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Physiological Reviews, Vol. 80, No. 4, October 2000, pp. 1291-1335
Copyright ©2000 by the American Physiological Society
Departments of Anesthesiology and Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York
I. INTRODUCTION
II. STRUCTURE AND CATALYTIC FUNCTION
A. Catalytic/
-Barrel
B. Hydrophobic Rim
C. X/Y-Spanning Sequence (Z Region)
D. PH Domain
E. EF-Hands
F. C2 Domain
G. Carboxy-Terminal Extension
III. THE PHOSPHOLIPASE C-ISOZYMES
A. Tissue Distribution and Expression
B. Control by G protein-Coupled Receptors
C. Serine/Threonine Phosphorylation
D. Polyphosphoinositide Synthesis: the Need to Resupply
E. Scaffolding and Lateral Organization
F. Nuclear Targeting
G. Studies in Transgenic Animals
IV. THE PHOSPHOLIPASE C-ISOZYMES
A. Expression in Adults and During Development
B. Activation by Receptor Tyrosine Kinase
C. Serine/Threonine Phosphorylation
D. Tyrosine Phosphorylation and the Control of Catalytic Activity
E. Regulation by Immunoglobulin/Cytokine Receptors
F. Regulation by Heptahelical Receptors
G. PI-Transfer Protein
H. Subcellular Distribution and Translocation
I. Functional Studies of the PLC-Isozymes
V. THE PHOSPHOLIPASE C-ISOZYMES
A. Yeast PLC
B. Slime Mold PLC
C. Plant PLC
D. Mammalian PLC-
VI. SUMMARY AND CONCLUSIONS
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ABSTRACT |
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Rebecchi, Mario J. and
Srinivas N. Pentyala.
Structure, Function, and Control of
Phosphoinositide-Specific Phospholipase C. Physiol. Rev. 80: 1291-1335, 2000.
Phosphoinositide-specific
phospholipase C (PLC) subtypes
,
, and
comprise a related
group of multidomain phosphodiesterases that cleave the polar head
groups from inositol lipids. Activated by all classes of cell surface
receptor, these enzymes generate the ubiquitous second messengers
inositol 1,4,5-trisphosphate and diacylglycerol. The last 5 years have
seen remarkable advances in our understanding of the molecular and
biological facets of PLCs. New insights into their multidomain
arrangement and catalytic mechanism have been gained from
crystallographic studies of PLC-
1 , while new modes of
controlling PLC activity have been uncovered in cellular studies. Most
notable is the realization that PLC-
, -
, and -
isoforms act in
concert, each contributing to a specific aspect of the cellular
response. Clues to their true biological roles were also obtained. Long
assumed to function broadly in calcium-regulated processes, genetic
studies in yeast, slime molds, plants, flies, and mammals point to
specific and conditional roles for each PLC isoform in cell signaling
and development. In this review we consider each subtype of PLC in
organisms ranging from yeast to mammals and discuss their molecular
regulation and biological function.
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I. INTRODUCTION |
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Phosphoinositide-specific phospholipase C (PLC) isozymes found in eukaryotes comprise a related group of proteins that cleave the polar head group from inositol phospholipids. Under the control of cell surface receptors, these enzymes hydrolyze the highly phosphorylated lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], generating two intracellular products: inositol 1,4,5-trisphosphate (InsP3), a universal calcium-mobilizing second messenger, and diacylglycerol (DAG), an activator of protein kinase C.
Historically, the PLC isozymes have been studied since the 1950s. Early observations by the Hokins (142), and later by Michell (243) and others, led to the recognition of PLC as a key enzyme in agonist-stimulated phosphoinositide metabolism and calcium signaling. The direct link between PLC and the release of intracellular calcium stores was finally forged with the publication of a seminal paper in 1983 by Streb et al. (351) describing the second messenger properties of InsP3 (reviewed in Ref. 25).
In the late 1980s and early 1990s, three mammalian PLC subtypes,
,
, and
, were isolated and their corresponding cDNA sequences determined (300). Paralleling this work, a number of PLC
regulators were identified, especially the GTP-binding (G)
q related subunits (349) (reviewed in Ref.
91) and protein tyrosine kinases (reviewed in Ref. 45). Isolation and
identification of these components allowed investigators to test
whether already recognized regulatory mechanisms controlled the PLC
subtypes. Their results gave rise to the current G protein and tyrosine
kinase models of PLC regulation.
PLCs are soluble multidomain proteins ranging in molecular
masses from 85 to 150 kDa. Four
-, two
-, four
-isoforms, and numerous spliced variants have been described in mammals. Those found
in yeasts, slime molds, filamentous fungi, and plants closely resemble
mammalian
. Comparisons of their DNA sequences suggest an
evolutionary relationship in which PLC-
appeared first in primitive
single-celled eukaryotes. The PLC-
and -
subtypes arose
later, after the split between fungi or plants and animals, but before
the parazoan-eumetazoan split, about 940 million years ago
(198); their delayed appearance coincides with the
diversification of other signaling components, such as G
subunits
and protein kinase C. Later duplications of each PLC subtype led to the
appearance of the numerous isoforms in animals.
At present, many of the players in phosphoinositide/calcium signaling are identified, some with three-dimensional pictures. On a cellular level, questions of which PLC isozymes go with which regulators are mostly answered. Despite this progress, our understanding of how and where PLC isozymes work in living cells is limited. New information suggests a higher level of organization than is implied by the current regulatory schemes, giving rise to a number of questions: Are these freely diffusing effector proteins or part of a highly organized network? Do these enzymes only act at the plasma membrane? Might they act in concert? Where is their substrate localized and how is it supplied? Finally, what are the physiological functions of the many isoforms and how is their expression controlled? In our review we attempt to address these questions (for other recent reviews see Refs. 91, 182, 299, 335).
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II. STRUCTURE AND CATALYTIC FUNCTION |
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The sequences of the eukaryotic PLC contain a string of modular
domains organized around a catalytic
/
-barrel formed from the
characteristic X- and Y-box regions (392). They
include a pleckstrin homology (PH) domain, EF-hand motifs, and a
single C2 domain that immediately follows the Y-box region (see
Fig. 1). Additional regulatory motifs are
present in the
- and
-subtypes, but absent in PLC-
. To
simplify the discussion of common domains, we draw comparisons to
1, the only eukaryotic PLC for which the three-dimensional structure is known (Fig. 1).
