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Ionic signal transduction in growth factor action
Abstract:
Growth factors are polypeptides which exert their mitogenic action through binding to specific high-affinity receptor molecules on the cell surface of target cells. This interaction leads to the rapid activation of a receptor-linked signal transduction system, involving the stimulation of an intrinsic receptor tyrosine phosphokinase activity, the breakdown of inositol lipids, and the production of ionic signals. In this contribution we have analysed the nature and origin of the ionic signals, and we have applied monoclonal antibodies against the receptor for epidermal growth factor (EGF), as well as tumour-promoting phorbol esters, to dissociate the early cellular responses to growth factors. Evidence is presented that the ionic signals are coupled to the breakdown of inositol lipids. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) would lead to the production of 1,2-diacylglycerol (DG) and inositol triphosphate (IP3). DG production results in the stimulation of protein kinase C, which causes the activation of Na+/H+ exchange by increasing its affinity for cytoplasmic H+. Consequently, a rise in cytoplasmic pH is observed. This response can be mimicked by the tumour promoter TPA, which can replace DG in activating the protein kinase C. Independently, IP3 production leads to the rapid mobilization of Ca2+ from intracellular stores. Monoclonal antibodies against the EGF receptor differed in their ability to evoke EGF-like responses upon binding to the EGF receptor. Of the three anti-EGF receptor IgGs tested, one was directed against the EGF-binding domain (2E9), and the others (2D11 and 2G5) were directed against sugar moieties not involved in EGF binding. Receptor tyrosine kinase activity could be stimulated by 2E9 as well as 2D11, but not by 2G5. Only 2D11 induced morphological changes similar to EGF. None of the antibodies was able to trigger the production of ionic signals, which implies probably that antibody binding to the receptor, even when they bind to the EGF binding domain, is an insufficient stimulus for the breakdown of inositol lipids. Most importantly, these monoclonal antibodies were also not able to induce DNA synthesis in quiescent human fibroblasts, not even after cross-linking of the EGF receptors by a second antibody. It may thus be concluded that the stimulation of the intrinsic receptor tyrosine phosphokinase activity can be dissociated from other early responses, and that none of the identified early responses is a sufficient trigger for the mitogenic action of growth factors.
Insights
Growth factors trigger cell responses via receptor binding, leading to ionic signals and lipid breakdown. However, stimulating receptor tyrosine kinase activity alone does not cause cell division, indicating complex signaling pathways are required for growth factor action.
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biochemistry
Background:
- Growth factors are polypeptides that bind to cell surface receptors, initiating signal transduction pathways.
- These pathways involve receptor tyrosine kinase activation, inositol lipid breakdown, and ionic signal production.
- Understanding the interplay of these early responses is crucial for elucidating growth factor-mediated mitogenesis.
Purpose of the Study:
- To analyze the nature and origin of ionic signals induced by growth factors.
- To differentiate early cellular responses to growth factors using monoclonal antibodies against the epidermal growth factor (EGF) receptor and phorbol esters.
- To determine if stimulating receptor tyrosine kinase activity is sufficient for initiating downstream signaling and cell proliferation.
Main Methods:
- Application of monoclonal antibodies targeting the EGF receptor (EGF-binding domain and sugar moieties).
- Use of tumor-promoting phorbol esters (TPA) to mimic diacylglycerol effects.
- Measurement of ionic signals (pH changes, calcium mobilization), protein kinase C activation, and DNA synthesis in human fibroblasts.
Main Results:
- Ionic signals are coupled to inositol lipid hydrolysis, producing diacylglycerol and inositol triphosphate.
- Diacylglycerol activates protein kinase C, leading to increased cytoplasmic pH via Na+/H+ exchange, a response mimicked by TPA.
- Monoclonal antibodies, including one against the EGF-binding domain, stimulated receptor tyrosine kinase activity but failed to induce ionic signals or DNA synthesis, even upon cross-linking.
Conclusions:
- Stimulation of intrinsic receptor tyrosine kinase activity can be dissociated from other early cellular responses like inositol lipid breakdown and ionic signaling.
- Antibody binding to the EGF receptor, even at the EGF-binding site, is insufficient to trigger the breakdown of inositol lipids or subsequent mitogenic responses.
- None of the early responses investigated, including receptor tyrosine kinase activation, are sufficient triggers for the mitogenic action of growth factors, suggesting a requirement for multiple coordinated signals.