CELL SIGNALING THROUGH PHOSPHOLIPID BREAKDOWN 
John H. Exton, M.D., Ph.D., Investigator 
I. Agonist and Guanine Nucleotide Regulation of 
Polyphosphoinositide Phospholipase C. 
It is commonly accepted that many hormones 
and neurotransmitters exert their effects on their 
target cells by stimulating the breakdown of a 
plasma membrane phospholipid, phosphatidyl- 
inositol 4,5-bisphosphate (PIP2), through activation 
of a specific phospholipase C to yield two intracel- 
lular signaling molecules, namely inositol 1,4,5- 
trisphosphate (IP3), which releases Ca^^ ions from 
an internal store, and 1,2-diacylglycerol (DAG), 
which activates protein kinase C. 
Key components in the signaling system are the 
hormone receptors, the PlP^-specific phospholipase 
C and the GTP-binding regulatory protein (G pro- 
tein) that links these. The V^-vasopressin receptor 
has been solubilized from liver plasma membranes 
and purified to apparent homogeneity; a key step 
in this process is affinity chromatography on a V^- 
vasopressin antagonist linked to agarose. The re- 
ceptor, which can be specifically crosslinked to 
[^^^I] vasopressin using disulfosuccinimidyl tartrate, 
is a 58 kDa protein that shows a selectivity for 
antagonists compared with antagonists. Peptides 
derived from the receptor are being sequenced to 
develop probes to begin cloning. 
Two PIP^ phospholipases have been identified in 
liver plasma membranes; one of them is apparently 
regulated by G^^ (see below) and has been purified 
to near homogeneity. This 148 kDa polypeptide 
cross-reacts with monoclonal antibodies to the type 
II or 7 form of the enzyme from brain. 
Several G proteins have been purified from rat 
liver plasma membranes as a (3 7 heterotrimers. 
Some of them have been identified as G and G 
1 s 
species by toxin labeling and reconstitution with 
SV9 eye" membranes, whereas others represent 
novel G proteins. Purification of some of these 
unidentified G proteins to homogeneity is being 
undertaken. The purified G proteins and their re- 
solved a-subunits are being analyzed by labeling 
with [^^PJazidoanilido GTP and by Western analy- 
sis, using polyclonal antisera to different peptide 
sequences in the a-subunits of known G proteins 
(G.J, G.^, G.J, G^, and T^) and to a sequence com- 
mon to most G protein a-subunits. They will be 
tested for the activity of G (the G protein regulat- 
ing PIP^ phospholipase C)ly 1) reconstitution with 
preparations of the vasopressin receptor and mea- 
surement of GTPase activity and GTP effects on va- 
sopressin binding; 2) reconstitution with PIP^ phos- 
pholipase C and assay of GTP7S stimulation of activ- 
ity; and 3) reconstitution with liver plasma 
membranes photolyzed with azidoanalido GTP, to 
inactivate endogenous G proteins, and assay for 
GTP7S stimulation of endogenous PIP^ phos- 
pholipase C. 
In another study, G protein a-subunits are being 
solubilized from liver plasma membranes previously 
treated with a GTP analogue (GTP7S), and are being 
purified to identify G^^ (the a-subunit of Gp). With 
this protocol a protein (presumably Gp^) has been 
shown to retain its ability to activate PIP^ phos- 
pholipase C through several chromatographic steps. 
This protein is being purified to homogeneity to 
provide partial sequence to begin cloning. 
II. Regulation of Phosphatidylcholine Breakdown in 
Isolated Hepatocytes. 
High-performance liquid chromatography (HPLC) 
analyses of the molecular species of DAG generated 
in hepatocytes in response to Ca^''^-mobilizing ago- 
nists have indicated that only part of the DAG accu- 
mulating in response to hormones is derived from 
the breakdown of phosphoinositides and that there 
is another major source. Fatty acid analyses of the 
DAG species have shown that the most likely 
source is phosphatidylcholine (PC). When this 
phospholipid is selectively labeled with [^H]alkyl- 
lyso-glycerophosphochofine or [^^C]lyso-PC, its 
breakdown to DAG and phosphatidic acid (PA) is 
stimulated by Ca^^ -mobilizing agonists. The re- 
sponse is mimicked by the Ca^"*^ ionophore A23187 
and is markedly reduced in Ca^^ -depleted cells, im- 
plying that it is secondary in part to the elevation in 
cytosolic Ca^^ induced by the agonists. The break- 
down of labeled PC is also induced by tumor-pro- 
moting phorbol esters, suggesting the involvement 
of protein kinase C. The molecular DAG species 
generated by A23187 are similar to those produced 
by the Ca^^-mobilizing agonists but differ from 
those induced by phorbol ester. This suggests that 
the mechanisms involving Ca^^ and protein kinase 
C are not identical. Current efforts are directed to- 
ward defining the specific phospholipases and 
other components involved in both mechanisms of 
regulation of PC hydrolysis. As a first approach, the 
characteristics and subcellular distributions of PC 
Continued 
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