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Beta PDGF receptor mutants defective for mitogenesis promote neurite outgrowth in PC12 cells
1George Williams Hooper Foundation, University of California, San Francisco 94143-0552, USA.
Background:
Platelet-derived growth factor (PDGF) promotes mitogenesis in fibroblast cell lines but stimulates neurite outgrowth in PC12 cells that ectopically express the beta PDGF receptor. To determine which substrates must associate with this receptor protein-tyrosine kinase in order to promote neurite outgrowth, we introduced into PC12 pheochromocytoma cells three mutant forms of the beta PDGF receptor that no longer associate with specific substrate proteins. We then assayed the ability of these receptor mutants to affect neurite extension.
Results:
Receptors lacking the kinase-insert domain did not associate with either phosphatidylinositol 3-kinase (PI 3-kinase) or Ras GTPase-activating protein (Ras-GAP) in PC12 cells. A carboxy-terminal truncation of the beta PDGF receptor eliminated the association of phospholipase C-gamma 1 (PLC-gamma 1) with the receptor and prevented phosphorylation of PLC-gamma 1 in PC12 cells. Finally, beta PDGF receptors that have tyrosine-to-phenylalanine point mutations at positions 708, 719, 977 and 989 did not associate with either PI 3-kinase or PLC-gamma 1. All three mutant forms of the beta PDGF receptor promoted PDGF-dependent neurite outgrowth in PC12 cells and elicited activation of mitogen-activated protein (MAP) kinases.
Conclusions:
PC12 cells expressing the beta PDGF receptor extend neurites in response to PDGF in the absence of signalling through PI 3-kinase, RasGAP, and PLC-gamma 1. This contrasts with the requirements for mitogenesis for epithelial and fibroblast cell lines, in which the association of PI 3-kinase with the beta PDGF receptor is essential. This receptor protein-tyrosine kinase therefore phosphorylates and activates a similar set of intracellular signalling molecules in the context of both mitogenesis and differentiation, but the importance of particular pathways for each phenotypic response is distinct.
Insights
Platelet-derived growth factor (PDGF) signaling in PC12 cells promotes neurite outgrowth independently of PI 3-kinase, RasGAP, and PLC-gamma 1. This differs from fibroblast mitogenesis, highlighting distinct pathway importance for different cellular responses.
Area of Science:
- Cellular Biology
- Molecular Biology
- Neuroscience
Background:
- Platelet-derived growth factor (PDGF) drives cell proliferation in fibroblasts but neurite outgrowth in PC12 cells expressing the beta PDGF receptor.
- Investigating specific substrate associations with the beta PDGF receptor is crucial for understanding neurite outgrowth.
- Mutant beta PDGF receptors were engineered to disrupt substrate binding and assess their impact on neurite extension.
Purpose of the Study:
- To identify essential substrate proteins that associate with the beta PDGF receptor for promoting neurite outgrowth in PC12 cells.
- To elucidate the distinct signaling pathways utilized by the beta PDGF receptor for differentiation versus proliferation.
Main Methods:
- Engineered three mutant forms of the beta PDGF receptor in PC12 cells, each lacking specific substrate interactions.
- Assessed the ability of these mutant receptors to mediate PDGF-dependent neurite outgrowth.
- Analyzed the activation of downstream signaling molecules, including MAP kinases, in response to PDGF stimulation.
Main Results:
- Receptors lacking the kinase-insert domain failed to associate with PI 3-kinase or Ras-GAP.
- Carboxy-terminal truncation abolished PLC-gamma 1 association and phosphorylation.
- Mutant receptors, despite lacking these specific substrate interactions, still promoted PDGF-dependent neurite outgrowth and MAP kinase activation.
Conclusions:
- PC12 cells extend neurites in response to PDGF without requiring signaling through PI 3-kinase, RasGAP, or PLC-gamma 1.
- This contrasts with fibroblast mitogenesis, where PI 3-kinase association is critical.
- The beta PDGF receptor activates similar intracellular signaling molecules for both mitogenesis and differentiation, but pathway importance varies by cellular outcome.