Related Experiment Videos
EGF receptor down-regulation attenuates ligand-induced second messenger formation
A Gilligan1, M Prentki, B B Knowles
1Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Abstract:
Epidermal growth factor (EGF)-induced increases in cytosolic Ca2+ and inositol polyphosphate production were compared in a human hepatocellular carcinoma-derived cell line, PLC/PRF/5, and in an EGF receptor-overexpressing subline, NPLC/PRF/5. Formation of these second messengers was correlated to EGF receptor display at the cell surface by monitoring ligand-induced EGF receptor down-regulation. Both cell lines exhibited a strikingly similar cytosolic Ca2+ increase upon exposure to EGF. The initial inositol phosphate responses were also similar in the two cell lines; inositol 1,4,5-trisphosphate increased within 10-15 s and returned to prestimulatory values after 2 min in both cell lines, while inositol tetrakisphosphate and inositol 1,3,4-trisphosphate were elevated after a 2-min exposure to EGF. At later times the responses were markedly different; NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate (maximum at 1-3 h) but PLC/PRF/5 cells showed decreased levels of these isomers after 10 min and a return to basal values by 1 h. Exposure of PLC/PRF/5 cells to EGF caused a progressive decrease in the amount of EGF receptor at the cell surface whereas such treatment did not change the surface receptor levels in NPLC/PRF/5 cells. Kinetic analysis of EGF receptor down-regulation showed that receptor internalization was rapid enough to account for the transient nature of the inositol phosphate response in PLC/PRF/5 cells. Thus, the divergent patterns of signaling exhibited by the two cell lines may reflect differences in the efficiency of EGF-induced down-regulation of surface receptors.
Insights
Epidermal growth factor (EGF) signaling differs between cancer cells. Overexpressing EGF receptors leads to sustained inositol phosphate production, unlike standard cancer cells where receptor down-regulation causes transient signaling.
Area of Science:
- Cellular signaling and cancer biology
- Molecular mechanisms of growth factor receptor regulation
Background:
- Epidermal growth factor (EGF) is a key regulator of cell growth and proliferation.
- EGF receptor (EGFR) signaling pathways involve second messengers like calcium and inositol polyphosphates.
- Dysregulation of EGFR signaling is implicated in various cancers, including hepatocellular carcinoma.
Purpose of the Study:
- To compare EGF-induced second messenger production in a human hepatocellular carcinoma cell line (PLC/PRF/5) and an EGF receptor-overexpressing subline (NPLC/PRF/5).
- To investigate the correlation between EGF receptor cell surface expression and the dynamics of intracellular signaling.
- To elucidate the role of EGF receptor down-regulation in modulating the duration of signaling responses.
Main Methods:
- Measurement of cytosolic Ca2+ and inositol polyphosphate production (inositol 1,4,5-trisphosphate, inositol tetrakisphosphate, inositol 1,3,4-trisphosphate) in response to EGF.
- Quantification of EGF receptor levels at the cell surface using ligand-induced down-regulation assays.
- Kinetic analysis of EGF receptor internalization and its correlation with signaling pathway duration.
Main Results:
- Both cell lines showed similar initial increases in cytosolic Ca2+ and inositol 1,4,5-trisphosphate upon EGF exposure.
- NPLC/PRF/5 cells exhibited prolonged production of inositol 1,3,4-trisphosphate and inositol tetrakisphosphate, while PLC/PRF/5 cells showed transient responses.
- EGF treatment progressively decreased surface EGF receptors in PLC/PRF/5 cells but not in NPLC/PRF/5 cells, with rapid internalization correlating to transient signaling.
Conclusions:
- The efficiency of EGF receptor down-regulation significantly influences the duration of downstream signaling events.
- Differences in EGF receptor internalization kinetics likely explain the divergent inositol polyphosphate signaling patterns observed.
- This study highlights the critical role of receptor trafficking in regulating cellular responses to growth factors in cancer.