MEMBRANE PROTEIN STRUCTURE/FUNCTION 
Leiand Ellis, Ph.D., Assistant Investigator 
The response of cells to the polypeptide hor- 
mone insulin begins with the binding of insulin to 
a specific cell surface receptor. The insulin receptor 
(IR) is synthesized as a large (1,355 amino acid) sin- 
gle polypeptide chain precursor, which undergoes 
post-translational processing to yield an integral 
transmembrane glycoprotein comprising two a- 
subunits (—135 kDa) and two P-subunits (~95 
kDa). Dr. Ellis and his colleagues wish to relate the 
structural features of the IR protein to the details of 
IR function. The size and relatively simple trans- 
membrane topology of the IR make it possible to 
engineer and study its two functional domains indi- 
vidually as soluble molecules: the extracellular do- 
main is secreted as a dimer that binds insulin with 
high affinity, and the cytoplasmic domain is an ac- 
tive monomeric protein tyrosine kinase (PTK). 
Ideas and hypotheses concerning receptor struc- 
ture/function have derived from this reductionist 
approach. Transfected mammalian cell lines and 
transgenic mice are being used to test these ideas in 
the context of the intact transmembrane receptor 
(in collaboration with Dr. Robert Hammer, HHMI, 
University of Texas Southwestern Medical Center at 
Dallas). 
The extracellular ligand-binding domain of the IR 
is a complex molecule: each half of the disulfide- 
linked (ap)^ dimer comprises 929 residues derived 
from both a- (735 aa) and (3-subunits (194 aa), with 
16 potential N-linked glycosylation sites and 41 cys- 
teines. Furthermore, little is presently known about 
how this domain folds during biosynthesis or how 
this domain interacts with insulin. The study of an 
extensive series of deletion mutants has revealed 
the location within the primary amino acid se- 
quence of independently folded soluble sub- 
domains, as well as their stability, efficiency of se- 
cretion, and interaction with both insulin and a 
panel of monoclonal antibodies specific for the ex- 
tracellular domain of the receptor. These land- 
marks now guide the future biochemical and mo- 
lecular dissections of this domain. 
The baculovirus insect cell (Sf9) system has been 
successfully employed to express an active soluble 
derivative of the cytoplasmic IR PTK domain. Re- 
sults demonstrate the utility of this approach for 
the study of functional domains of large membrane 
proteins. The availability of milligram quantities of 
the protein now renders feasible the use of bio- 
physical methods such as circular dichroism (CD) 
and nuclear magnetic resonance (NMR) spectros- 
copy to study the interaction of small molecules 
(metal ions, ATP, peptide substrates) with the en- 
zyme in solution. Thus new avenues are available 
with which to explore the function of the enzyme. 
The NMR studies (in collaboration with Dr. Barry 
Levine, Oxford University) have provided the first 
look at catalysis by a PTK in real time, as it is now 
possible to follow in solution the binding of pep- 
tide substrates to the enzyme and the phosphoryla- 
tion of individual tyrosine residues of peptide sub- 
strates (especially the order of phosphorylation of 
multiple tyrosines in peptide substrates). The role 
of individual amino acid residues of the peptide on 
its binding to the enzyme and its kinetics of phos- 
phorylation can also be studied. These functional 
studies complement efforts to obtain the three-di- 
mensional structure of the enzyme by x-ray crystal- 
lography (in collaboration with Dr. Wayne A. 
Hendrickson, HHMI, Columbia University College 
of Physicians and Surgeons). 
Dr. Ellis's interest in membrane proteins and 
their structural organization stems from a general 
interest in cell membranes and cell-cell interac- 
tions, especially in the developing nervous system. 
After the cessation of mitosis, neurons enter a stage 
of development during which cell processes (axons 
and dendrites) are elaborated and connections 
(synapses) with appropriate target cells are estab- 
lished. Such elongating processes end in a terminal 
enlargement, the nerve growth cone. The sprouting 
neuron responds to a number of extracellular sig- 
nals, including grov^^h factors and hormones, com- 
ponents of the extracellular matrix, and its target 
cell. The biochemical and molecular components of 
the neuronal plasmalemma (especially the nerve 
growth cone) that mediate(s) such critical develop- 
mental events must be described before this stage 
of neuronal differentiation can be understood. 
The growth cone particle (GCP) fraction pre- 
pared from fetal (17 day gestation) rat brain con- 
sists of pinched-off cellular fragments that exhibit 
all of the ultrastructural features of nerve growth 
cones. GCPs are utilized to identify and characterize 
membrane-associated proteins expressed during 
neuronal sprouting. The initial biochemical analysis 
of membranes prepared from GCPs revealed three 
major polypeptides: pp46, p38, and p34. Subse- 
quently pp46 was shown by others to be the 
growth-associated protein, GAP-43, a protein of un- 
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