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Biochemical Society Transactions (2003) Volume 31, part 5
Bi-directional signalling from the InsP3 receptor:
regulation by calcium and accessory factors
H.L. Roderick1 and M.D. Bootman
The Babraham Institute, Babraham, Cambridge CB2 4AT, U.K.
Abstract
Calcium is a pleiotropic messenger controlling a diverse array of intracellular events from fertilization to cell
death. One of the main mechanisms by which intracellular calcium is elevated is through InsP3 [Ins(1,4,5)P3 ]induced mobilization of calcium from its receptor on the endoplasmic reticulum calcium store. The activity of
the InsP3 R (InsP3 receptor) is subject to regulation by many factors other than InsP3 , most notably calcium
itself, which regulates the channel in a bell-shaped dependent manner. InsP3 R sensitivity is also regulated
by post-translational modifications such as phosphorylation and by binding of accessory proteins. Taken
together it appears that the InsP3 R can be regarded as a cellular sensor for many signalling pathways,
qualitatively and quantitatively regulating intracellular calcium signals with consequences for downstream
cellular physiology.
Calcium is a central player in cell physiology, controlling
a diverse array of cellular processes including fertilization,
muscle contraction and development. Prolonged increases in
cytosolic calcium levels, however, may be toxic and result
in cell death. Thus cytosolic calcium levels are maintained
at a resting level of around 100 nM, from which they transiently increase following agonist stimulation into the
low micromolar range. The source of this calcium is
either from the ER (endoplasmic reticulum)/sarcoplasmic
reticulum or from the extracellular space. In non-excitable
tissues the classical mechanism of calcium release is
through agonist stimulation of Ins(1,4,5)P3 production by
PLC (phospholipase C) cleavage of PtdIns(4,5)P2 . InsP3
subsequently diffuses to its cognate receptor on the ER,
resulting in channel opening and calcium release [1]. In
addition to requiring InsP3 , InsP3 Rs (InsP3 receptors) are
regulated in a biphasic manner by calcium, i.e. calcium
is activatory at low concentrations (up to 0.3 µM) and
inhibitory at higher concentrations (0.5–1 µM) [2]. This strict
regulation of the receptor by calcium is mediated by both
a direct interaction of calcium with the receptor as well as
by calcium-regulated protein–protein interactions and posttranslational modification. In addition, InsP3 R activity is
regulated by calcium-independent accessory proteins, Mg2+ ,
redox potential and ATP [3]. These properties of the InsP3 R
place it at the centre of a bi-directional web of signalling
pathways (Figure 1). The characteristics, and hence the regulation of the InsP3 R by this web, however, may change according to InsP3 R isoform and splice variants thereof as well
Key words: bi-directional signalling, calcium, inositol 1,4,5-trisphosphate receptor [Ins(1,4,5)P3
receptor], macromolecular complex.
Abbreviations used: ER, endoplasmic reticulum; InsP3 R, InsP3 receptor; PLC, phospholipase C;
CaM, calmodulin; CaBP, calcium-binding protein; PKG, protein kinase G; TRP, transient receptor
potential; FKBP12, 12-kDa FK506-binding protein; PKA, cAMP-dependent protein kinase; RyR,
ryanodine receptor.
1
e-mail [email protected]
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as the cellular complement of signalling proteins. Thus a celltype-specific regulation of the InsP3 R is possible with specific
consequences downstream of the calcium signal. This mini
review will focus on the regulation of the InsP3 R by accessory
proteins, secondary modification and, furthermore, how
InsP3 Rs themselves may act as signal-transducing proteins.
One of the most studied interacting partners of the InsP3 R
is the tetra-EF-hand-containing protein CaM (calmodulin).