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A. Catalytic
/
-Barrel
In the crystallographic structure of PLC-
1, the
catalytic domain is formed from the X and Y regions, 147 and 118 residues, respectively (88, 90). The domain
is comprised of alternating
-helices and
-strands and resembles
an incomplete triose phosphate isomerase (TIM),
/
-barrel. Like
similar structures, the catalytic residues of PLC-
1 are
located at one end of the barrel. In this case, the site which is
partly rimmed by hydrophobic residues is formed by a shallow cavity at
the carboxy-terminal end. The unfinished lip of the barrel forms a
spoutlike structure that may allow entry and egress of substrate or
product at the membrane surface. The intervening sequence joining the X
and Y halves of the barrel (43 residues) is highly disordered and not
an integral part of the structure, although it may have an important
regulatory function (see discussion below).
Eukaryotic and prokaryotic forms of PLC catalyze hydrolysis of the O-P bond connecting phosphoinositol to DAG. The requirement for inositol is absolute, because the substrate, through the 2-position hydroxyl, participates in nucleophilic attack on the phosphorous, resulting in a cyclic intermediate. Catalysis proceeds by an in-line sequential mechanism involving the cyclic 1,2-phosphodiester intermediate, which can be further hydrolyzed to myo-inositol 1-phosphomonoester (41, 146, 147, 221, 378). Eukaryotic forms readily hydrolyze this intermediate, although the relative amounts of the cyclic and noncyclic product depend on the particular isozyme, substrate, pH, and calcium concentration (188).
Various features of the polar head group affect substrate preference. Although the prokaryotic forms of PLC prefer phosphatidylinositol (PI) and PI-glycans, the eukaryotic enzymes have an order of preference that is generally PI(4,5)P2 > phosphatidylinositol 4-phosphate [PI(4)P] > PI. Unlike their secreted prokaryotic counterparts, they are incapable of cleaving the polar head group of PI-glycan anchors. Neither forms are capable of hydrolyzing the 3-phosphorylated phosphoinositides.
Within the PLC-
1 catalytic site, a network of hydrogen
bonds and salt-bridges ligate the inositol ring substituents and
generally account for the observed substrate preference. Lys-438 and
Lys-440 of the first half of the
/
-barrel, and Ser-522 and
Arg-549 of the second half, ligate the phosphomonoesters at positions 4 and 5 of the PI(4,5)P2 polar head group. These
are conserved in the
- and
-isozymes. Interestingly, single amino
acid substitutions of Arg-549 do not abolish catalytic activity but
switch substrate preference from PI(4,5)P2 to
PI (52, 383).
The catalytic residues, conserved in all eukaryotic PLCs, include
His-311, His-356, Glu-341, Asp-343, and Glu-390 (89). A
single calcium ion is bound to the active site coordinated by the side
chains of Asn-312, Glu-341, Asp-343, and Glu-390 of
PLC-
1. The 2-position hydroxyl of the inositol ring and
the exocyclic phosphodiester oxygen also appear to contact this metal
which plays an essential role in catalysis, lowering the
pKa of the attacking hydroxyl and the negative
charge of the transition state. In the current model of the reaction
(89), an active site base, possibly Glu-390 in a charge
relay system with His-392, strips a proton from the 2-position hydroxyl
of the inositol ring, promoting intramolecular attack on the
phosphorous and cyclization. His-311 is too far removed to abstract a
proton but instead stabilizes the developing charge on the initial
pentacovalent transition state. His-356 participates in general
acid/base catalysis, protonating the DAG leaving group during the
formation of the cyclic 1,2-phosphoinositol intermediate. Acting as a
general base, this residue then abstracts a proton from water which
attacks the cyclic phosphodiester intermediate. Consistent with this
model, amino acid substitutions of His-311 and His-356, as well as the
calcium binding residue Glu-341, have been shown to reduce or abolish
catalytic activity (52, 85).
Although other domains in PLC have the potential to bind calcium, the
single catalytic calcium ion seems to be the only essential metal. This
is supported by studies of a PLC-
1 mutant missing other
calcium binding sites located in the C2 domain (121). This mutated enzyme which has the same activation constant
(Kact) for calcium (~1.4 µM) as the
wild-type PLC. The in vitro results also agree with calcium
activation constants obtained in permeabilized cells (2).
Interestingly, the dissociation constant (Kd)
for calcium binding to the catalytic site of PLC-
1,
measured by isothermal titration calorimetry, is ~ 30-50 µM,
in the absence of phospholipid (121). Although this is
substantially greater than its Kact, the
crystallographic structure shows that the
PI(4,5)P2 polar head group helps coordinate
the metal ion, accounting, at least in part, for the weak affinity
measured in the absence of substrate. The affinities of the
- and
-catalytic sites for calcium have yet to be determined, but their
Kact is generally less than those reported for
(see Refs. 165 and 379 for examples). Thus the
- and
-, but
not the
-isoforms, are expected to be active at resting cytoplasmic
calcium concentrations.
B. Hydrophobic Rim
Surrounding the active site is a ridge of hydrophobic residues,
Leu-320, Tyr-358, Phe-360, Leu-529, and Trp-555 (88); a similar ridge or rim is found in the prokaryotic forms
(130). Such a ridge could insert into the membrane surface
in a process required for full enzymatic activity. This proposal is
based on studies of PLC-
1, -
2,
-
1, and -
1 in which raising the surface pressure of phospholipid monolayers to levels equivalent to, or slightly beyond, the packing densities found in membrane bilayers profoundly inhibits catalytic activity (33,
160, 161, 294). One notable
exception is the PLC-
isoform found in turkey erythrocytes, which
exhibits a pressure optimum that is nearly equivalent to bilayer
packing density.
