The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers

subsequent binding of β-arrestin.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C
Generates Two Messengers
Cyclic AMP is not the only second messenger employed by 7TM receptors and the G proteins. We turn now to another
ubiquitous second-messenger cascade that is used by many hormones to evoke a variety of responses. The
phosphoinositide cascade, like the adenylate cyclase cascade, converts extracellular signals into intracellular ones. The
intracellular messengers formed by activation of this pathway arise from the cleavage phosphatidyl inositol 4,5bisphosphate (PIP2), a phospholipid present in cell membranes. The binding of a hormone such as vasopressin to a 7TM
receptor leads to the activation of the β isoform of phospholipase C. The G protein that activates phospholipase C is
α
called G q. The activated enzyme then hydrolyzes the phosphodiester bond linking the phosphorylated inositol unit to
α
the acylated glycerol moiety. The cleavage of PIP2 produces two messengers: inositol 1,4,5-trisphosphate, a soluble
molecule that can diffuse from the membrane, and diacylglycerol, which stays in the membrane.
Comparison of the amino acid sequences of different isoforms of phospholipase C as well as examination of the known
three-dimensional structures of phospholipase components reveal an intriguing modular structure (Figure 15.11).
This analysis reveals the basis for both phospholipase enzymatic activity and its regulation by signal-transduction
pathways. The catalytic core of these enzymes has an α β barrel structure similar to the catalytic core of triose phosphate
isomerase and other enzymes (Section 16.1.4). This domain is flanked by domains that interact with membrane
components. At the amino terminus is a pleckstrin homology (PH) domain. This ~120-residue domain binds a lipid head
group such as that of PIP2 (Figure 15.12). The PH domain is joined to the catalytic domain by a set of four EF-hand
domains. Although EF-hand domains often take part in calcium-binding (Section 15.3.2), the EF-hand domains of
phospholipase C lack many of the calcium-binding residues. On the carboxyl-terminal side of the catalytic domain is a
C2 domain (for protein kinase C domain 2). This ~130-residue domain is a member of the immunoglobulin domain
superfamily (Chapter 33) and plays a role in binding phospholipid headgroups. Such interactions, often but not always,
require the presence of bound calcium ions.
The binding of a G protein brings the enzyme into a catalytically active position. The β isoform of phospholipase C has
an additional domain at its carboxyl terminus a domain that interacts with the α subunit of Gq in its GTP form.
Because this G protein is linked to the membrane by its fatty acid anchor, this interaction helps pull the β isoform of
phospholipase to the membrane. This interaction acts in concert with the binding of the PH and C2 domains of
phospholipase C to membrane components to bring the active site in the catalytic core into a position against a
membrane surface that is favorable for cleavage of the phosphodiester bond of PIP2 (Figure 15.13). Some of these
interactions and the enzymatic reaction itself also depend on the presence of calcium ion. Phospholipase isoforms that
lack the carboxyl-terminal regulatory domain do not respond to these signal-transduction pathways. The two products of
the cleavage reaction, inositol 1,4,5-trisphosphate and diacylglycerol, each trigger additional steps in the signal-
transduction cascades.
15.2.1. Inositol 1,4,5-trisphosphate Opens Channels to Release Calcium Ions from
Intracellular Stores
What are the biochemical effects of the second messenger inositol 1,4,5-trisphosphate? These effects were delineated by
microinjecting IP3 molecules into cells or by allowing IP3 molecules to diffuse into cells whose plasma membranes had
been made permeable. Michael Berridge and coworkers found that IP causes the rapid release of Ca
3
intracellular stores
2+
from
the endoplasmic reticulum and, in smooth muscle cells, the sarcoplasmic reticulum. The elevated
2+
in the cytosol then triggers processes such as smooth muscle contraction, glycogen breakdown, and
level of Ca
vesicle release. In Xenopus oocytes, the injection of IP3 suffices to activate many of the early events of fertilization.
Inositol 1,4,5-trisphosphate is able to increase Ca2+ concentration by associating with a membrane protein called the IP
3
-gated channel or IP receptor. This receptor, which is composed of four large, identical subunits, forms an ion channel.
3
The ion-conducting channel itself is likely similar to the structurally characterized K+ channel (Section 13.5.6). At least
three molecules of IP3 must bind to sites on the cytosolic side of the membrane protein to open the channel and release
Ca2+. The highly cooperative opening of calcium channels by nanomolar concentrations of IP enables cells to detect
3
and amplify very small changes in the concentration of this messenger. The cooperativity of IP3 binding and channel
opening is another example of the role of allosteric interactions.
How is the IP3-initiated signal turned off? Inositol 1,4,5-trisphosphate is a short-lived messenger because it is rapidly
converted into derivatives that do not open the channel (Figure 15.14). Its lifetime in most cells is less than a few
seconds. Inositol 1,4,5-trisphosphate can be degraded to inositol by the sequential action of phosphatases or it can be
phosphorylated to inositol 1,3,4,5-tetrakisphosphate, which is then converted into inositol by an alternative route.
