MOLECULAR BIOLOGY OF GROWTH FACTOR RECEPTOR SIGNAL TRANSDUCTION 
Lewis T. Williams, M.D., Ph.D., Investigator 
Experiments performed in Dr. Williams's labora- 
tory this year provided insight into the mechanism 
by which the platelet-derived growth factor (PDGF) 
receptor functions in signal transduction. Although 
most of these studies focused on the PDGF p-recep- 
tor, preliminary experiments with the closely re- 
lated PDGF a-receptor suggest that it functions in a 
similar manner. The data suggest a model in which 
the binding of PDGF to the extracellular domain 
stimulates the following sequence of events: the re- 
ceptor forms a dimer that becomes autophos- 
phorylated; the conformation of the cytoplasmic 
domain is altered by autophosphorylation; the re- 
ceptor then physically associates with at least three, 
and possibly four, cytoplasmic signaling molecules; 
the receptor tyrosine kinase phosphorylates the sig- 
naling molecules on tyrosine residues. Experiments 
done in Dr. Williams's laboratory by Dr. Deborah 
Morrison showed for the first time that the biologi- 
cal activity of one of the signaling molecules, the 
Raf-1 protein, was altered by the direct phosphory- 
lation of the protein by the receptor. Thus tyrosine 
phosphorylation of signaling molecules may regu- 
late their activities, and the physical association of 
these molecules with the receptor may localize 
their activities. 
I. PDGF-induced Receptor Association and Tyrosine 
Phosphorylation of Signaling Molecules. 
For the last three years Dr. Williams's group has 
attempted to identify the molecules that associate 
with the ligand-activated PDGF receptor and serve 
as substrates of its tyrosine kinase. Their first prog- 
ress occurred last year, when Drs. Shaun R. Cough- 
lin (HHMI, University of California at San Francisco) 
and Jaime Escobedo showed that a 3'-phos- 
phatidylinositol kinase was physically associated 
with the PDGF-stimulated receptor. This work was 
extended this year to the study of other signaling 
molecules. 
A. Raf-1. The Raf-1 protein is a 74 kDa serine/threo- 
nine kinase that has an oncogenic counterpart de- 
scribed previously by Dr. Ulf Rapp at the National 
Institutes of Health. Dr. Morrison showed that the 
Raf-1 protein coimmunoprecipitated with the PDGF 
receptor when extracts of PDGF-stimulated cells 
were precipitated with either antireceptor or anti- 
Raf antibodies. Extracts of unstimulated cells 
showed no association of receptor and Raf-1 pro- 
tein. Immobilized, highly purified autophosphor- 
ylated PDGF (B-receptor (produced by Dr. Escobedo 
using a baculovirus expression system) was used to 
adsorb the PDGF receptor from 3T3 cell extracts 
in vitro. Dr. Morrison also showed that when the 
receptor was dephosphorylated by phosphatase 
treatment it lost its ability to bind to Raf-1, suggest- 
ing that only the phosphorylated form of the recep- 
tor was capable of interacting with the Raf-1 pro- 
tein. In other experiments, insect cells were 
co-infected with recombinant PDGF receptor bac- 
ulovirus (constructed by Dr. Escobedo) and recom- 
binant Raf- 1 baculovirus (constructed in the labora- 
tory of Dr. Thomas Roberts, using the Raf-1 cDNA 
provided by Dr. Rapp). The Raf-1 protein in these 
cells became phosphorylated on tyrosine in re- 
sponse to PDGF, and the receptor was physically as- 
sociated with the Raf-1 protein. In more direct in 
vitro experiments, highly purified PDGF p-receptor 
protein was incubated directly with highly purified 
Raf-1 protein. In the presence of ATP the receptor 
directly phosphorylated the Raf protein on tyro- 
sines and caused an increase in Raf-1 kinase activity 
of four- to sixfold in vitro. 
A mutant PDGF ^-receptor protein that had a 
large deletion of the domain that interrupts the ty- 
rosine kinase-coding sequence was also expressed 
by Dr. Escobedo and Sutip Navankasattusas and 
was immunopurified for in vitro experiments. This 
kinase insert (KI) deletion mutant had the same au- 
tophosphorylating activity as the wild-type (J-recep- 
tor but failed to phosphorylate the Raf-1 protein 
and failed to activate Raf kinase in vitro. When ex- 
pressed in intact cells the KI deletion mutant recep- 
tor was defective in its phosphorylation of Raf-1 ki- 
nase and stimulation of DNA synthesis. These 
experiments show that the Raf-1 protein is a sub- 
strate of the PDGF receptor kinase and that regula- 
tion of the Raf kinase activity is an important step in 
PDGF-induced mitogenesis. 
B. Phospholipase C-y. Working in Dr. Williams's 
laboratory. Dr. David Kaplan showed that phos- 
pholipase C-7, an enzyme that hydrolyzes phos- 
phatidylinositol to produce diacylglycerol and ino- 
sitol phosphate second messengers, associates with 
ligand-activated PDGF receptor in intact cells and 
with highly purified and autophosphorylated re- 
ceptor in vitro. Phospholipase C-'y, in contrast to 
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