Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

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Abstract
We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of [125I]EGF to A431 cells reached a plateau after a 30 minute incubation at 37°C but was undetectable in UMR-106 cells. Degradation of [125I]EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound [125I]EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4°C, [125I]EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37°C reduced subsequent cell surface binding of [125I]EGF at 4°C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of [125I]EGF binding was observed within 5–15 minutes and was due to a reduction in the affinity of cell surface receptors of [125I]EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 μM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 μM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in [125I]EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p < 0.0005), 39% (p < 0.0002), and 35% (p < 0.002), respectively. We concluded that activation of protein kinase C decreases the affinity of UMR-106 EGF receptors and that this action may be opposed by the activation of cyclic AMP-dependent protein kinase.
Funding Information
  • National Institutes of Health (T32-AM07298-06A2, 2-R01-AM32196, BRSG SO7-RR05364)

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