title: Neurite outgrowth in dorsal root neuronal hybrid clones modulated by ganglioside GM1 and disintegrins.
abstract: Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.
authors.affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106.
authors.forename: E
authors.initials: E
authors.lastname: Barletta
authors.forename: E G
authors.initials: EG
authors.lastname: Bremer
authors.forename: L A
authors.initials: LA
authors.lastname: Culp
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D005353:Fibronectins / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism / Q000502:physiology*; D005727:Ganglia, Spinal; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism; D006822:Hybrid Cells; D016023:Integrins / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology*; D009842:Oligopeptides / Q000502:physiology; D010978:Platelet Factor 4 / Q000378:metabolism; D051381:Rats; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000502:physiology
chemical_list: D002772:Cholera Toxin; D011508:Chondroitin Sulfate Proteoglycans; D003513:Cycloheximide; D005353:Fibronectins; D005677:G(M1) Ganglioside; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D016023:Integrins; D009842:Oligopeptides; D010978:Platelet Factor 4; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; C046617:arginyl-glycyl-aspartyl-serine