Inhibition by lateral pressures implies that the enzyme must do work to penetrate the membrane surface, bringing the substrate into register with catalytic residues. The presence of hydrophobic residues surrounding the active site further suggests that this ridge inserts into the acyl-chain region. Mutagenesis of the rim, involving replacement of bulky nonpolar residues with alanine, reduces the effects of increased surface pressure in monolayers, without affecting PI(4,5)P2 hydrolysis in detergent-mixed micelles (84). These results point to an important interaction between the hydrophobic rim and the membrane bilayer and are consistent with a hydrophobic insertion model (a smaller area of penetration reduces the negative slope of the pressure/activity relation). Nevertheless, the identity of the protein sequence and the depth to which it penetrates are unknown. Moreover, these enzymes need only dip between the polar head groups to effectively engage substrate.1 At this depth they would still experience the lateral pressures exerted in the monolayer experiments.
C. X/Y-Spanning Sequence (Z Region)
Among the PLC subtypes, sequences linking the X- and Y-box regions are poorly conserved and are not required for catalysis (39, 83, 318), suggesting a role in subtype-specific regulation. These regions are also susceptible to proteolysis (59, 83, 318), consistent with the idea that they are highly flexible.
Unlike the
- and
-subtypes, the X/Y-spanning polypeptide in
PLC-
(also known as the Z region) is extensive, consisting of
multiple adaptor domains (Table 3). The
-sequences contain two Src
homology (SH)2, an SH3, and a single PH domain that engage both protein
and lipid binding partners. Although these domains are critical to
extrinsic regulation of the
-isoforms, they also exert an intrinsic
control on catalytic activity (see sect. IV). In contrast,
the comparable sequences of PLC-
and -
subtypes lack any
identifiable regulatory motifs. Nonetheless, the relatively short
sequences in the
- and
-subtypes also appear to exert an
intrinsic control over the catalytic core (318), raising
the possibility that Z-region sequences are key to a general
mechanism for controlling PLC catalytic activity.
D. PH Domain
The PH domain was originally described as a novel protein motif of
~100-amino acid residues, repeated twice in the protein, pleckstrin
(platelet and leukocyte C kinase substrate) (128, 239). These motifs have now been identified in >100 other
proteins (reviewed in Refs. 219, 296). Most can be grouped by function into a few classes: Ser/Thr protein kinases, Tyr protein kinases, small
G protein regulators, endocytic GTPases,
phosphoinositide-metabolizing enzymes, and
cytoskeletal-associated proteins. Many feature a catalytic site
(e.g., protein kinase) and additional adaptor domains. Because PH
domains lack any obvious catalytic properties and are found in proteins
associated in some way with the membrane, it was suggested that these
domains function as adaptors or tethers, linking their host proteins to
the membrane surface (99). Principal binding partners are
phosphoinositides and the 
-subunits of heterotrimeric G proteins.
Most eukaryotic PLCs contain a single PH domain of ~130 residues
located in the amino-terminal region. An additional PH motif, found
in the Z region of PLC-
1 and -
2, is split
by two SH2 and a single SH3 domain. PH sequences are not well conserved
among the PLCs, suggesting their association with subtype-specific
regulation. Interestingly, some PLCs lack any PH domain (Tables
1 and 2; see PLC in higher plants and PLC-
4 spliced variants).
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The
PH domain binds the polar head group of
PI(4,5)P2 (112, 220)
and is archetypical. The domain is required for PLC-
1 to
processively hydrolyze its substrate (59,
228, 402), suggesting that it tethers the
enzyme to the membrane surface during
catalysis.2 This is further
supported by equilibrium binding measurements (52,
58, 280, 295). Photolabeling
studies of the whole protein also point to a single high-affinity
PI(4,5)P2 binding site (180,
361).
The crystallographic structure of the PLC-
1 PH domain,
bound to Ins(1,4,5)P3 (Fig. 1), provides a
molecular view of this high-affinity site (98). The
whole structure is highly dipolar, with the positively charged surface
surrounding the binding cavity where nine residues ligate the 4 and 5 position phosphomonoesters through hydrogen bonds and salt bridges.
This remarkable specificity for PI(4,5)P2 is
also found in the related, but noncatalytic,
InsP3/PI(4,5)P2 binding protein
(IP3BP130) (179, 413) and the PH domain of
PLC-
4 (254). Comparable sequences of the
primitive
-isoforms (in Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Dictyostelium
discoidium) bear little resemblance to their mammalian counterparts.
Instead of PI(4,5)P2, PLC-
isoforms
bind the higher order polyphosphoinositide, phosphatidylinositol
3,4,5-trisphosphate [PI(3,4,5)P3], in vitro
(10, 94) and in living cells (94), forging a direct link between PI 3-kinase activation
and recruitment of PLC-
. The Kd for binding
the amino-terminal PH domain of PLC-
1 to this lipid
is ~1 µM (94). Mutating a sequence localized to the
putative loop between
-strands 3 and 4 of the
1 PH
domain, a region also important in the binding of
PI(4,5)P2 by PLC-
1, blocks binding.
Although the PH domains of the
-isoforms may also serve as membrane
tethers, they are not polyphosphoinositide specific. PLC-
1 and -
2 bind strongly to membranes
regardless of the presence of these lipids (165,
309). Moreover, PLC-
1, -
2,
and -
3 fail to bind InsP3 or other
inositol polyphosphate or polyphosphoinositide analogs, with
measurable affinity (361) (see point 1 in
NOTE ADDED IN PROOF). The isolated PH domains, like the
full-length enzymes, bind with moderate affinity to artificial
membrane bilayers, but with relatively little specificity for the
phospholipid head group (387). These nonspecific lipid
interactions can account, at least in part, for the membrane binding of
the intact enzyme (see also sect. IIG).
