Insulin Receptor Structure and Function 
Leland Ellis, Ph.D. — Assistant Investigator 
Dr. Ellis is also Assistant Professor of Biochemistry at the University of Texas Southwestern Medical Center 
at Dallas. He received his Ph.D. degree from the University of North Carolina at Chapel Hill with Aldo 
Rustioni and was a postdoctoral fellow at Columbia University College of Physicians and Surgeons with 
Karl Pfenninger and at the University of California, San Francisco, with William Rutter. 
THE response of cells to the polypeptide hor- 
mone insulin begins with the binding of insu- 
lin to a specific cell surface receptor; this results 
in the rapid autophosphorylation of the cytoplas- 
mic protein tyrosine kinase domain of the recep- 
tor specifically on several tyrosine residues. Al- 
though the postreceptor mechanisms involved 
in the mediation of the multitude of physiologi- 
cal responses to insulin remain largely obscure, 
this initial transmembrane signaling event is re- 
quired for all known insulin-dependent cellular 
responses. 
To begin to relate the structural features of the 
insulin receptor protein to the details of its func- 
tion, we have established two experimental ap- 
proaches. First, the availability of cDNAs that en- 
code the insulin receptor protein, together with 
methods to introduce mutations into receptor 
coding sequences and to express these altered re- 
ceptors in heterologous cell expression systems, 
has provided the tools with which to test ideas 
concerning the functional roles of particular re- 
ceptor residues. Second, the size and membrane- 
associated nature of the protein have made it 
possible to exploit the relatively simple trans- 
membrane topology of the receptor to study its 
two functional domains individually as soluble 
molecules: the extracellular domain is secreted 
as a heterotetramer that binds insulin with high 
affinity, and the cytoplasmic domain is an active 
protein tyrosine kinase. 
The extracellular ligand-binding domain of the 
insulin receptor is a complex molecule: each 
half- receptor comprises 929 residues derived 
from both a- (735 amino acids) and (8-subunits 
(194 amino acids) and includes 16 potential 
asparagine-linked glycosylation sites and 4 1 cys- 
teines. There is little information at present as to 
how this domain folds during biosynthesis or 
how it interacts with insulin. The study of an ex- 
tensive series of deletion mutants has revealed 
sites within the receptor primary sequence at 
which truncation results in the generation of in- 
dependently folded soluble subdomains. These 
sites now provide landmarks that guide further 
biochemical and molecular dissections of this 
complex domain. Furthermore, the establish- 
ment of a heterologous cell expression system 
that provides tens of milligrams of a secreted solu- 
ble derivative of the extracellular domain pro- 
vides sufficient protein for its further biochemi- 
cal dissection and renders feasible efforts to 
crystallize this functional domain of the receptor, 
a prerequisite for the elucidation of its three-di- 
mensional structure by x-ray crystallography, in 
collaboration with Wayne Hendrickson (HHMI, 
Columbia University College of Physicians and 
Surgeons) . 
The cytoplasmic protein tyrosine kinase do- 
main of the receptor has been expressed indepen- 
dently of the cell membrane and as a soluble 
monomeric insulin-independent enzyme (401 
amino acids) . To be relevant to the study of the 
function of the kinase in the wild-type mem- 
brane-associated insulin receptor, the soluble ki- 
nase must exhibit functional properties charac- 
teristic of the wild-type receptor. In fact, this 
soluble enzyme is recognized by a panel of con- 
formation-sensitive antireceptor monoclonal anti- 
bodies, it exhibits a low level of basal activity 
toward exogenous substrates that increases upon 
autophosphorylation, and the sites of autophos- 
phorylation of the enzyme are typical of those 
observed following insulin-dependent autophos- 
phorylation of the wild-type receptor in intact 
cells. Thus, although synthesized free of the cell 
membrane and now insulin-independent, this sol- 
uble derivative of the protein tyrosine kinase do- 
main exhibits the two functional states observed 
for the kinase in the context of the wild-type 
transmembrane receptor. 
An efficient heterologous cell expression sys- 
tem renders feasible the use of biophysical meth- 
ods for functional studies of this soluble enzyme, 
which are not yet possible for the wild-type 
membrane-associated insulin receptor. For exam- 
ple, by the use of nuclear magnetic resonance 
(NMR) spectroscopy (in collaboration with Barry 
Levine, Oxford University), we have begun to ex- 
plore the interaction of small molecules (metal 
ions, ATP, peptide substrates) with the enzyme 
and to study catalysis by the kinase in solution in 
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