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. Agents that act this way in- 
clude regulators of heart function and blood 
flow, such as epinephrine, norepinephrine, ace- 
tylcholine, angiotensin, and vasopressin; other 
neurotransmitters, such as serotonin, neuroten- 
sin, and substance P; and agents that control cer- 
tain pituitary and pancreatic secretions, food di- 
gestion, bladder and uterine contraction, platelet 
aggregation, and certain responses to trauma and 
infection. 
We initially established that many hormones 
and neurotransmitters act by increasing the intra- 
cellular concentration of calcium ions. The next 
phase of our work involved the demonstration 
that the increase is due to both mobilization of 
calcium ions from internal stores and stimulation 
of their inflow across the cell membrane. We also 
demonstrated that the receptors for calcium-me- 
diated agents are located on the outer surface 
of their target cells. Thus these cells must 
have some means of signaling from the receptors 
to the internal calcium stores, and our eff^orts 
were directed toward elucidating the signaling 
mechanism. 
Initially we tested the hypothesis that the sig- 
nal is generated by the breakdown of phosphati- 
dylinositol, a phospholipid in the cell mem- 
brane. However, this was found to be too slow to 
account for the changes in calcium. Attention was 
then focused on a related phospholipid, phos- 
phatidylinositol 4,5-bisphosphate (PIP2), which 
breaks down more rapidly in response to hor- 
mones. The situation became clearer when inosi- 
tol 1,4,5-trisphosphate (IP3) was identified as 
the signaling molecule for intracellular calcium 
release. This compound is generated when PIP2 is 
broken down by the enzyme phospholipase C. The 
other compound produced is 1 ,2-diacylglycerol 
(DAG), which is also a signaling molecule, since 
it activates a specific protein-phosphorylating 
enzyme, protein kinase C. 
The present activities of the laboratory encom- 
pass two 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 proteins from liver cell 
membranes and reconstituting them with other 
components of the signaling system. Reconstitu- 
tion of the G proteins with PIP2 phospholipase C 
has been achieved, and the system has been used 
to purify the G proteins to homogeneity in both 
the complete form {a^y heterotrimers) and in 
the form of free a-subunits. Two a-subunits (42 
and 43 kDa) have been identified, and both have 
been shown immunologically to be members of 
the Gq family of G proteins. Partial sequencing of 
tryptic peptides of both proteins has confirmed 
that they are Gaq and Gq:,i. 
Purification of the G protein activators of the 
phospholipase C in the heterotrimeric form has 
likewise yielded two G proteins corresponding to 
Gq and Gn. Using Gofq and Gofn activated by a 
GTP analogue, we have shown that the specific 
isozyme form of the PIP2 phospholipase C con- 
trolled by these G proteins in both liver and brain 
is the 148-kDa -isozyme. Evidence that Gq 
and G,] are coupled to receptors for calcium- 
mobilizing agents has also been obtained. Three 
calcium-mediated agents (vasopressin, epineph- 
rine, and angiotensin) specifically stimulate the 
labeling of Gaq and Ga,, by a radioactive, light- 
reactive analogue of GTP in liver cell mem- 
branes. Binding of a GTP analogue is enhanced by 
an acetylcholine analogue when Gq and G,j are 
reconstituted with the M, (calcium-mobilizing) 
muscarinic receptor but not with the M2 recep- 
tor. Co-reconstitution of the purified Mj recep- 
tor, Gq/Gi,, and phospholipase C-/3i permits 
agonist-stimulated hydrolysis of PIP2 in a GTP ana- 
logue-dependent manner. The activation of the 
phospholipase is paralleled by activation of the G 
proteins, as measured by their binding of the 
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