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On substrata coated with laminin or native collagen (Types I/III), PC12 cells employ an active adhesion mechanism (i.e., one inhibited at low temperature, by azide or in the absence of divalent cations) to attach and extend neurites; on substrata coated with wheat germ agglutinin (WGA) or polylysine, by contrast, PC12 cells attach via a passive mechanism and fail to extend neurites (Turner et al., 1987). This paper reports the isolation of 2 monoclonal antibodies (3A3 and 1B1) that promote retraction of neurites extended on laminin and collagen. In studies of initial cell attachment, 3A3 inhibited active attachment to laminin or collagen but not passive attachment to WGA or polylysine, whereas 1B1 inhibited both active and passive attachment. The more potent of the antibodies, 3A3, precipitates 2 radioactive protein bands (of approximately 185 and 125 kDa) from 1% Nonidet P-40 extracts of metabolically labeled PC12 cells. The properties of these proteins suggest that the antigen recognized by 3A3 is a member of the integrin family of matrix receptors. The other monoclonal antibody, 1B1, reacts with many PC12 proteins, including both bands precipitated by 3A3. The available data strongly suggest that an integrin with specificity for both laminin and collagen mediates PC12 adhesion to the substratum at both the cell body and the neurite growth cone.

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Abstract. We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (~<24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inacfive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth.Comparison of collagen Iv's effects to those of laminin reveal~ that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize fl~ integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the ot~/31 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.

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