PH domains of PLC and other host proteins also bind to
so-called WD-40 proteins, which fold into
-propeller structures
whose surfaces harbor binding sites for other proteins
(326). One of these, the G-
subunit, has special
relevance for the PLC-
2 and -
3 isoforms
that are activated by G
heterodimers. Although the affinities of
most PH domains for these G subunits are low (296), their
colocalization with PLC at the membrane surface could translate
relatively weak binding into specific lateral interactions. Using
resonance energy transfer to measure these interactions, we obtained
evidence of specificity in the binding of G
subunits to the PH
domains of several PLC isotypes (387). The order of
affinity is PLC-
2 >
1 and
1. Moreover, the
2 PH domain is
sufficient for G
binding and enzyme activation, since exchanging
the PH domain of PLC-
1, a G
-insensitive subtype for the corresponding domain of
2, results in a chimera
that is highly activated by G
subunits (385). These
data are consistent with an earlier study that mapped the sequence for
G
activation to the amino-terminal two-thirds of the
PLC-
2 isoform (395). Although the PH domain
seems sufficient, other G
interaction sites have also been
considered. One promising candidate encompasses the sequence 580-641
of PLC-
2, located within the well-conserved Y half
of the catalytic
/
-barrel (202, 313,
406). These observations support the view that PLC-
isoforms engage G
subunits through multiple sites, but the
critical determinants reside in the PH domain.
E. EF-Hands
PLC isoforms have up to four EF-hand motifs, each consisting
of a helix-loop-helix structure. In PLC-
1, as in other
EF-hand proteins such as calmodulin and tropinin C, the motifs are
divided into pairwise lobes (88). This striking similarity
to calmodulin extends to their main chain conformations that are nearly
superimposable. As originally noted (88), the conformation of the
second lobe and the EF-hand/C2 interface correspond closely to the
calcium-saturated form of calmodulin bound to its target
polypeptide. The interesting juxtaposition to the catalytic
/
-barrel suggests the second lobe is not part of a calcium
switch, but is instead an integral part of the enzyme's core
structure. Indeed, deletions in this region completely inactivate
mammalian PLC-
1 (257).
The first two EF-hands present in mammalian, yeast
(22, 107, 283, 337,
411), and D. discoideum isoforms
(79) possess residues that would appear capable of binding
calcium or magnesium ions, whereas EF-hands 3 and 4 do not. There
is no evidence, however, that the first two motifs actually bind metal
ions, since none is found in crystals of the enzyme soaked in calcium
or its analogs (90); calcium binding to these motifs is
also not discernible in solution, whereas a single calcium binding
site, corresponding to the catalytic
/
-barrel, is readily
detected (121). Furthermore, EF-hands 1 and 2 do not
influence calcium sensitivity, since substitution of the putative
binding residues in PLC-
from D. discoideum is without
effect (78). This rule may extend to other PLC subtypes as
well, since
- and
-isoforms retain the four helix-loop-helix motifs but lack residues critical to metal binding.
Although the EF-hand region may have an important regulatory
function, it has yet to be identified. In fact, the first two EF-hands, as well as the amino-terminal PH domain, have been
dispensed with entirely in higher plants. Of four
Arabidopsis PLC sequences, two are also missing a portion of
the third EF-hand (125, 139). Similarly,
two spliced variants of PLC-
4 from mammalian retina lack
the PH domain and first EF-hand motif (102).
F. C2 Domain
C2 motifs, ~120 residues in length, have been identified in
numerous proteins, many of which function in lipid-signaling
pathways, including PLC (reviewed in Refs. 258, 303). The C2 domain
from PLC-
1 (88, 90,
120), like that of synaptotagmin-I (SytIA) (325, 357), consists of eight antiparallel
-strands arranged as a sandwich; their main chains can be
superimposed. In the
1-domain, three loops at one end of
the
-sandwich form the binding sites for up to three calcium ions
(90). The calcium binding regions of the
1
C2 domain, designated (CBR) 1(643-653), 2 (675-680), and 3 (706-714), are well
conserved in the various
-isoforms found in organisms ranging from
yeast to humans, suggesting that some important function has been retained.
Because each coordination complex is completed by water, the binding
affinity is assumed to be weak in the absence of membranes, in
agreement with the low calcium affinity measured by isothermal calorimetric titration (121). Moreover, disruption of
these calcium sites in PLC-
1 fails to affect the
calcium-dependent hydrolysis of PI(4,5)P2
in detergent micelles and phosphatidylcholine bilayers. Despite this
evidence, additional ligands (lipid or protein) could contribute to the
coordination of calcium in living cells, forming a stable complex that
modulates PLC catalytic activity at lower calcium
concentrations.3
More recent results indicate that PLC-
1 forms a
functional ternary complex with phosphatidylserine (PS) and calcium
(EC50 ~ 8 µM) through its C2 domain, in vitro
(227). The inability of phosphatidic acid (PA) to
substitute suggests the involvement of specific phosphoserine
determinants. Importantly, the complex is highly activating, but only
when the membrane concentration of PI(4,5)P2
is limiting (~1 mol%). These results support the tether and fix
model (88), wherein the PH domain bound to PI(4,5)P2 tethers the enzyme, whereas the
low-affinity binding of the C2 domain orients and fixes the
catalytic core to the membrane surface. Thus PS and calcium bound to
the rigid C2 domain could enhance surface sampling by the tethered
catalytic core, facilitating processive substrate hydrolysis when the
density of substrate is on the order of 1 mol% or less. While
attractive, this idea remains to be tested.
Both
- and
-subtypes also contain C2 domain motifs, yet the key
residues involved in calcium ligation are not conserved, a situation
reminiscent of some PKC and synaptotagmin subtypes whose C2 domains are
also unable to bind calcium. Although these domains may have been
retained as an integral part of the PLC catalytic core, they could also
function in recognition of other regulatory lipids and proteins. The
later possibility is consistent with our recent finding that the
PLC-
1 C2 domain binds specifically to GTP-charged
q, its physiological activator (386). As
discussed below, the C2 domain of PLC-
appears to operate in concert
with the carboxy-terminal extension to effectively engage this
protein. Whether comparable determinants are present in the C2 domains of the
-isoforms is not known.
G. Carboxy-Terminal Extension
A single C2 domain and short peptide cap the carboxy-terminal
ends of
- and
-isozymes, whereas
-subtypes have extensions of
~400-amino acid residues that contain sequences important to membrane
binding, nuclear localization, and their activation by G protein
subunits (165, 185, 274,
393). Deletion of this entire carboxy-terminal region
from PLC-
1 does not destroy catalytic activity but
abolishes activation by G
q and related proteins in vitro
(274) and in living cells (393) (see sect.
