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Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP
Author(s) -
Borst Stephen E.,
Catherwood Bayard D.
Publication year - 1989
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650040209
Subject(s) - ibmx , forskolin , epidermal growth factor , receptor , endocrinology , medicine , phorbol , cell culture , chemistry , cell surface receptor , a431 cells , microbiology and biotechnology , biology , cell , biochemistry , protein kinase c , signal transduction , cell cycle , genetics , molecular medicine
We studied the binding and degradation of 125 I‐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 [ 125 I]EGF to A431 cells reached a plateau after a 30 minute incubation at 37°C but was undetectable in UMR‐106 cells. Degradation of [ 125 I]EGF proceeded at a 50‐fold higher rate in A431 cells on a per cell basis, but receptor‐bound [ 125 I]EGF was internalized and degraded at a 3.5‐fold higher rate by UMR‐106 cells on a per receptor basis. At 4°C, [ 125 I]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 [ 125 I]EGF at 4°C a maximum of 80% with an IC 50 of 1.25 ng/ml. Maximal TPA reduction of [ 125 I]EGF binding was observed within 5–15 minutes and was due to a reduction in the affinity of cell surface receptors of [ 125 I]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 [ 125 I]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.