Signalling by PLC and Ca2+
Prof Colin W Taylor
Aims
To provide, via lectures and critical reading of the literature, an understanding of Ca 2+
signalling and phospholipase C
Learning outcomes
At the end of these lectures, you should:
i. Understand key features shared by all signalling pathways and the importance of
proteins in transmitting information within signalling pathways
ii. Be familiar with evidence demonstrating the role for PLC in Ca2+ signalling
iii. Understand the many roles of PI(4,5)P2 and how its concentration at the inner leaflet
of the plasma membrane is maintained and regulated
iv. Understand the regulatory mechanisms for PLCs from different families
v. Understand the processes responsible for Ca2+ transport across the plasma membrane,
and the ER and mitochondrial membranes
vi. Understand the mechanisms whereby receptors stimulate release of Ca 2+ from
intracellular stores and Ca2+ entry across the plasma membrane. You should be
familiar with IP3 and ryanodine receptors; with current models for store-operated Ca2+
entry; with the spatial organisation of Ca2+ signals; and with examples of decoding Ca2+
signals via conserved Ca2+-binding motifs.
vii. You should be familiar with techniques used to analyse Ca2+ signalling pathways
Lectures
Lecture 1 Key features of intracellular signalling pathways. The importance of interactions
between proteins. Historical development of the links between
phosphoinositides and Ca2+ signalling.
Lecture 2 Stimulation of PLC by receptors is causally linked to Ca 2+ signals. PI(4,5)P2 is
maintained by regulated enzymes and PI-transfer proteins. PLCs catalyze
hydrolysis of PIP2 to IP3 and DG. PLC families differ in how regulators cause the
inhibitory XY-linker to be displaced (‘interfacial activation’ and/or allosteric
regulation).
Lecture 3 Conserved Ca2+-binding motifs detect Ca2+ signals. Ca2+ transport across the
membranes of organelles and the plasma membrane allow cells to maintain a
low cytosolic [Ca2+]. Abundant buffers slow Ca2+ diffusion in cytosol.
Lecture 4 IP3 receptors and ryanodine receptors are large-conductance, weakly selective
cation channels adapted to allow rapid release of Ca2+ from intracellular stores.
Lecture 5 Ca2+-mediated interactions between IP3R allow elementary Ca2+ release events to
propagate. ‘Ca2+ synapses’ allow local decoding of elementary Ca2+ release
events.
Lecture 6 Cells can decode Ca2+ spikes. CaMKII remembers and is adapted to decode Ca2+
spikes. Store-operated Ca2+ entry links IP3-evoked emptying of stores to Ca2+
entry across the plasma membrane; STIM1 and Orai are the essential proteins.
Background Reading:
Cell Signalling Biology, M J Berridge, 2015, http://www.cellsignallingbiology.org
Cell Signalling, J T Hancock, 2017, Chapter 9: Intracellular Ca2+ ions – control of their
concentrations and roles in signalling
References (starred references are particularly important for understanding)
Intracellular signalling: background
Lim, W, Mayer, B & Pawson, T (2015) Cell Signalling. Principles and mechanisms. Garland
Science, New York, pp 400. A good textbook. Useful for essential background and/or
broader perspectives.
,Berridge, MJ (2015) Cell Signalling Biology. www.cellsignallingbiology.org. This has become a
monumental interactive source of information on all aspects of cell signalling. The figures
(some used in my lectures) are a particular strength. Again useful for breadth and
background. It is freely available.
Goodsell, DS (2009) The machinery of life. Ignore the text, just admire the watercolours of
cells and molecules and the impression they leave of biochemistry operating in very
crowded environments. He also has images on-line:
http://mgl.scripps.edu/people/goodsell/
Ellis, RJ (2001) Macromolecular crowding: obvious but underappreciated. Trends Biochem
Sci. 26, 597-604. Short discussion of how biochemistry in a crowded cell may be very
different from that in a test tube.
Bray, D (1995) Protein molecules as computational elements in living cells Nature 376, 307-
312. Beautifully written discussion of how proteins - through their abilities to bind several
different molecules - can assemble to form computational networks. Well worth reading,
as is his more recent book (I have a copy you can borrow): Bray D, (2009) Wetware: a
computer in every living cell, Yale University Press, New Haven, pp 267.