III). Deletions of the carboxy-terminal extensions of
PLC-
1 or -
2 or portions thereof, also
block binding to acidic phospholipids in vitro (165), association of PLC-
1 with the cell's particulate
fraction (185, 393), and transfer to the
nucleus (185). The carboxy-terminal region is also
required to stimulate the intrinsic GTPase activity of
G
q/11 and to link the
-isoforms to
membrane-associated scaffolding proteins (see sect.
IIIE).
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III. THE PHOSPHOLIPASE C- ISOZYMES |
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Four
-isotypes and additional spliced variants have been
identified in mammals.
-Homologs also have been found in turkey (PLC-
tk)(382), Drosophila (NorpA
and PLC-21)(32, 191, 330, 421), Xenopus (105), sponge, and
hydra (198) (Table 1); each isoform has distinctive
sequences outside the canonical X and Y regions.
PLC-
isoforms are regulated by heterotrimeric GTP-binding
proteins in a manner that generally fits the adenylyl cyclase control paradigm (see Refs. 91, 92, 335 for previous reviews), yet the coupling
of G proteins to PLC involves some features of note. Of special
interest is the high GTPase stimulating (GAP) activity of PLC itself,
which appears to require an opposing activity of agonist-bound
receptor to generate an InsP3/calcium signal (see sect.
IIIB5).
Although PLC regulation has been intensively studied, a number of
questions remain concerning the determinants of specificity in the
coupling of different receptor subtypes to individual
-isoforms and
the lateral organization of these signaling components on the plasma
membrane surface. Their presence in the nucleus and participation in
the nuclear PI cycle further reflects our limited understanding of what
they do. While transgenic animal experiments have provided some
insights, they have also raised many questions concerning the
biological role of each isoform. Especially relevant are recent studies
of NorpA in Drosophila phototransduction and the phenotypes
of transgenic mice lacking particular PLC isoforms or their regulatory
G protein subunits. The various signaling and developmental phenotypes
associated with their disruption suggest a few well-circumscribed
functions for each isoform.
A. Tissue Distribution and Expression
Mammalian PLC
-isoforms are differentially distributed,
with each pattern of expression reflecting, to some degree, the
functions identified in transgenic work. PLC-
1 is most
widely expressed, with the highest concentrations found in specific
regions of the brain (114, 145,
246, 302, 353). This PLC is
prominent in the pyramidal cells of the hippocampus and, to a lesser
extent, in the granule cells of the dentate gyrus, the reticular,
mediodorsal, and anteromedial thalamic nuclei (307).
PLC-
1 mRNA levels are highest in the cerebellar Purkinje
and granule cells, frontal and pyriform cortex, hippocampus, and
dentate gyrus; hindbrain structures have relatively low levels of this isoform.
PLC-
1 exists as alternatively spliced variants
1a and
1b (14). The
1b-variant replaces 75 carboxy-terminal residues of
the original PLC-
1 cDNA with a unique 32-amino acid
sequence. Both are abundant in brain, although the
1a-variant more so. Neither variant is detected in
kidney or stomach. Interestingly, most cell lines fail to express any
detectable PLC-
1; exceptions include C6Bu-1, PC-12, and
NIH-3T3 cells. One of these, the rat C6Bu-1 glioma cell line, expresses
both spliced variants (15). Whether the variants are
coexpressed in specific neuronal tracts is not yet known.
PLC-
2, first isolated from an HL-60 cDNA library
(200, 272), is expressed at highest levels in
cells of hematopoeitic origin (211). This pattern of
expression is consistent with the part PLC-
2 plays in
leukocyte signaling and host defenses (see sect. IIIG).
PLC-
3 protein, originally isolated from rat brain, is
widely expressed, with the highest concentrations found in brain,
liver, and parotid gland (166). In brain, its mRNA is
discretely distributed, with the highest levels found in cerebellar
Purkinje and granule cells, and the pituitary gland (362).
PLC-
4 was first isolated from cerebellum
(244, 245) and retina (173,
210). Its mRNA is highly concentrated in cerebellar Purkinje and granule cells (308, 362), the
median geniculate body (308), whose axons terminate in the auditory
cortex, and the lateral geniculate nucleus, where most retinal axons
terminate in a visuotopic representation of each half of the visual
field. This pattern of expression may be highly relevant to the
phenotypes of PLC-
4 null animals (see sect.
IIIG).
Several alternatively spliced variants of PLC-
4 have
been identified. One PLC-
4 protein, designated the
"b" form, is missing the carboxy-terminal 162 amino acids
(190). The sequence is replaced by a unique 10-residue
peptide. This isoform, which is also found in brain, is recovered
exclusively in the cytoplasmic fraction, unlike the "a" variant.
Additional variants have been described in the retina
(101, 102). They are divided into two groups:
PLC-
4 class "I" and "II," each containing a and
b variants that lack the PH domain and first EF-hand motif. These
sequences are replaced by a unique amino-terminal region in class
II forms. The Ib and IIb variants have an additional 12-amino acid
insert within the sequence connecting the X and Y halves of the
/
-barrel, the result of alternative splicing. More recently,
another retinal variant has been described that contains 14 unique
amino-terminal residues (3); we term this form variant
III (see Table 1).
The Drosophilia PLC-
4 homolog, NorpA, was
originally described as an eye-specific gene product
(32), but hybridization with cRNA probes in Northern blots
showed that the gene encodes at least four transcripts ranging in size
from 5 to 7.5 kb (421). These transcripts are expressed in
adult body and early stages of development. The various size
transcripts of the NorpA gene are accounted for by
alternative splicing of two forms of exon 4 which encode slightly
different sequences between residues 130 and 155 at the
carboxy-terminal boundary of the PH domain motif (191). Termed subtypes 1 and 2, NorpA subtype 1 is eye
specific, whereas type 2 is diffusely present in brain and leg and is
at high levels in thorax and abdomen. Subtype 1 is required for normal photoreceptor cell development, as well as the normal phototransduction process itself, whereas specific functions have yet to be assigned to
the second subtype.
Drosophila PLC-21 differs from NorpA (330) and
is expressed as two alternatively spliced variants. More recently,
-homologs have been identified in sponge (PLC-
S) and hydra
(PLC-
H1 and
H2) (198).
