Neuron-specific interactions with two neurite-promoting fragments of fibronectin

Proteolytic fragments of fibronectin were used to identify regions of the molecule that support neurite extension and to investigate further the differential behavior of central and peripheral nervous system neurons in response to fibronectin (Rogers, S. L., P. C. Letourneau, S. L. Palm, J. B. McCarthy, and L. T. Furcht (1983) Dev. Biol. 98: 212-220). Fibronectin fragments with differing biological activities were produced by proteolytic digestion with trypsin and cathepsin D and sequential affinity chromatography on gelatin-agarose and heparin-Sepharose. The resulting fragments (described by Smith, D. E., D. F. Mosher, R. B. Johnson, and L. T. Furcht (1982) J. Biol. Chem. 257: 5831-5838; Smith, D. E., and L. T. Furcht (1982) J. Biol. Chem. 257: 6518-6523 included an NH2-terminal 27,000-dalton peptide that weakly binds heparin, a 46,000-dalton gelatin-binding fragment, a series of fragments (80,000 to 125,000 daltons) from the center of the molecule containing previously described cell-binding activity, two major peptides of Mr = 33,000 and 66,000 that bind heparin strongly and are thought to originate from the A and B chains, respectively, of plasma fibronectin, and a 31,000-dalton COOH-terminal peptide containing a free sulfhydryl from the A chain of the molecule. Tissue culture dishes were treated with these proteolytic preparations, and dissociated embryonic chick peripheral (PNS) and central nervous system (CNS) cells were cultured on each experimental substratum in serum-free medium. The fibronectin fragments were evaluated for ability to promote cell attachment, neurite initiation, and maintenance of neurite growth. The 27,000-, 46,000-, and 31,000-dalton preparations did not promote cell attachment or neurite extension. Both PNS and CNS neurons attached to and extended stable neurites upon the COOH-terminal heparin-binding preparation containing the 33,000- and 66,000-dalton peptides. A differential response of the neurons to the 80,000- to 125,000-dalton "cell-binding" peptides was observed: whereas PNS neurons maintained neuritic growth on this preparation for at least 48 hr, CNS neurons extended neurites during the first 24 hr of culture but, by 48 hr, withdrew these neurites and became increasingly clumped. On the basis of (1) the observed neuronal responses to the heparin binding and "cell binding" regions, and (2) the different ligand-binding properties of these regions, we propose that cell attachment and neurite extension can be mediated and/or modulated by two separate regions of fibronectin and that cellular response to the intact molecule may involve multivalent interactions.