duced receptor internalization occurred in cells in 
which protein kinase C activity had been 
downregulated by prolonged treatment with phor- 
bol esters, implying that the process is independent 
of protein kinase C. Pretreatment of the cells with 
compounds such as trifluoperazine, chlorproma- 
zine, and W7 blocks the effects of EGF on hormone 
internalization. 
Additional studies suggest that several other EGF- 
stimulated responses are also desensitized after 
treatment of A43 1 cells with high concentrations of 
EGF. These include EGF-stimulated PI turnover and 
EGF-stimulated tyrosine protein kinase activity. De- 
sensitization of EGF-stimulated PI turnover oc- 
curred rapidly at 37°C, with half-maximal effect at 
15 min. The desensitization was homologous, or 
agonist specific, in nature. Treatment of cells with 
EGF decreased the responsiveness of the cells to 
EGF, whereas treatment with bradykinin (another 
compound capable of stimulating PI turnover in 
these cells) did not alter the ability of the cells to 
respond to EGF. Like desensitization of ^^^I-EGF in- 
ternalization, desensitization of EGF-stimulated PI 
turnover occurred in cells that had been rendered 
protein kinase C-deficient by prolonged incubation 
with TPA. This again suggests that desensitization of 
the EGF receptor is not mediated by protein kinase 
C. Treatment of the cells with trifluoperazine could 
block desensitization of PI turnover. 
The EGF receptor is present on the cell surface 
as a monomer. Upon binding EGF, the monomeric 
receptor dimerizes; it is this dimeric form of the re- 
ceptor that has been hypothesized to be involved 
in signal transduction. Studies in Dr. Pike's labora- 
tory have shown that EGF receptor dimerization is 
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inhibited in desensitized cells. This is true both 
when whole cells are treated with EGF and the 
monomers and dimers isolated by sucrose density 
gradient centrifugation and when the capacity of 
isolated monomers to form dimers is assessed in 
vitro. 
Not all treatments that affect EGF receptor sensi- 
tivity lead to alterations in receptor dimer forma- 
tion. Treatment of cells with TPA stimulates the ac- 
tivity of protein kinase C, which phosphorylates the 
EGF receptor. This leads to a loss of EGF binding 
and a decrease in the ability of EGF to stimulate re- 
sponses via its receptor. However, treatment of 
cells with TPA does not alter the ability of EGF to 
induce EGF receptor dimers, indicating that the ef- 
fects on dimerization are specific for EGF-depen- 
dent desensitization. 
In many cases desensitization of receptors has 
been associated with the phosphorylation of the re- 
ceptor by cytosolic kinases. Dr. Pike's laboratory 
has therefore begun to investigate the possibility 
that a cytosolic kinase in A431 cells is capable of 
phosphorylating and desensitizing the EGF recep- 
tor. Preliminary data show that A43 1 cells contain a 
cytosolic kinase that is capable of phosphorylating 
affinity-purified EGF receptors. The activity of the 
enzyme appears to be stimulated by EGF and inhib- 
ited by trifluoperazine. Studies are in progress to 
purify and characterize this kinase and determine 
whether it is involved in desensitization of the EGF 
receptor. 
Dr. Pike is also Assistant Professor of Biochemis- 
try and Molecular Biophysics at the Washington 
University School of Medicine. 
Article 
Kuppuswamy D., and Pike, L.J. 1989. Ligand-induced desensitization of ^^^I-epidermal growth factor inter- 
nalization. J Biol C;?7em 264:3357-3363- 
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