CELL SIGNALING THROUGH PHOSPHOLIPID BREAKDOWN 
John H. Exxon, M.D., Ph.D., Investigator 
G Protein-mediated Regulation 
of Phosphoinositide Phospholipase C 
Many hormones and neurotransmitters exert their 
effects by stimulating the breakdown of plasma 
membrane phosphatidylinositol 4,5-bisphosphate 
(PIP2) in their target cells, through activation of a 
specific phospholipase C to yield inositol 1 ,4,5-tris- 
phosphate (IP3), which releases Ca^^ ions from in- 
ternal stores, and 1 ,2-diacylglycerol (DAG), which 
activates protein kinase C. 
Key components in the signaling system are the 
hormone receptors, the PIP2-specific phospholi- 
pase C, and the specific GTP-binding regulatory 
protein (G protein) that links these. Dr. Exton and 
his colleagues have identified two G proteins from 
bovine liver plasma membranes that specifically reg- 
ulate the -isozyme of phospholipase C and have 
purified them to homogeneity in both the a-subunit 
and heterotrimeric {a(iy ) forms. The heterotrimeric 
proteins have a subunits of 42 and 43 kDa, which 
have been identified by sequencing as and 
two novel G protein a subunits identified by molec- 
ular cloning by Dr. Melvin Simon (California Insti- 
tute of Technology) . 
Studies of the PIP2 phospholipase C isozyme spec- 
ificity for activation by the G proteins have indi- 
cated unequivocally that the -isozyme is activated 
by both the 42- and 43-kDa a subunits, whereas the 
7i- and 5, -isozymes are not. This is different from 
the isozyme specificity for phosphorylation by 
growth factor receptor tyrosine kinases. 
In collaborative studies with Dr. Elliott Ross (Uni- 
versity of Texas Southwestern Medical Center at 
Dallas), Dr. Exton and his colleagues have reconsti- 
tuted Gq and Gji purified from bovine liver with 
recombinant Ml muscarinic receptor in lipid vesi- 
cles and have demonstrated cholinergic agonist acti- 
vation of the G proteins demonstrated by stimula- 
tion of the binding of the labeled GTP analogue 
[^^S]GTP7S. In contrast, the M2 receptor was a poor 
activator of Gqy,,. The activation of GTP7S binding 
to Gq/n induced by agonist in the presence of the 
Ml receptor was paralleled by an increased ability 
of the G proteins to activate the /3j -isozyme of PIP2 
phospholipase C. Co-reconstitution of this receptor 
with Gq/i , and the phospholipase demonstrated gua- 
nine nucleotide-dependent agonist activation of 
PIP2 hydrolysis. 
During these studies, the GTPase activity of Gqyn 
was low relative to that of most other G proteins, 
even when activated by agonist and receptor. This 
activity could be greatly stimulated by phospholi- 
pase C-/3, in the presence of the Ml receptor and 
cholinergic agonist. These studies show that the 
phospholipase C acts as a GTPase-activating protein 
(GAP) for its G protein regulator Gq/,,. This would 
provide a mechanism for the rapid termination of 
agonist activation of the lipase. 
In a search for other G proteins that activate other 
PIP2 phospholipase C isozymes, a novel cytosolic 
phospholipase was found in liver and brain that was 
not related to the other known (jS, 7, 6) isozymes. 
This was stimulated by a mixture of G proteins acti- 
vated by GTP7S. Purification of the G protein sub- 
unit responsible for the stimulation revealed the 
surprising finding that it was the 187 complex. Al- 
though this complex produced a very large stimula- 
tion of the soluble phospholipase, it was ineffective 
on the 5j- and 7; -isozymes and produced a small 
stimulation of the jSi-isozyme. A 25-nanomolar con- 
centration of brain or liver was necessary for 
half-maximal stimulation of the soluble enzyme. 
The activation was completely reversed by excess 
The findings demonstrate a novel mechanism of 
G protein activation of a phospholipase, namely via 
the 187 complex. 
Regulation of Phosphatidylcholine 
Hydrolysis 
Many growth factors and other agonists stimulate 
the breakdown of phosphatidylcholine (PC) in 
cells, through activation of phospholipase C and D. 
The products are DAG and phosphatidate (PA) . One 
target of DAG is protein kinase C, but this consists of 
many isozymes (a-77) . The activation and transloca- 
tion of specific isozymes in response to PC and PIP2 
breakdown induced by a-thrombin is being studied 
in IIC9 fibroblasts. The function(s) of PA remains 
obscure, but one possible target is protein kinase 
C^ This enzyme was previously identified only at 
the cDNA level, and its properties were not well 
defined. Its purification from several mammalian 
tissues was undertaken, and it was finally purified to 
homogeneity from bovine kidney. Its molecular 
weight at 78,000 and its kinetic properties and in- 
sensitivity to Ca^^ were established. More impor- 
tantly, it was shown not to bind phorbol esters or 
respond to these compounds. However, it responds 
to certain lipids, including phosphatidylserine, PA, 
and unsaturated fatty acids. Potential mechanisms of 
control of this ubiquitous kinase are being studied. 
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