Although their domain organization seems identical to the mammalian and
Drosophila isoforms, there is currently no information on
their distribution or function.
B. Control by G protein-Coupled Receptors
PLC-
isoforms function as effector enzymes for receptors
belonging to the rhodopsin superfamily of transmembrane proteins that
contain seven transmembrane spanning (heptahelical) segments (169). They are activated by a wide range of
stimuli, from photons and tiny odorant molecules, to full-sized
proteins and require specific combinations of G
and G
subunits
to couple to their effectors. In the standard G protein model of PLC
activation, binding of agonist triggers receptor-catalyzed exchange
of GTP for bound GDP on the
-component of the heterotrimer. The
GTP-charged subunit then dissociates in the plane of the membrane,
and either the
-subunit monomer, the 
-heterodimer, or both
bind to PLC-
, increasing its catalytic activity and thereby
amplifying the initial receptor stimulus (Fig.
2). Because the evidence supporting this model has been extensively reviewed before, we focus on some of the
unsettled issues and some of the newer developments that have led to a
more complete view of how these enzymes participate in signaling
events.
|
1. Activation by G
subunits
Before the identification of specific PLC isotypes or the G
proteins involved, both pertussis toxin (PTX)-sensitive and
-insensitive G proteins were implicated in phosphoinositide/calcium
signaling (36, 93). Differential sensitivity
to PTX suggested that G protein-activated forms of PLC might be
heterogeneous, which was substantiated by the identification of the
four different mammalian
-isotypes. Concurrent with their
identification, the Gq subfamily of PTX-insensitive
-subunits (
q,
11,
14,
15, and
16) were isolated and characterized.
q and
11 are
found in nearly all tissues (350), whereas
14,
15, and
16 are
generally restricted to cells of hematopoeitic origin
(390).
When reconstituted into artificial vesicles, these G
subunits
activate PLC-
isoforms but fail to stimulate PLC-
1 or
1 (91, 334); comparable
results are obtained in cotransfection experiments. Nonetheless, the
-isoforms are not uniformly responsive to these subunits, with
PLC-
2 being considerably less sensitive in vitro
(134, 166, 310,
343). Coexpression of PLC-
isotypes 1, 2, or 3 and
various
q subfamily members produces a similar pattern
(173, 199, 208,
395). Interestingly, a recently identified
4-variant, which is missing a portion of the
carboxy-terminal region (G box), is insensitive to
q
stimulation (190). Hence, PLC-
1,
-
3, -
tk, and most PLC-
4
variants seem to be controlled by
q-related proteins,
whereas the less sensitive
2-isoform is not.
Nonetheless, this simple state of affairs is unlikely to pertain when
these proteins are expressed in their natural setting, as discussed below.
Although the PLC-
isoforms are differentially sensitive to
G
q-related subunits, the subunits themselves are nearly
interchangeable in their activation of individual isoforms, whether
examined by reconstitution in artificial membranes (134,
208, 255, 394) or in
cotransfection experiments (173, 199,
208, 397). This promiscuity is also observed,
with few exceptions in the coupling of various receptor types to PLC
through
q-related subunits (399).
2. Activation by 
-subunits

-Heterodimers are now recognized as regulators of many
effectors, including selective potassium and calcium ion channels, several isotypes of adenylyl cyclase, and PLC (reviewed in Ref. 61).
The realization that these heterodimers activate PLC helps explain the
disruption of phosphoinositide/calcium signaling by PTX and integrates
toxin-sensitive G proteins of the Gi subfamily into the
overall scheme of PLC regulation.
When reconstituted into artificial and biological membranes, G
subunits strongly activate mammalian PLC-
2, -
3
(42, 43, 273), and
-
tk (37, 382).
PLC-
1 is weakly stimulated (42), whereas
PLC-
4 is completely insensitive (209). Both
3 and
tk can be stimulated by
q in the presence of saturating amounts of free G
,
suggesting the formation of a ternary complex (343, 382). Although the dual activation of these PLC isoforms
resembles the regulation of adenylyl cyclase, the effector types differ significantly since the G
-sensitive cyclase isoforms, AC II and
IV, require concurrent activation by both G
and GTP-charged
s (363).
Although PLC-
3 is strongly activated by G
in
vitro, this isoform is only weakly stimulated in coexpression
experiments in COS cells (170). The reason for this
discrepancy is unclear, but other work demonstrates that some receptors
are coupled through PTX-sensitive pathways to this isoform
(250-252). In smooth muscle, adenosine A1, M2
muscarinic, somatostatin, and µ-,
-, and k-opioid receptors
can couple through Gi and Go to PLC, although
the extent varies, with some receptors coupling through
G
q related proteins as well. These cells contain the
PLC-
isoforms 1, 2, and 3. Interestingly, the
Gi/Go-mediated component is blocked by
antibodies against PLC-
3, but not
2 or
1; the extent of inhibition correlates with the extent
of PTX sensitivity. Presumably, high concentrations of free

-subunits arise from the normally abundant Gi
heterotrimers, thereby stimulating PLC-
3. If true, then
PLC-
2, which is also present, should have been
stimulated, but was not. These results imply that PLC-
2
may be activated by G
q or its associated G
subunits, but not the G
arising from Gi/o. This seems
to contradict the trend determined in artificial and biological
reconstitution assays. How these differences arise, when the PLCs are
expressed in their natural settings, is unknown.
There are at least 16 distinct G
, 6 G
, and 12 G
subunits,
yielding more than 1,300 different heterotrimer combinations. Although
not all combinations are possible, the number is huge, giving support
to the notion of "right" 
-subunit combinations. The
possibility that specific 
-combinations are required to activate
particular PLC-
isoforms has been investigated. Of the limited
number of
- and
-subunits tested, however, most are completely
interchangeable when reconstituted with PLC in artificial vesicles
(35, 368) or coexpressed in living cells
(389). The few exceptions are G
5, which is
most effective in stimulating PLC-
2 when cotransfected
with the G
2 subunit (389), and retinal G
, which is less effective than other subunit combinations in stimulating PLC (368). The relevance of the
G
5 activity is questionable, since tissue expression of
this subunit and PLC-
2 do not coincide. Moreover, weak
stimulation by the retinal heterodimer appears to be caused by the
attachment of a farnesyl rather than a geranylgeranyl group to the
retinal
-subunit. Thus when normal patterns of expression and lipid
modification are considered, there is little evidence of effector
selectivity for 
-subunit combinations; rather, this specificity
appears at the level of receptor/G protein coupling. This raises the
question of specificity in the pathway from receptor to PLC, and the
origin of the 
-subunits. What are the actual concentrations of

-subunits liberated by activated receptors in living cells? Are
other factors necessary to enhance the potency of 
-subunits? Are
the right 
-subunit combinations really necessary? Are these
subunits sequestered with, or directed to, a particular isoform? These
questions remain unresolved.
