Molecular Mechanisms Involved in the Actions 
of Calcium-mediated Hormones 
John H. Exton, M.D., Ph.D. — Investigator 
Dr. Exton is also Professor of Molecular Physiology and Biophysics and of Pharmacology at the Vanderbilt 
University School of Medicine. He received his medical degree from the University of New Zealand and his 
Ph.D. degree in biochemistry from the University of Otago, New Zealand. His postdoctoral research was 
done in the Department of Physiology at Vanderbilt University School of Medicine with Charles Park, 
where he has remained as a faculty member. His honors include the Lilly Award of the American Diabetes 
Association and the M.D. degree with distinction from the University of Otago. 
THE major objective of my laboratory is to elu- 
cidate the mechanisms of action of hor- 
mones, neurotransmitters, and other agents that 
transmit information in the nervous system and 
other organ systems by altering membrane lipids 
and increasing the concentration of calcium ions 
in their target cells. A large number of agents act 
this way. They include regulators of heart func- 
tion and blood flow, such as epinephrine, nor- 
epinephrine, acetylcholine, angiotensin, and 
vasopressin; other neurotransmitters, such as se- 
rotonin, neurotensin, and substance P; and agents 
that control certain pituitary and pancreatic se- 
cretions, food digestion, bladder and uterine con- 
traction, platelet aggregation, and certain re- 
sponses to trauma and infection. 
We initially established that many actions of 
hormones and neurotransmitters are not due to 
changes in the cellular levels of cyclic nucleotide 
second messengers but result from an increase 
in the level of calcium ions. The next phase of 
the work involved the demonstration that these 
calcium-mediated agents act both by mobilizing 
calcium ions from internal stores and by stimulat- 
ing the inflow of these ions across the cell mem- 
brane. We also demonstrated that the receptors 
for these agents are located on the outer surface 
of their target cells. Thus these cells must have 
some means of signaling from the receptors to 
their internal calcium stores. 
Efforts were directed toward elucidating the 
signaling mechanisms. Initially we tested the hy- 
pothesis that the signal was generated by the 
breakdown of phosphatidylinositol, a phospho- 
lipid in the cell membrane. However, this break- 
down was too slow to account for the changes in 
calcium. A related phospholipid, phosphatidyl- 
inositol 4,5-bisphosphate (PIP2), broke down 
more rapidly in response to hormones, but the 
change was transient. The situation became 
clearer when inositol 1 ,4,5-trisphosphate (IP3) 
was identified as the signaling molecule for cal- 
cium. This compound is generated when PIP2 is 
broken down by the enzyme phospholipase C. 
The other compound produced is 1,2-diacylgIy- 
cerol (DAG), which is also a signaling molecule, 
since it activates a specific protein kinase (pro- 
tein kinase C) . 
The present activities of the laboratory encom- 
pass three major research areas. The first involves 
elucidating how calcium-mediated agents stimu- 
late the breakdown of PIP2 . A major discovery has 
been the finding that a G protein (a regulatory 
protein that binds the nucleotide GTP) is in- 
volved in coupling the receptors for these agents 
to the phospholipase C enzyme that breaks down 
PIP2. Our work has involved purifying and char- 
acterizing the relevant G protein from liver cell 
membranes and reconstituting it with other com- 
ponents of the signaling system. Reconstitution 
of the G protein with PIP2 phospholipase C has 
been achieved, and the system has been used to 
purify the protein to homogeneity in both the 
complete form (^a^y heterotrimer) and in the 
form of the free a-subunit. The a-subunit (molec- 
ular weight 42,000) has been shown immuno- 
logically to be a member of the Gq family of G 
proteins. 
The a-subunit activated by a GTP analogue has 
been used to identify the specific isozyme form of 
the PIP2 phospholipase C controlled by the G 
protein. In both liver and brain, it is the l48-kDa 
18-isozyme. Evidence that the purified G protein is 
coupled to at least one receptor for a calcium- 
mobilizing agent has been obtained by the dem- 
onstration that binding of a GTP analogue to the 
G protein heterotrimer is enhanced by its recon- 
stitution with the Ml (calcium-mobilizing) mus- 
carinic receptor for acetylcholine, but not with 
the M2 form of this receptor. In addition, three 
calcium-mediated agents (vasopressin, epineph- 
rine, and angiotensin) specifically stimulate the 
labeling of two G proteins by a radioactive, light- 
reactive analogue of GTP, azidoanilido GTP, in 
liver cell membranes. These proteins (apparent 
molecular weights 42,000 and 43,000) have also 
been shown immunologically to be members of 
the Gq family of G proteins. It is likely that the 
smaller protein is identical to the a-subunit 
(42,000 molecular weight) that activates PIP2 
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