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Biochem. J. (2003) 374, 281–296 (Printed in Great Britain)
REVIEW ARTICLE
Mechanisms of cross-talk between G-protein-coupled receptors resulting
in enhanced release of intracellular Ca2+
Tim D. WERRY*, Graeme F. WILKINSON† and Gary B. WILLARS*1
*Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, U.K., and †Molecular Pharmacology,
Enabling Science and Technology, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
Alteration in [Ca2+ ]i (the intracellular concentration of Ca2+ ) is a
key regulator of many cellular processes. To allow precise regulation of [Ca2+ ]i and a diversity of signalling by this ion, cells
possess many mechanisms by which they are able to control
[Ca2+ ]i both globally and at the subcellular level. Among these are
many members of the superfamily of GPCRs (G-protein-coupled
receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate
PLC (phospholipase C) enzymes cause release of Ca2+ from
intracellular stores and influence Ca2+ entry across the plasma
membrane. It has been well documented that Ca2+ signalling by
one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated
heterologous desensitization between two different PLC-coupled
GPCRs. This is not surprising, given our current understanding of
negative-feedback regulation and the likely shared components
OVERVIEW OF CROSS-TALK
As our understanding of cellular signalling pathways has advanced, it has become increasingly apparent that, far from regulating linear transduction pathways, receptor activation can result
in much more complex patterns of signalling within cells. Indeed,
it has been known for some time that different intracellular
signal transduction pathways can interact to provide an additional
layer of complexity to their regulation. Such complexity of signalling and signal regulation allows for interactions between
intracellular events thought previously to be independent. There
is considerable evidence, for example, that signalling via GPCRs
(G-protein-coupled receptors) that couple preferentially to one
signalling pathway can be regulated by inputs from GPCRs
coupling to other pathways. Such interactions may affect coupling
specificity and efficacy, thereby having important implications for
the pathophysiological consequences of receptor activation and
potentially providing novel targets for therapeutic intervention.
Heterologous interactions or ‘cross-talk’ between receptors
can result in loss of function (desensitization), and also gain or
enhancement of function. In the context of this review, we will
consider such cross-talk that results in an enhanced release of
Ca2+ from intracellular stores. A review considering the broader
aspects of cross-talk between GPCRs has been published recently
of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling
preferentially to different signalling pathways, which result in a
potentiation of Ca2+ signalling. Such interactions have important
implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently
no single mechanism that adequately accounts for all examples
of this type of cross-talk. Indeed, many studies either have not
addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible
mechanisms to convey their potential diversity and to provide a
basis for further experimental investigation.
Key words: cross-talk, G-protein-coupled receptor (GPCR),
intracellular Ca2+ release, Ins(1,4,5)P3 , potentiation, synergy.
[1]. As will become clear during the course of this review, although
there are many examples of cross-talk leading to enhanced Ca2+
mobilization, mechanistic information is often lacking. There
is not a single model that adequately describes all examples
of this type of cross-talk, and there are inherent difficulties
in testing hypotheses definitively. Furthermore, there has often
been a tendency to look no further than a limited range of proposed models even when alternatives could adequately, if not
more appropriately, explain the data. The aim of the present
review is to highlight currently favoured mechanisms. Just as
importantly, we will discuss other areas that are currently less well
explored, but that nevertheless provide valid models for cross-talk
between different GPCRs that results in the enhanced release of
intracellular Ca2+ . We will focus mainly (but not exclusively) on
Ins(1,4,5)P3 -sensitive stores.
Enhanced mobilization of intracellular Ca2+ resulting from
cross-talk between different GPCRs can be conveniently illustrated by our demonstration that the recombinant Gα i -coupled
chemokine receptor, CXCR2 (C-X-C chemokine receptor 2 or
IL-8RB), only elevates [Ca2+ ]i (the intracellular concentration
of Ca2+ ) in HEK-293 cells if endogenous P2Y2 nucleotide
receptors are also activated [2] (Figure 1). This Ca2+ signalling
is independent of extracellular Ca2+ , clearly showing that this
cross-talk results from enhanced release from an intracellular
Abbreviations used: [Ca2+ ]i , intracellular Ca2+ concentration; cADPR, cyclic ADP-ribose; CCR, C-C chemokine receptor; CICR, Ca2+ -induced Ca2+
release; CXCR, C-X-C chemokine receptor; ER, endoplasmic reticulum; GABA, γ-aminobutyric acid; GAP, GTPase-activating protein; GPCR, G-proteincoupled receptor; GRK, G-protein-coupled receptor kinase; 5-HT, 5-hydroxytryptamine (serotonin); mGluR, metabotropic glutamate receptor; NAADP,
nicotinic acid–adenine dinucleotide phosphate; NCS, neuronal Ca2+ sensor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PKG,
protein kinase G (G-kinase); PLC, phospholipase C; PTH, parathyroid hormone; PTX, pertussis toxin; RGS, regulator of G-protein signalling; SERCA,
sarco/endoplasmic reticular Ca2+ -ATPase.