Abell, E et al. (2011) Parallel adaptive feedback enhances reliability of the Ca 2+ signalling
system. Proc. Natl. Acad. Sci. USA 108, 14485-14490. A very general problem, that we too
often ignore, is to understand how a cell configures its complex interacting signalling
machinery to allow reliable signalling. How does it get just the right number of proteins
into exactly the right place (or does it)? This paper at least frames the question.
Links between phosphoinositides and Ca2+ signalling: background and history
*
Rossi AM & Taylor CW (2019) IP3 receptors – lessons from analyses ex cellula. J. Cell. Sci. In
press. Recent review, with a focus on methods, that briefly summarises key steps on the
way to understanding how IP3 receptors work. I will leave copy of the manuscript on
Moodle.
Michell, RH (1975) Inositol phospholipids and cell surface receptor function. Biochim
Biophys Acta 415, 81-147. The seminal review that shaped work leading to the discovery
of IP3. A shorter perspective from the same author is more useful for setting the
historical context: Michell, RH (2009) First came the link between phosphoinositides and
Ca2+ signalling, and then a deluge of other phosphoinositide functions. Cell Calcium 45,
521-526.
Burgess, GM et al (1983) Calcium pools in saponin-permeabilized guinea pig hepatocytes. J.
Biol. Chem. 258, 15336-15345. Elegant and convincing demonstration that in
unstimulated cells, ER is the major intracellular Ca 2+ store. Well worth reading.
Berridge MJ & Fain, JN (1979) Inhibition of phosphatidylinositol synthesis and the
inactivation of calcium entry after prolonged exposure of the blowfly salivary gland to 5-
hydroxytrpytamine. Biochem. J. 176, 59-69. A classic showing that PLC lies upstream of
Ca2+ signals.
Berridge, MJ (1983) Rapid accumulation of inositol trisphosphate reveals that agonists
hydrolyse polyphosphoinosides instead of phosphatidylinositol. Biochem J. 212, 849-
858. More evidence from the blowfly: this time showing that IP 3 and DG are the first
products of receptor-activated PLC. Paved the way to testing whether IP 3 release Ca2+
from stores.
Streb, HP et al. (1983) Release of Ca2+ from a nonmitochondrial store of pancreatic acinar
cells by inositol-1,4,5-trisphosphate. Nature 306, 67-69. The first report of IP3-evoked
Ca2+ release. Very important at the time, but now of historical interest. A more useful
insight into the immediate aftermath of the discovery of IP3 is provided in the review:
Berridge, MJ & Irvine RF (1984) Inositol trisphosphate, a novel second messenger in
cellular signal transduction. Nature 312, 315-321.
Berridge, MJ (2005). Unlocking the secrets of cell signaling. Annu. Rev. Physiol. 67, 1-21. A
leisurely walk down memory lane for Mike Berridge.
Putney, JW (1986) A model for receptor-regulated calcium entry. Cell Calcium 7, 1-12 The
first clear exposition of store-operated Ca2+ entry as the link between receptor-
stimulated PI turnover and Ca2+ entry. Developed further in: Putney, JW (1986)
Capacitative calcium entry revisited. Cell Calcium 11, 611-624. Much more in the lecture
on SOCE.
,Moran, MM et al. (2011) Transient receptor potential channels as therapeutic targets.
Nature Rev. Drug. Discov. 10, 601-620. A flavour of mammalian trp channels. More in
lectures from others.
PI(4,5)P2: roles and metabolism
Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol.
Rev. 93, 1019-1137. Massive and authoritative review. Enormously useful as a source of
specific information, but perhaps a bit overwhelming to take in one-go. Particularly
useful: Table 2 (proteins regulated by PIP2), p1028 (concerns about inositol-depletion
hypothesis of Li and bipolar disorder), Figs 9 and 10 (PLC and PITP families).