3. Evidence of combinatorial specificity
Like the PLC-
isoforms, G protein-coupled receptors also
discriminate poorly among G
q subfamily members and
various 
-subunit combinations when reconstituted into artificial
membranes or overexpressed in cultured cells (399).
Nonetheless, evidence of combinatorial specificity in receptor/effector
coupling can be obtained in living cells expressing their normal
receptor/G protein complement. The evidence is based mainly on
antisense RNA experiments involving the suppression of individual G
subunits or their combinations. In the case of the M1
muscarinic receptor coupling to PLC, several preferred combinations
have been identified (G-
q or
11,
1 or
4, and
4)
(76). Comparable results have been obtained for other
signaling pathways. For example, efficient coupling of M4 muscarinic and somatostatin receptors to the inhibition of
voltage-gated calcium channels requires two completely different
heterotrimers,
o1
3
4 and
o2
1
3, respectively
(195).
What confers this remarkable specificity? Clearly, determinants
intrinsic to receptor and effector are insufficient, since these bestow
selectivity only among
-subunit classes. Determinants found in other
factors, such as the RGS proteins (see sect.
IIIB5), also fail most tests of specificity,
although combining different determinants (receptor/G
protein/RGS/effector) may help in the selection process. Alternatively,
a physical sorting mechanism may sequester specific G-subunit
combinations with their cognate receptors. The findings that receptors,
G
and 
subunits, PLC isoforms and their substrates, are
laterally organized lend support to this idea (see sect.
IIIE). Another plausible mechanism would involve
unique phosphorylation states of the receptor itself, modulating its
ability to couple through distinct heterotrimers and PLC-
isoforms.
Evidence for this mechanism was obtained in an examination of the
PKA-mediated phosphorylation of the
2-adrenergic receptor and its coupling to Gs and Gi
(reviewed in Ref. 218).
4. How do G protein subunits activate PLC?
Although regions of PLC-
that are important to their
interactions with G proteins have been identified (see sect.
II), the molecular basis of their activation is still
unknown. G
subunits do not serve as membrane tethering devices for
the
-isozymes (165, 309), nor do they
affect the penetration of the membrane by PLC (249), nor
do they increase its sensitivity to calcium (28,
30). The same appears to be true for G
subunits.
This would suggest that the mechanisms involve acceleration of some step in the catalytic cycle itself, yet attempts to measure the effects
of G-subunit activation on substrate or product affinities or the
catalytic rate constants have been inconclusive. Part of the problem
lies in deriving mechanistic inferences from the kinetics of
lipid-hydrolyzing enzymes. These catalysts operate at a
membrane/solution interface, forcing the investigator to deal with
artificial membrane binding and exchange steps that may be independent
of the affinities of the enzyme for substrate and product. Fortunately,
water-soluble glycero-1-phosphoinositol 4,5-bisphosphate and
similar compounds are now known to be hydrolyzed by PLC
(398). This should remove some of the complications,
permitting a mechanistic understanding of G protein stimulation.
5. GTPase activating proteins and phosphoinositide signaling
Members of the G
q subfamily have a slow intrinsic
GTPase activity in vitro (~0.8 min
1)
(103). This leisurely rate is increased dramatically by
the so-called regulators of G protein signaling (RGS) proteins
(reviewed in Ref. 24), and the PLC-
isoforms themselves. First
discovered in yeast (Sst2), at least 19 different RGS proteins have
been identified in mammals. Of those tested, RGS2 (137),
RGS3 (259), and RGS4 (133, 149)
interact most effectively with G
q and block activation
of PLC-
1. RGS4 activates
q GTPase
by ~25-fold, whereas G
-interacting protein (GAIP), which interacts
strongly with
i-related subunits, stimulates only about
2-fold at the equivalent RGS4 concentrations (133). In
artificial vesicles, RGS2, which is widely expressed (49),
is more selective than RGS4 for
q, and is 10-fold more
potent at inhibiting PLC-
1 activation in vitro (137). Like RGS4, RGS2 is also an effective GTPase
activating protein (GAP) for
i and suppresses
Gi-dependent signaling in living cells (157).
Thus RGS proteins that exclusively recognize the
q-class
have not emerged.
Nonetheless, some RGS proteins are able to suppress calcium signaling
in an agonist-selective manner, suggesting they do discriminate among
receptor/G
q complexes. In permeabilized pancreatic
acinar cells, RGS4 inhibits PLC activation and calcium release from
internal stores, but the amounts required to suppress carbachol,
bombesin, and CCK differ by more than 10-fold (400);
similar results are obtained in intact cells where RGS4 decreases the
frequency and amplitude of calcium oscillations and raises the
threshold for stimulation. While RGS1 and RGS16 are also potent
inhibitors of muscarinic receptor signaling, they weakly suppress
CCK-induced calcium release. In this case, the degree of
selectivity for muscarinic compared with CCK receptor is 100- to 1,000 fold. In contrast, RGS2 is equivalent in its suppression. This
differential sensitivity is not due to the expression of different
levels of receptor or Gq-related proteins.