CaM has been shown to play a role both in the activation and
inhibition of the InsP3 R, although the activation of InsP3 R
by CaM remains contentious. CaM can bind to InsP3 Rs in
its calcium-bound (Ca2+ /CaM) or calcium-free form (apoCaM). Binding of apo-CaM to the InsP3 R inhibits InsP3
binding, and Ca2+ /CaM binding may facilitate the calciumdependent inactivation of the receptor. Due to sequence
differences of the three InsP3 R isoforms and splice variants,
the action of CaM may be cell- and tissue-specific (for reviews
see [4,5]; Figure 1). Recently, a member of the neuronal
calcium-sensor family of proteins (CaBP, or calcium-binding
protein) has also been shown to bind to N-terminal 600
amino acids of the InsP3 R, which is also where the calciumindependent CaM-binding site is located [6] (Figure 1).
However, unlike CaM, CaBP was shown to activate calcium
release, even in the absence of InsP3 . This function of CaBP
could provide a novel mechanism of InsP3 R activation, for
example following ionotropic stimulation or depolarization
of neurones. The putative activatory novel role for CaBP in
InsP3 R regulation, however, contrasts with our observation
that heterologous expression of these proteins on a null
background causes inhibition of InsP3 -mediated calcium
release [7].
In addition to their direct effect, Ca2+ /CaM and calcium
synergize with other signalling pathways, including phosphorylation by CaM kinase II, cGMP-dependent kinase
[cGMP kinase/PKG (protein kinase G)], protein kinase C
and dephosphorylation by calcineurin to modulate InsP3 R
Calcium Oscillations and the 5th UK Calcium Signalling Conference
Figure 1 Mechanisms of InsP3 R regulation
Some of the components of the InsP3 R signalling complex. Due to space constraints and a lack of information regarding
the location of all the protein-interacting regions and sites of secondary modification not all the described regulators of the
receptor are represented. The ER localization of the InsP3 R is shown. Domain boundaries between the N-terminal inhibitory
domain (1–224). InsP3 -binding domain (amino acids 225–576), regulatory domain (amino acids 576–2275), ER membranespanning domains (amino acids 2275–2590) and C-terminal domain (amino acids 2590–2749) of the type 1 mouse InsP3 R
are shown. The starting positions of the SI (amino acids 318–332), SII (1692–1732) and SIII (amino acids 901–910) splice
sites are indicated by dotted lines. PKA-phosphorylation sites, serines 1589 and 1755, and the PKG-phosphorylation site,
serine 1589, are indicated by a red P. Calcium-binding sites are indicated by red circles. Interacting proteins are also
indicated. The red dumbbell represents CaM or CaBP. Abbreviations: CN, calcineurin; CLNX, calnexin; CRT, calreticulin; mGluR,
metabotropic glutamate receptor; IP3 , InsP3 .
opening [8] (Figure 1). The recruitment of some of these
protein kinases/phosphatases to their site of action on the
InsP3 R is often dependent on scaffolding proteins which act
as bridges between both sets of proteins. The regulation of
muscle tone by the NO/cGMP/PKG has been shown to
involve a decrease in InsP3 -mediated calcium release. The
proposed mechanism of this decreased calcium release has
been determined to be in part through phosphorylation of
the receptor by PKG. In order for PKG to elicit its effect, the
presence of the recently cloned InsP3 R-associated cGMP
kinase substrate (IRAG) is also required. This protein acts
to tether PKG to its InsP3 R substrate [9].
B-cell activation through antigen receptor binding is associated with the activation of protein tyrosine kinases such
as Lyn and Syk, which phosphorylate many downstream
substrates. PLC-γ 2 is a major downstream effector of B-cell
receptor engagement and is responsible for the generation of
InsP3 and resultant calcium mobilization from internal stores.
In addition to activation of PLC-γ 2 and resultant InsP3 production, B-cell receptor engagement increases the sensitivity
of InsP3 Rs to activation by tyrosine phosphorylation. The
kinase responsible for this phosphorylation event is Lyn,
which is anchored to the InsP3 R and the B-cell receptor by
the multiple ankyrin-repeat-containing protein BANK [10].
