One regulatory site of phosphorylation, Thr^^^, is 
a substrate for protein kinase C. Phosphorylation of 
the EGF receptor at this site causes an inhibition of 
the receptor tyrosine kinase activity and accounts 
for the inhibition of signal transduction caused by 
phorbol ester. However, this mechanism does not 
account for the physiologically relevant desensitiza- 
tion of the receptor that occurs after the treatment 
of cells with EGF. During the past year Dr. Davis's 
laboratory demonstrated that a serine phosphoryla- 
tion site located within the carboxyl-terminal do- 
main of the receptor (Ser'°^^/^) is required for EGF- 
stimulated desensitization of the EGF receptor. 
Mutation of the receptor at Ser'""*^/^ blocks the ho- 
mologous desensitization of the receptor tyrosine 
kinase activity and causes potentiation of signal 
transduction. Significantly, the in vitro phosphory- 
lation of the EGF receptor at Ser'"'*'^^^ caused an inhi- 
bition of the receptor tyrosine kinase activity. These 
studies demonstrate that there are two independent 
mechanisms of regulation of EGF receptor function 
by phosphorylation. Because autocrine and para- 
crine stimulation of the EGF receptor can contribute 
to tumor growth, the ability to manipulate EGF re- 
ceptor function by multisite phosphorylation has 
implications for the design of novel antitumor 
drugs. 
Disease Potential of the erbB Oncogene 
Direct evidence for the involvement of the phos- 
phorylation site Ser'°'"'^^ in oncogenic transforma- 
tion was obtained from studies of erbB. Retroviruses 
can cause erythroleukemia after a long latent period 
by inserting within the c-erbB gene. This insertion 
results in the expression of a truncated EGF receptor 
with constitutive protein-tyrosine kinase activity. 
The insertionally activated erbB oncogene stimu- 
lates the self-renewal of erythrocytic progenitor 
cells and is exclusively leukemogenic. In contrast, 
acute transforming viruses that carry the erbB onco- 
gene can induce both sarcomas and erythroleuke- 
mias. This expanded oncogenic potential of erbB 
can be attributed to structural alterations in the erbB 
protein. Comparison of the deletions present within 
the erbB gene in a series of viruses that exhibit an 
expanded disease tissue tropism indicates that the 
deletion of the phosphorylation site Ser'"'*^/^ is a 
common event. 
To test the hypothesis that the mutational removal 
of this phosphorylation site is relevant to the in- 
creased disease potential of erbB, Dr. Davis and his 
colleagues constructed recombinant retroviruses 
with defined mutations. Infection of animals with 
the recombinant viruses demonstrated that the re- 
placement of Ser'°^^^^ with Ala residues causes the 
rapid formation of sarcomas. Thus the loss of the 
negative regulatory phosphorylation site Ser'°^*^/^ 
causes an expansion of the tissue specificity of tu- 
mor formation by the erbB oncogene. Together 
these data establish an important functional role for 
the Ser'°^^^^ phosphorylation site. 
Function of the Transferrin Receptor 
The major mechanism that accounts for the accu- 
mulation of iron by cells is the receptor-mediated 
internalization of a serum iron-binding protein, 
transferrin. The ubiquitous expression of the trans- 
ferrin receptor by proliferating cells reflects the re- 
quirement of iron for mitogenesis. An understand- 
ing of the molecular mechanisms that account for 
the function of the transferrin receptor is a goal of 
this research. The disruption of the transferrin re- 
ceptor gene has been achieved in Dr. Davis's labora- 
tory in tissue culture cells. These cells provide a 
suitable model system for the expression of mutant 
forms of the transferrin receptor. During the past 
year the efi'ects of point mutations and deletions 
within the cytoplasmic tail of the transferrin recep- 
tor have been systematically analyzed. The results 
demonstrated that only a small region of the recep- 
tor cytoplasmic tail is required for endocytosis. Stud- 
ies in progress are designed to characterize cellular 
proteins that interact with the internalization signal 
sequence present within the tail of the transferrin 
receptor. This project was supported by a grant from 
the National Institute of General Medical Sciences, 
National Institutes of Health. 
Dr. Davis is also Associate Professor of Molecu- 
lar Medicine and of Biochemistry and Molecular 
Biology at the University of Massachusetts Medi- 
cal School, Worcester. 
Articles 
Countaway, J.L., Nairn, A.C., and Davis, R.J. 1992. 
Mechanism of desensitization of the epidermal 
growth factor receptor protein-tyrosine kinase./ 
Biol Chem 261 ■.\\29-\\iO. 
Girones, N., Alvarez, E., Seth, A., Lin, I.-M., Latour, 
D.A., and Davis, R.J. 1 99 1 . Mutational analysis of 
the cytoplasmic tail of the human transferrin re- 
ceptor. Identification of a sub-domain that is re- 
quired for rapid endocytosis. / Biol Chem 
266:19006-19012. 
Gonzalez, F.A., Raden, D.L., and Davis, R.J. 1991 . 
Identification of substrate recognition determi- 
nants for human ERKl and ERX2 protein kinases./ 
Biol Chem 266:22159-22163. 
CELL BIOLOGY AND REGULATION 45 