*Chang, C-L & Liou, J (2016) Homeostatic regulation of the PI(4,5)P 2-Ca2+ signaling system at
ER-PM junctions. Biochem. Biophys. Acta. 1861, 862-873. Short, accessible review that
briefly covers the history and then store-operated Ca2+ entry and PI/PA transfer proteins.
Lemmon, MA (2008) Membrane recognition by phospholipid-binding domains. Nat. Rev.
Mol. Cell Biol. 9, 99-111. Nice review.
Mehta, ZB et al. (2014) The cellular and physiological functions of the Lowe syndrome
protein, OCRL1. Traffic 15, 471-487. For more information on Lowe's syndrome and the
less severe Dent's disease, which also results from loss-of-function mutations in the PIP 2
5-phosphatase, OCRL1.
Voronov, SV et al. (2008) Synaptojanin 1-linked phosphoinositide dyshomeostasis and
cognitive deficits in mouse models of Down's syndrome. Proc. Natl. Acad. Sci. USA 105,
9415-9420. Primary report linking trisomy of synaptojanin 1 (a PI(4,5)P 2 5-phosphatase)
to Down's syndrome. Meticulous.
Berridge, MJ Module 12 in www.cellsignallingbiology.org provides the best summary the
inositol-depletion hypothesis as a possible mechanism to explain treatment of bipolar
disorder with Li or valproate.
Simmen,T & Tagaya, M (2017) Organelle communication at membrane contact sites (MCS):
from curiosity to center stage in cell biology and biomedical research Adv. Exp. Med.
Biol. 997, 1-12. Good overview of MCS: the introductory chapter to a book on this
burgeoning topic.
Kim S et al. (2015) Phosphatidylinositol-phosphatidic acid exchange bv Nir2 at ER-PM
contact sites maintains phosphoinositide signaling competence. Dev. Cell. 33, 549-561.
Evidence that Nir2 (a PITP) sustain signalling from PIP2 by transferring PI/PA between
ER/PM membranes PA and transferring PI between membranes.
Chang, C-L et al. (2013) Feedback regulation of receptor-induced Ca2+ signaling mediated by
E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions. Cell Rep. 5, 813-
825. Primary report suggesting that Ca2+ binds to an extended synaptotagmin in the ER
(E-Syt11) causing ER-PM contact sites to narrow, allowing recruitment of the PI transfer
protein (Nir2), which they suggest then replenishes PI to sustain signalling. Attractive
model, but a role for the interactions seems to be evident only after heroic stimulation.
Worth reading (with a critical eye).
Giordano, F et al. (2013) PI(4,5)P2-dependent and Ca2+-regulated ER-PM interactions
mediated by extended synaptotagmins. Cell 153, 1494-1509. Published just before Chang
et al. (2013) and some common ground between them (Ca2+-recruitment of E-Syts
causing narrowing of ER-PM junctions), but they argue that the requirement for PIP 2 for
recruitment means that loss of PIP2 diminishes recruitment.
Fernandez-Busnadiego, R et al. (2015) Three-dimensional architecture of extended
synaptotagmin-mediated endoplasmic reticulum-plasma membrane contact sites. Proc.
Natl. Acad. Sci.USA E2004-2013. EM tomography of MCS formed by E-Syts. Worth looking
at the pictures.
Phospholipases C
*Kadamur, G & Ross, EM (2013) Mammalian phospholipase C. Annu. Rev. Physiol. 75, 127-
154. Clear well-written overview. The best current introduction to the topic. Useful
section in Balla (2013, see above) too.
Essen, L-O et al. (1997) Structural mapping of the catalytic mechanism for a mammalian
phosphoinositide-specific phospholipase C. Biochem. 36, 1704-1718. Heavy duty
structural approaches to the enzymatic mechanism - worth a look if you're chemistry-
oriented.
, *Hicks, SN et al. (2008) General and versatile autoinhibition of PLC isozymes. Mol. Cell 31,
383-394. Evidence that the X-Y linker is auto-inhibitory in all PLCs (PLCζ is the exception).
Shows also that rac1 activation of PLCβ2 is mediated by its ability to bring PLC to the PM
in the correct orientation (rather than by allosteric activation). Worth reading for the
development of ideas that bringing PLC to the PM may be sufficient for negatively
charged PM lipids to displace the acidic X-Y linker and relieve auto-inhibition (interfacial
activation).