In addition to RGS proteins,
q GTPase is stimulated by
PLC-
1, a property that extends to all PLC-
isoforms
(E. Ross, personal communication). This activity was first demonstrated
by reconstitution of M1 muscarinic receptors,
Gq, and PLC-
1 (26). In these
artificial membranes, muscarinic agonist stimulates exchange of GDP for
GTP on G
q , whereas PLC and agonist increase the
steady-state GTPase activity up to 20-fold. Thus the GAP activity
of PLC-
1 is balanced by receptor-catalyzed GDP/GTP
exchange activity, yielding a population of G
q-GTP that
increases the steady-state PLC activity by 90-fold (28) which explains the old observation that
hydrolysis-resistant analogs, but not GTP itself, supports
PLC/receptor coupling in biological membranes. Interestingly, G
subunits also play an important role, suppressing the GAP activities of
both PLC and RGS (54). This would further expand the
number of points where G
subunits could mediate or regulate
PLC/receptor coupling.
The remarkable activities of PLC and RGS proteins, and the opposing
exchange activity of the receptor, have important implications for the
kinetics of signal generation and termination, the stimulus threshold,
and the specificity of receptor/G protein coupling to PLC. Their GAP
activities suggest PLC and RGS proteins rapidly dissipate the
amplification cascade, and control the "noise" of agonist-independent activation, raising the threshold for
stimulus-response coupling. Beyond their capability as noise
suppressors, RGS proteins have been found to act on specific
receptor/
q complexes rather than the isolated
subunits, helping to account for the agonist-specific nature of the
PI calcium signal. Moreover, differential RGS expression could enforce
a tissue-specific response pattern to a given set of agonists.
Depending on the complement of receptors and RGS proteins, multiple
different receptors could generate temporally distinct calcium signals
in the same cell, while drawing on a common pool of G protein subunits
and effector enzymes. The spatial distribution and sensitivity of
elementary calcium release and refilling events could further magnify
these differences.
C. Serine/Threonine Phosphorylation
Phosphorylation of PLC-
by cAMP-dependent protein kinase
(PKA) and protein kinase C (PKC) link the
-isoforms to heterologous and homologous receptor pathways that modulate phosphoinositide/calcium signals. Reciprocal and synergistic links between cAMP and
InsP3/calcium paths have long been recognized. Most notable
are the findings that calcium stimulates some forms of adenylyl cyclase
(356), permitting a wide range of cellular responses to
agonists that engage these pathways.
The results of most studies show that increasing cAMP suppresses PLC
activation, although a few have reported potentiation (reviewed in Ref.
106). The ability of cAMP to inhibit PLC is thought to occur at the
level of PLC-
phosphorylation. For example, PKA directly
phosphorylates PLC-
2, in vitro and in cotransfection experiments, thereby inhibiting its activation by G
subunits (224). One of the putative phosphorylation sites, Ser-954,
is located in the carboxy-terminal P-box region. Similar results have been reported for PLC-
3 (1).
Interestingly, phosphorylation by PKA uncouples receptors that activate
PLC-
3 through Gi/o, while preserving the
activation by receptors that utilize Gq. The results are
entirely consistent with the independent and simultaneous activation of
this PLC isoform by G
and
q, previously observed in vitro (343). On the other hand, PKA-mediated
phosphorylation has been reported to partially block the activation of
PLC-
3 by G
q (416). Here the
PLC-
3 phosphorylation has been mapped to a single site,
Ser-1105, located in the G-box region. Mutation of this Ser to Ala
confers resistance to PKA inhibition. Whether this might be related to
activation by G
subunits derived from Gq, rather than
the G
q itself, is unclear.
Various heptahelical receptors linked to PLC can be downmodulated
by PKC through the generation of DAG and the rise in cytoplasmic calcium (106). This rapid desensitization may occur at the
levels of receptor, G protein, or effector, or some combination
thereof. Importantly, the PLC-
isoforms themselves are substrates
for PKC. Studies of PC12, C6Bu1, and NIH-3T3 cells, which contain PLC-
1, -
1, and -
1, show
that treatment with tetradecanoylphorbol 13-acetate (TPA) stimulates
phosphorylation of PLC-
1, but not the other subtypes
(312). Phosphorylation of PLC-
1 by PKC in vitro results in a stoichiometric incorporation of phosphate at Ser-887, but without any measurable effect on PLC activity. This result
was surprising since PKC-mediated desensitization seems to
correlate with the level of PLC-
phosphorylation. More recently, significant inhibition of the partially purified enzyme was reported, but G
q stimulation was unaffected (223).
Suppression of this calcium-stimulated G
q
independent activity requires an as yet unidentified cofactor.
Comparable results have been reported for PLC-
tk
reconstituted in erythrocyte membranes (104). Here the stimulation by P2Y agonist and guanosine
5'-O-(3-thiotriphosphate) (GTP
S) is partly
suppressed by phosphorylation. On artificial surfaces, however,
activation of the purified enzyme by G
q is hardly
affected and stimulation by G
subunits is completely insensitive.
These results would suggest that other factors, such as the receptor
and RGS proteins, are necessary to observe the PKC-mediated inhibition.
PLC-
2, which is closely related to
tk, is
not phosphorylated when coexpressed with PKC isoforms, which themselves
fail to inhibit PLC activation by G
(224). On the
other hand, PKC-dependent phosphorylation of PLC-
3
has been observed. Phosphorylation correlates with uncoupling from
platelet-activating factor receptors, which are linked to
Gq, but not formyl-Met-Leu-Phe (FMLP) receptors, which are
linked to Gi/o. These results suggest the same covalent modifications of PLC produce functionally distinct consequences that
depend on the particular receptor and its cognate G protein. This might
appear to contradict the generally held idea of heterologous desensitization, in which engagement of second messenger-activated protein kinases by one receptor should desensitize other receptors, assuming a common set of consensus phosphorylation sites. Yet heptahelical receptor desensitization is mediated by many different protein players and can occur at many steps on the path to PLC activation, some of which are receptor specific. Differential phosphorylation of the receptors or associated proteins (RGS proteins,
-arrestins), could affect how receptor-specific signaling
components are uncoupled from their effector, laterally segregated, and
internalized (218).
D. Polyphosphoinositide Synthesis: the Need to Resupply
During the sustained phases of receptor activation of PLC-
isoforms, the mass of InsP3 produced often exceeds the fall
in cellular PI(4,5)P2 levels by severalfold.
In some cases, levels of this lipid fail to decrease or even rise. The
entire agonist-sensi