Abstract
Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.
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Abbreviations
- EGF:
-
epidermal growth factor
- FGF:
-
fibroblast growth factor
- PBS:
-
phosphate buffered saline
- PDBu:
-
4β-phorbol-12,13-dibutyrate
- PDGF:
-
platelet-derived growth factor
- PK-C:
-
protein kinase-C
- TGF-β:
-
transforming growth factor-β
- TPA:
-
12-0-tetradecanoylphorbol-13-acetate
References
AssoianRK, FrolikCA, RobertsAB, MillerDM and SpornMB (1984) Transforming growth factor-β controls receptor levels for epidermal growth factor in NRK fibroblasts. Cell 36: 35–41.
BeguinotL, HanoverJA, ItoS, RichertND, WillinghamMC and PastanI (1985) Phorbol esters induce transient internalization without degradation of unoccupied epidermal growth factor receptors. Proc. Natl. Acad. Sci. USA 82: 2774–2778.
BjorgeJD and KudlowJE (1987) Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. J. Biol. Chem. 262: 6615–6622.
BurrJG, DreyfussG, PenmanS and BuchananJM (1980) Association of the src product of Rous sarcoma virus with cytoskeletal structures of chicken embryo fibroblasts. Proc. Natl. Acad. Sci. 77: 3484–3488.
CantorC and SchimmelPR. In “Biophysical Chemistry” part 3, “Behavior of Biological Macromolecules”; W.H. Freeman and Co. San Francisco, 1980, p. 861.
CarpenterG, LembachKJ, MorrisonMM and CohenS (1975) Characterization of the binding of 125I-labeled epidermal growth factor to human fibroblasts. J. Biol. Chem. 250: 4297–4304.
ClarkAJL, IshiiS, RichertN, MerlinoGT and PastanI (1985) Epidermal growth factor regulates the expression of its own receptor. Proc. Natl. Acad. Sci. 82: 8374–8378.
CochetC, GillGN, MeisenhelderJ, CooperJA and HunterT (1984) C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem. 259: 2553–2558.
CollinsMKL and RozengurtE (1982) Binding of phorbol esters to high affinity sites on murine fibroblastic cells elicits a mitogenic response. J. Cell Physiol. 112: 42–50.
DavisRJ and CzechMP (1985) Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654. Proc. Natl. Acad. Sci. USA 82: 1974–1978.
HunterT, LingN and CooperJA (1984) Protein kinase-C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nature (Lond.) 311: 480–483.
JettenAM (1980) Retinoids specifically enhance the number of epidermal growth factor receptors. Nature 284: 626–629.
KawamotoT, SatoJD, LeA, PolikoffJ, SatoGH and MendelsohnJ (1983) Growth stimulation of A-431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc. Natl. Acad. Sci. 80: 1337–1341.
LarrickJW and CresswellP (1979) Modulation of Cell Surface Iron Transferrin Receptors by Cellular Density and State of Activation. J. Supramolecular Structure 11: 579–586.
LeeL-S and WeinsteinIB (1978) Tumor-promoting phorbol esters inhibit binding of epidermal growth factor to cellular receptors. Science 202: 313–315.
LeofEB, OlashawNE, PledgerWJ and O'KeefeEJ (1982) Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor. Biochem. Biophys. Res. Comm. 109: 83–91.
McCaffreyPG, FriedmanBA and RosnerMR (1984) Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor. J. Biol. Chem. 259: 12502–12507.
MiyataY, NishidaE, KoyasuS, YaharaI and SakaiH (1989) Protein kinase C-dependent and-independent pathways in the growth factor-induced cytoskeletal reorganization. J. Biol. Chem. 264: 15565–15568.
OlashawNE, O'KeefeEJ and PledgerWJ (1986) Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters. Proc. Natl. Acad. Sci. USA 83: 3834–3838.
RizzinoA and CrowleyC (1980) Growth and differentiation of embryonal carcinoma cell line F9 in defined media. Proc. Natl. Acad. Sci. USA 77: 457–461.
RizzinoA, KazakoffP, RuffE, KuszynskiC and NebelsickJ (1988a) Regulatory effects of cell density on the binding of transforming growth factor β, epidermal growth factor, platelet-derived growth factor, and fibroblast growth factor. Cancer Res. 48: 4266–4271.
RizzinoA, KazakoffP and NebelsickJ (1990) Density-induced down regulation of epidermal growth factor receptors. In Vitro Cell Dev. Biol. 26: 537–542.
RizzinoA, OrmeLS and DeLarcoJE (1983) Embryonal carcinoma cell growth and differentiation: Production of and response to molecules with transforming growth factor activity. Exp. Cell Res. 143: 143–152.
RizzinoA, RuffE and KazakoffP (1988b) Isolation and characterization of A-431 cells that retain high EGF binding capacity and respond to EGF growth stimulation. Cancer Res. 48: 2377–2381.
Rodriguez-PenaA and RozengurtE (1984) Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem. Biophys. Res. Comm. 120: 1053–1059.
ScatchardG (1949) The attraction of proteins for small molecules and ions. Ann. NY Acad. Sci. 51: 660–672.
ScottCD and BaxterRC (1987) Insulin-like growth factor-II receptors in cultured rat hepatocytes: Regulation by cell density. J. Cell. Phys. 133: 532–538.
ShoyabM, DeLarcoJ and TodaroG (1979) Biologically active phorbol esters specifically alter affinity of epidermal growth factor membrane receptors. Nature 279: 387–391.
VeomettG, KuszynskiC, KazakoffP and RizzinoA (1989) Cell density regulates the number of cell surface receptors for fibroblast growth factor. Biochem. Biophys. Res. Comm. 159: 694–700.
WrannM and FoxCF (1980) Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor. Science 210: 1363–1364.
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Rizzino, A., Huebert, C., Kuszynski, C. et al. Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors. Cytotechnology 7, 85–92 (1991). https://doi.org/10.1007/BF00350914
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DOI: https://doi.org/10.1007/BF00350914