Lyon, AM et al. (2013) Full-length Gαq-phospholipase C-β3 structure reveals interfaces of
the C-terminal coiled-coil domain. Nat. Struct. Mol. Biol. 20, 355-362. Must rank as the
dullest title, but evidence that αq-GTP directly displaces the X-Y-linker region from its
position occluding the active site. Review from the same group provides a broader view:
Lyon AM & Tesmer, JJG (2013) Structural insights into phospholipase C-β function. Mol.
Pharm. 84, 488-500. Read this ahead of the primary paper (more digestible).
Nomikos, M et al. (2011) Novel regulation of PLCζ activity via its X-Y linker. Biochem. J. 438,
427-432. Evidence that PLCζ is the odd one out among PLCs in having an X-Y linker that is
positively-charged and not autoinhibitory. More (and more refs) on PLCζ in lecture on
Ca2+ oscillations.
Smrcka, AV et al. (2012) Role of phospholipase Cε in physiological phosphoinositide
signaling networks. Cell. Signal. 24, 1333-1243. For information on PLCε.
*Gresset, A et al. (2010) Mechanism of phosphorylation-induced activation of
phospholipase C-γ enzymes. J. Biol. Chem. 285, 35836-35846. Evidence that within the
more complicated XY-linker of PLCγ (which includes 2 SH2 motifs), the cSH2 motif
contributes to auto-inhibition; it is then pulled away when nearby residues (eg Y 783) are
phosphorylated. Hence nSH2 recruits PLC to the receptor (via phosphotyrosine residues),
while intramolecular binding of cSH2 to phosphotyrosine relieves autoinhibition. Neat! A
review from these authors summarises much of their work: Gresset, A et al. (2012) The
phospholipase C isozymes and their regulation. Subcell. Biochem. 58, 61-94.
Ca2+-binding motifs
Gifford, JL et al. (2007) Structures and metal-ion-binding properties of the Ca 2+-binding helix-
loop-helix EF-hand motifs. Biochem. J. 405, 199-221. In depth review of EF-hands.
Dickson, VK et al. (2014) Structure and insights into the function of a Ca2+-activated Cl-
channel. Nature 516, 213-218. Fig. 3b for a beautiful pentagonal bipyramid Ca2+-binding
motif in an ion channel structure.
Gerke, V, Creutz, CE, Moss, SE (2005). Annexins: linking Ca 2+ signalling to membrane
dynamics. Nat Rev Mol Cell Biol 6, 449-461.
Nalefski, EA, Falke, JJ (1996). The C2 domain calcium-binding motif: structural and functional
diversity. Protein Sci 5, 2375-2390.
Celio, MR (1996) Guidebook to the calcium-binding proteins, OUP, pp238. A comprehensive
listing of Ca2+-binding proteins – useful only if you want to chase details of a specific
protein or motif.
Ca2+ extrusion and uptake mechanisms
*Gadsby, DC (2009) Ion channels versus ion pumps: the principal difference in principle.
Nat. Rev. Mol. Cell Biol. 10, 344-352. Beautifully written review that considers the
fundamental differences between pumps and ion channels. Clearly describes why
channels are much faster than pumps. Worth reading.
Lytton, J (2007). Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport.
Biochem J 406: 365-382. Comprehensive review. Predates recognition that an NCX
also mediates Na+/Ca2+ exchanger across the mitochondrial inner membrane.
Bano, D et al. (2005) Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitoxicity.
Cell 120, 275-285. Original report on the role of calpain-cleavage of NCX3 in
excitoxicity. But see: Gerencser, AA et al. (2009) Real-time visualization of cytoplasmic
calpain activation and calcium deregulation in acute glutamate toxicity. J. Neurochem.
110, 990-1004. This work challenges whether calpain activation, although it occurs,
contributes appreciably to dysregulation of neuronal Ca2+ homeostasis.
Liao, J et al. (2012) Structural insight into the ion-exchange mechanism of the
sodium/calcium exchanger. Science 335, 686-690. First insight into how the Na+/Ca2+