Thus B-cell receptor engagement provides a dual mechanism
to maximize calcium release through the InsP3 R which may
be required for the proliferation of B-cells following antigen
binding. During T-cell activation, InsP3 Rs are also tyrosine
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Biochemical Society Transactions (2003) Volume 31, part 5
phosphorylated by the non-receptor protein tyrosine kinase
Fyn which was found to be physically associated by the
receptor. This results in increased InsP3 R activity at higher
calcium concentrations, suggesting a change in the inactivation characteristics of the receptor [11].
In neuronal cells, InsP3 Rs are tethered to group 1 metabotropic glutamate receptors by the Homer protein, thus
linking the source of InsP3 production with its site of
action [12] (Figure 1). These Homer proteins are highly
enriched at the postsynaptic density where they may play
a role in synaptic metabotropic receptor function. Several
members of the Homer family, however, possess an extended
C-terminus which contains a coiled-coil domain. These
coiled-coil-containing Homer proteins oligomerize, bringing
Homer-interacting proteins such InsP3 Rs and metabotropic
receptors into multi-protein complexes. These complexes can
be disrupted by Homer 1a which lacks the coiled-coil domain
of other Homer family members. Homer 1a was originally
described as an immediate early gene which is up-regulated
during synaptic activity. This activity-dependent expression
of Homer 1a would provide a mechanism to down-regulate
metabotropic-agonist-induced InsP3 R signalling. Homerinteracting motifs were also reported for the α-adrenergic
receptor, RyR (ryanodine receptor) calcium-release channels
and the TRP (transient receptor potential) calcium-influx
channels (Figure 1). Thus in neuronal tissue Homer proteins
may scaffold multiple proteins involved in calcium signalling
into large macromolecular complexes.
The Ca2+ /CaM-activated protein phosphatase calcineurin
(PP2b) has also been shown to interact with the InsP3 R via an
immunophillin, FKBP12 (12-kDa FK506-binding protein).
Dephosphorylation of the InsP3 R by calcineurin resulted
in decreased calcium flux [13]. More recently a complex
containing Bcl-2, InsP3 R and calcineurin has been observed
in neuronal tissue (Figure 1). In this complex, calcineurin was
found to bind both Bcl-2 and InsP3 Rs. A direct interaction
between Bcl-2 and InsP3 Rs was, however, not detected.
The presence of an interaction between calcineurin and its
two substrates was increased following a rise in intracellular
calcium, as occurs during periods of ischaemia [14]. A more
direct role for FKBP12 in InsP3 R regulation has also been
reported. Application of FKBP12 to InsP3 Rs reconstituted
into lipid bilayers resulted in an increase in the duration of
their open time as well as an increase in co-ordinated gating
between receptors [15]. However, a rigorous biochemical
investigation of FKBP12 interaction with the InsP3 R could
not identify any interaction between these two proteins,
although a positive interaction between FKBP12 and RyRs
was observed [16].
InsP3 R is also regulated by PKA (cAMP-dependent
protein kinase). Depending on the splice variant of the type 1
InsP3 R, phosphorylation by PKA has been shown to either
increase [17] or decrease [18] its activity. This is due to
differential phosphorylation of two PKA-sensitive serines in
the type 1 InsP3 R (Figure 1). The neuronal type 1 InsP3 Rs
are phosphorylated by PKA on both serine residues whereas
peripheral InsP3 Rs are phosphorylated on only one residue
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[19] (Figure 1). In pancreatic acinar cells, the fingerprint of
cytosolic calcium transients is agonist-specific. This may be
as a result of receptor coupling through different messenger
pathways [20]. Another explanation for this is that the level
of PKA phosphorylation of the InsP3 R is dependent on the
agonist used, which would therefore result in agonist-specific
regulation of receptor activity [21]. The protein phosphatases
1 and 2a (PP1 and PP2a) have both been found to co-purify
with PKA and the InsP3 R, reminiscent of their interaction with the RyR [22]. This protein complex of phosphatase, kinase and substrate permits the rapid regulation of
InsP3 R activity by phosphorylation/dephosphorylation.
The cellular distribution of InsP3 Rs may also be a
critical determinant of its activity and functional effects.
