type I sequences in the amino-terminal domain of fibronectin. Mutagenesis will also be done to introduce a cysteine into which a heterobifunctional photoactivatable crosslinking reagent can be introduced. Transgluataminase-mediated and photoaffinity crosslinking of the series of recombinant proteins should allow identification and """"""""mapping"""""""" of molecules at cell surface assembly sites. Experiments will be done to test the hypothesis that the most aminoterminal and carboxylterminal type III sequences, III-1 and III-17, interact with one another during assembly to position idiosyncratic pairs of vicinal cysteines next to the disulfides of the type II homology sequences, thus allowing thiol-disulfide exchange. Interactions between the type III sequences will be probed with monoclonal and polyclonal antibodies to epitopes in the two sequences. Evidence of the disulfide exchange between protamers will be sought in cyanogen bromide digests of assembled fibronectin. Parallel digests of transglutaminase- crosslinked fibronectin should give further insight into the disposition of fibronectin in fibrils. Accomplishment of these aims may reveal new ways in which cells interact with extracellular molecules.
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