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PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor
Abstract:
Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF)-nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity.
Insights
Mutant PC12 cells lacking nerve growth factor (NGF) high-affinity receptors fail to internalize NGF and exhibit altered responses. This suggests a link between high-affinity NGF receptors, NGF internalization, and cellular responsiveness.
Area of Science:
- Cell Biology
- Neuroscience
- Molecular Biology
Background:
- PC12 pheochromocytoma cells are a standard model for studying nerve growth factor (NGF) responses.
- NGF is crucial for neuronal development and survival, mediating its effects through specific receptors.
- Understanding the mechanisms of NGF signaling is vital for neurobiology and potential therapeutic interventions.
Purpose of the Study:
- To investigate the role of high-affinity NGF receptors in NGF-mediated cellular responses.
- To characterize the phenotype of PC12 cells lacking functional high-affinity NGF receptors.
- To explore the relationship between NGF receptor binding, internalization, and downstream signaling.
Main Methods:
- Selection of nerve growth factor (NGF)-nonresponsive PC12nnr cell lines using a double selection procedure.
- Assessing various NGF-dependent cellular responses, including neurite outgrowth, proliferation, and molecular changes.
- Analyzing NGF binding kinetics, internalization mechanisms, and responses to other growth factors like epidermal growth factor (EGF).
Main Results:
- Four PC12nnr cell lines were identified, exhibiting a complete lack of NGF responsiveness.
- PC12nnr cells failed to internalize bound NGF via the high-affinity mechanism and only bound NGF at low-affinity sites.
- These cells showed normal responses to epidermal growth factor, indicating a specific defect in NGF signaling, not a general uptake issue.
Conclusions:
- The presence of high-affinity NGF receptors is causally linked to NGF responsiveness and receptor-mediated internalization.
- PC12nnr cells provide a valuable model for dissecting the molecular mechanisms of NGF signal transduction.
- Potential connections between the regulation of cyclic AMP (cAMP) responses and NGF signaling pathways warrant further investigation.