Neonatal cardiac myocytes from mice deficient in the cytoskeletal protein ankyrin do not possess the typical striated
distribution of InsP3 Rs and are not able to contract [23].
In support of these findings ankyrin binding to the InsP3 R
and the presence of ankyrin-binding motifs on the InsP3 R has
been reported [24] (Figure 1). However, the possible ER
luminal location of the ankyrin-binding region of the InsP3 R
may prevent its interaction with ankyrin. In Caenorhaditis
elegans InsP3 Rs were shown to interact with myosin II.
Disruption of the functional interaction between the two
proteins results in a loss of ability of the worm to up-regulate
pharyngeal pumping in response to food, which is a known
signalling role of InsP3 Rs [25]. Several studies have shown
that agonist stimulation may result in a redistribution of
InsP3 Rs within the cell [26,27]. In the study by Vermassen
et al. [27] a role for the cytoskeleton and protein kinase C
was shown in this process. Ubiquitination of the InsP3 R
occurs in many cell types following chronic agonist stimulation. The conjugation of the ubiquitin moiety to the InsP3 R
targets it for degradation by the proteosome with halfmaximal clearance of InsP3 R1 in SHY5Y cells being achieved
within 1 h. Up to 90% of cellular InsP3 Rs may be removed
by this process which will ultimately reduce the ability of the
cell to respond to agonist [28]. This agonist-mediated decrease
in receptor abundance may be a mechanism to protect the
cell from prolonged increases in intracellular calcium levels
[29].
The protein 4.1N has recently been shown to be responsible for the re-distribution of InsP3 Rs in Madin–Darby
canine kidney cells from the typical perinuclear region to the
plasma membrane following the formation of a confluent cell
monolayer [30] (Figure 1). The functional consequence of this
redistribution is not clear but it may serve to isolate calcium
signalling from the nucleus.
InsP3 Rs may also be regulated from the ER lumen
[31,32]. In addition to having a luminal calcium-binding site
[33], calcium regulated protein–protein interactions in the
ER lumen may regulate InsP3 R activity. Both calnexin and
calreticulin, ER luminal calcium-binding lectin chaperones,
have been shown to interact with InsP3 Rs. This interaction
appeared to be isoform-specific since calreticulin did not
interact with type 1 InsP3 Rs [34] (Figure 1). Both lectin
chaperones possess calcium-binding regions within their
Calcium Oscillations and the 5th UK Calcium Signalling Conference
sequence. However, only the low-affinity nature of the
calcium-binding sites present in calreticulin would enable
calcium to play a role in regulating its interaction with
InsP3 Rs in a manner similar to the regulation of RyRs
by calsequestrin [35]. Calnexin and calreticulin both bind
N-linked glycans which are present in the luminal domain
of InsP3 Rs. By virtue of this interaction calnexin and calreticulin may modulate InsP3 R function through structural
modification. An interaction between the luminal domain
of InsP3 Rs and the secretory vesicle calcium binding protein
chromogranin A has also been observed [36,37].
In addition to regulating calcium release from the ER,
InsP3 Rs may be involved in transducing the state of filling
of the ER to plasma membrane calcium-influx channels. A
‘conformational coupling’ mechanism has been proposed to
activate calcium influx following store depletion. In support
of this hypothesis, an interaction between InsP3 Rs and plasma
membrane calcium-influx channels of the TRP family has
been reported (Figure 1). Furthermore, depletion of ER
calcium has been shown to increase the interaction between
InsP3 Rs and TRP channels [38]. Since TRP channels contain a
Homer-binding motif, the interaction between TRP channels
and InsP3 Rs may be augmented in certain cell types by
Homer proteins. Homer protein oligomerization may thus
serve to form a bridge between TRP and InsP3 Rs.
In summary, functional InsP3 Rs not only consist of a
tetramer of InsP3 R subunits, but form a macromolecular
complex of many different proteins, which serve to tune
calcium-release activity according to the presence of inputs
from multiple signalling pathways. Furthermore, the fingerprint of the calcium signal may be due to cell-typespecific expression of various signalling proteins and InsP3 R
isoforms.
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