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Cell Signalling Biology Michael J. Berridge Module 2 Cell Signalling Pathways 2 52

4. DAG is phosphorylated by the Ca 2 + -sensitive enzyme Phospholipase Cβ (PLCβ)
DAG kinase to form phosphatidic acid (PA) or it is hy- The PLCβ isoforms are mainly activated by Gprotein-
drolysed by DAG lipase to form monoacylglycerols coupled receptors (GPCRs).PLCβ1and PLCβ3 are fairly
(MAGs) such as 2-arachidonylglycerol (2-AG), which ubiquitous, whereas PLCβ2and PLCβ4 have more lim-
is one of the endocannabinoids. The 2-AG is hydro- ited tissue distributions.
lysed by monoacylglycerol lipase (MAGL) to glycerol
and arachidonic acid.
5. PA is transferred from the plasma membrane to the ER PLCβ isoform distribution and function
where it interacts with CTP to form CDP/DAG by a PLCβ1 is highly concentrated in brain (pyramidal cells of
CDP/DAG synthetase. The inositol, which can be pro- hippocampus, Purkinje cells and granule cells). The knock-
duced through three mechanisms (de novo synthesis, out phenotype is characterized by seizures leading to sud-
recycling or uptake of dietary inositol), is attached den death. These seizures are similar to those seen dur-
to CDP/DAG by the PtdIns synthetase [cytidine di- ing epilepsy. Many of the defects are found in the central
phosphate (CDP)/diacylglycerol (DAG):myo-inositol nervous system (CNS). Activation of PLC by muscarinic
3-phosphatidyltransferase] located on the endoplasmic receptors is suppressed in the temporal lobe, cerebellum
reticulum (ER) to form PtdIns. and hippocampus, and this could decrease the inhibitory
6. The PtdIns is then transported from the ER back to the tone, leading to the seizures.
plasma membrane by a PtdIns transfer protein (PITP). PLCβ2 is mainly expressed in cells of the immune sys-
Cells express two PITPs, an α and a β isoform, pro- tem. Knockout mice show some disruption of chemokine
duced by separate genes. The latter appears to be the signalling. For example, neutrophils fail to respond to
housekeeping isoform that is essential for cell survival, the chemoattractant fMet-Leu-Phe with the usual changes
whereas the α isoform has a more specialized function. in PLC activation, Ca 2 + release and superoxide radical
When PtdIns is added back to the plasma membrane, it (O 2 −• ) production. However, the response to lipopolysac-
can once again be converted through the two phos- charide (LPS) is normal. Despite this absence of PLC activ-
phorylation reactions to maintain the supply of the ation in leucocytes, chemotactic responses are enhanced,
PtdIns4,5P 2 that is the precursor for the phosphoin- suggesting that this signalling pathway may normally ant-
ositide signalling pathway. agonize the signalling pathways normally used to control
7. Cells can also use plasma inositol, which is taken up chemotaxis.
by a sodium-dependent myo-inositol cotransporter-1 PLCβ3 is found mainly in brain, parotid, smooth
(SMIT1). muscle and liver. Disruption of this gene is lethal, with
the mice dying by day 2.5. The embryos are highly dis-
organized and have low cell numbers, suggesting a role
Phospholipase C (PLC) for this isoenzyme in cell division. This isoform has two
Phospholipase C (PLC) hydrolyses the lipid precursor sites (Ser-26 and Ser-105) that are phosphorylated by cyclic
PtdIns4,5P 2 to produce both InsP 3 and DAG. It is made GMP-dependent protein kinase (PKG) resulting in a de-
up of five subclasses, which have variable isoforms PLCβ crease in enzyme activity (Module 7: Figure smooth muscle
(β1--β4), PLCδ (δ1--δ4), PLCγ (γ1and γ2), PLCε and cell cyclic GMP signalling).
PLCζ (Module 2: Figure PLC structure and function). All PLCβ4 is found in cerebellum and granule cells. Knock-
forms of the enzyme have an absolute requirement for out mice appear to have defects in the cerebellum, result-
Ca 2 + that plays a critical role in the catalytic site. The ing in poor motor co-ordination due to a decrease in PLC
PtdIns4,5P 2 is cleaved through two sequential reactions: stimulation through metabotropic glutamatergic and mus-
first, the phosphodiester bond is cleaved to DAG and in- carinic receptors. There also are defects in the processing
ositol 1,2-cyclic phosphate, the latter is then hydrolysed to of visual information.
give the acyclic InsP 3 that is released into the cytoplasm. The primary activation mechanism of the PLCβ iso-
The domain structure of the different PLC isoforms re- forms is through the G q family of heterotrimeric G pro-
veals a number of common structural features related to teins (G q ,G 11 ,G 14 ,G 15 and G 16 )(Module 2: Figure PLC
the way the enzyme associates with the membrane and structure and function). While G q and G 11 are found
functions to hydrolyse PtdIns4,5P 2 . The catalytic domain in most tissues, the other three are restricted to cells of
is made up from the X and Y regions. They have at least haematopoetic origin. The function of these G proteins
two potential lipid-binding domains: the pleckstrin homo- is complicated because the PLCβ isoforms are sensitive
logy (PH) domain and the C2 domain (Module 6: Figure to both the α and βγ components of the heterotrimeric
modular lipid-binding domains). In the case of PLCδ1, the complex. PLCβ1and PLCβ4 are most sensitive to stim-
enzyme may first associate with the membrane through its ulation through α subunits, whereas βγ is more effective
PH domain, which has a high affinity for PtdIns4,5P 2 .Fur- at activating PLCβ2and PLCβ3. This sensitivity to βγ
ther interactions may then occur through the C2 domain, subunits may explain the pertussis-toxin-sensitive stimu-
which has an extensive interface with the catalytic domain lation of PLC by the G i family of G proteins . For example,
and may thus enable the catalytic site to integrate itself into the adenosine A 1 , muscarinic M 2 , somatostatin and μ-, δ-
the membrane to hydrolyse the lipid. The main difference and κ-opioid receptors can couple to PLC through G i and
between these PLC isoforms concerns the way in which G o .Ofthe fiveGβ and 11 Gγ subunits, there appears to
they are activated. be little specificity with regard to the activation of PLC.

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