Lithium ion, widely used to treat bipolar affective disorder, may act by inhibiting the recycling of inositol 1,3,4trisphosphate.
15.2.2. Diacylglycerol Activates Protein Kinase C, Which Phosphorylates Many Target
Proteins
Diacylglycerol, the other molecule formed by the receptor-triggered hydrolysis of PIP2, also is a second messenger and
it, too, activates a wide array of targets. We will examine how diacylglycerol activates protein kinase C (PKC), a protein
kinase that phosphorylates serine and threonine residues in many target proteins. The amino acid sequences and the
results of three-dimensional structural studies of protein kinase C isozymes reveal an elegant modular architecture
(Figure 15.15). The α, β, and γ isozymes of protein kinase C have in common a structurally similar catalytic domain,
homologous to that in PKA, at the carboxyl terminus. Adjacent to the catalytic domain is a C2 domain that is related to
the C2 domain of phospholipase C and that interacts with membrane phospholipids. On the other side of the C2 domain
is a pair of C1 domains, each organized around two bound zinc ions. These two domains, particularly the second (C1B)
domain, bind diacylglycerol (Figure 15.16A). Finally, at the amino terminus is a sequence of the form -A-R-K-G-A-L-RQ-K-. This sequence is striking because the consensus sequence for PKC substrates is X-R-X-X-(S,T)-Hyd-R-X in
which Hyd refers to a large, hydrophobic residue. This sequence from PKC is referred to as a pseudosubstrate sequence
because it resembles the substrate sequence except that it has an alanine residue in place of the serine or threonine
residue; so it cannot be phosphorylated. Recall that an analogous pseudosubstrate sequence is present in the regulatory
chain of PKA and that it prevents activity by occluding the active site in the R2C2 tetramer of PKA (Section 10.4.2).
Similarly, the pseudosubstrate sequence of PKC binds to that enzyme's active site and prevents substrate binding.
With this structure in mind, we can now understand how PKC is activated on PIP2 hydrolysis (Figure 15.16B). Before
activation, PKC is free in solution. On PIP2 hydrolysis in the membrane by phospholipase C, the C1B domain of PKC
binds to diacylglycerol. This binding and the interaction of the C2 domain with membrane phospholipids anchors the
enzyme to the membrane. The interaction between the C2 domain and the phospholipids, especially phosphatidyl serine,
requires calcium ions. The binding of the C1A domain to diacylglycerol pulls the pseudosubstrate out of the active site;
the released pseudosubstrate, which is quite positively charged, probably interacts with the negatively charged
membrane surface as well. The result of the conformational transitions is the binding of PKC to diacylglycerol-rich
regions of the membrane in an active state, ready to phosphorylate serine and threonine residues in appropriate sequence
contexts. Note that diacylglycerol and IP3 work in tandem: IP3 increases the Ca2+ concentration, and Ca2+ facilitates
PKC activation as well as phospholipase C activity.
Diacylglycerol, like IP3, acts transiently because it is rapidly metabolized. It can be phosphorylated to phosphatidate
(Section 26.1) or it can be hydrolyzed to glycerol and its constituent fatty acids (Figure 15.17). Arachidonate, the C20
polyunsaturated fatty acid that usually occupies the position 2 on the glycerol moiety of PIP2, is the precursor of a series
of 20-carbon hormones, including the prostaglandins (Section 22.6.2). Thus, the phosphoinositide pathway gives rise to
many molecules that have signaling roles.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.11. Modular Structure of Phospholipase C. The domain structures of three isoforms of phospholipase C
reveal similarities and differences among the isoforms. Only the β isoform, with the G-protein-binding domain, can be
stimulated directly by G proteins. For phospholipase Cγ, the insertion of two SH2 (Src homology 2) domains and one
SH3 (Src homology 3) domain splits the catalytic domain and a PH domain into two parts.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.12. Pleckstrin Homology Domain. PH domains facilitate the binding of proteins to membrane lipids,
particularly PIP2. In regard to phospholipase C, the PH domains help to localize the enzyme near its substrate,
PIP2.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.13. Phospholipase C Acts at the Membrane Surface. The PH and C2 domains of phospholipase help to
position the enzyme's catalytic site for ready access to the phosphodiester bond of the membrane lipid substrate, PIP2.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.14. Metabolism of IP3. The IP3 signal is terminated by the metabolism of the compound into derivatives
lacking second-messenger capabilities.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.15. Modular Structure of Protein Kinase C. Seven isozymes of PKC can be divided into two classes on the
basis of their domain organization.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers
Figure 15.16. Protein Kinase C Activation. (A) The C1 domain of PKC, structurally organized around two bound zinc
ions, binds diacylglycerol. (B) When the C1 domains bind to diacylglycerol in the membrane, the pseudosubstrate
is pulled from the active site, permitting catalysis. Calcium-binding C2 domains help to localize PKC to the
membrane.
II. Transducing and Storing Energy
15. Signal-Transduction Pathways: An Introduction to Information Metabolism
15.2. The Hydrolysis of Phosphatidyl Inositol Bisphosphate by Phospholipase C Generates Two Messengers