1
To whom correspondence should be addressed (e-mail [email protected]).
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T. D. Werry, G. F. Wilkinson and G. B. Willars
letion of a shared intracellular Ca2+ store (or depletion of some
other shared component of the signalling pathway) is responsible
for such inhibitory interactions [10], it is unclear how potentiating
cross-talk is achieved. This review aims to draw together many
findings related to this area in order to formulate a number of
possible models, some established, others more speculative, in the
hope that this will assist in future investigation of the mechanisms
underlying the enhancement of intracellular Ca2+ release as a
consequence of cross-talk between GPCRs.
POTENTIAL MECHANISMS OF CROSS-TALK
Regulation of PLC (phospholipase C) activity
Figure 1 Stimulation by UTP reveals a Ca2+ response to interleukin-8 (IL-8)
in HEK-293 cells with stable expression of recombinant human CXCR2
Representative traces show fluo-3 fluorescence recorded (using Ca2+ imaging equipment) as
an index of [Ca2+ ]i . Challenge of cells with 100 µM UTP results in a rapid and robust elevation
of [Ca2+ ]i (black and grey traces). Following removal of UTP, the addition of 30 nM IL-8 does
not evoke an elevation of [Ca2+ ]i (grey trace). However, if the presence of UTP is maintained,
addition of 30 nM IL-8 causes a marked elevation of [Ca2+ ]i (black trace). Bars and labels
indicate the onset and duration of stimulation by 100 µM UTP (which elevates [Ca2+ ]i via a
P2Y2 receptor in these cells) and 30 nM IL-8.
store. Not all studies have addressed the source of Ca2+ for
potentiated signalling, but it is worthy of note that there are
cases in which extracellular Ca2+ is required [3–8]. For example,
the ability of Gα q/11 -coupled neurokinin 1 receptors to potentiate
Ca2+ signalling by Gα i -coupled CXCR1/2 in neutrophils [3] and
the ability of Gα s -coupled prostaglandin E2 receptors to potentiate
Ca2+ signalling by Gα q/11 -coupled bradykinin receptors in neonatal rat dorsal root ganglion neurons [5] are both dependent
upon extracellular Ca2+ . Such observations demonstrate clearly
that GPCR signalling is able to converge at the level of Ca2+ entry
in order to enhance agonist-mediated changes in [Ca2+ ]i , but the
mechanisms involved are beyond the scope of this review.
There are many examples of cross-talk between GPCRs that
results in enhanced release of intracellular Ca2+ , and Table 1
presents some of these; others are discussed throughout the text.
In addition to those studies showing enhanced Ca2+ release, others
have reported enhanced phosphoinositide responses from which
it could be reasonably predicted that Ca2+ signalling would be
potentiated. All of the examples within Table 1 reflect alterations
in signalling as a consequence of either simultaneous activation of
the interacting GPCRs or acute (seconds to minutes) activation
of the ‘priming’ receptor prior to activation of the second receptor
type. There are, however, examples of such cross-talk that require
extended periods of exposure to the priming agonist. For instance,
interleukin-1β potentiates both phosphoinositide hydrolysis and
Ca2+ signalling by the bradykinin B2 receptor in canine tracheal
smooth muscle cells, but this requires many hours of exposure
to the interleukin [9]. This potentiation is dependent on protein
synthesis, and may be due to the up-regulation of bradykinin
receptor expression. Such examples clearly illustrate a mechanism
of cross-talk, but are not considered further in this review, in which
we discuss the acute regulation of Ca2+ signalling.
Interactions between GPCRs that result in enhanced mobilization of intracellular Ca2+ are in stark contrast to the interactions
that are often observed between different Gα q/11 -coupled GPCRs,
where ongoing activation of one often markedly inhibits Ca2+
release due to activation of the other. While it is likely that dep
c 2003 Biochemical Society
Regulation of the activity of PLC is perhaps one of the most
obvious and straightforward means of potentiating Ca2+ signalling. Hydrolysis of PtdIns(4,5)P2 by PLC results in the generation of Ins(1,4,5)P3 and the subsequent release of Ca2+ from
intracellular stores. Clearly, increased production of this second
messenger could enhance Ca2+ signalling. The idea that potentiation can be brought about by increased PLC activity is suggested
by observations that phosphoinositide hydrolysis is enhanced in
some examples of cross-talk (see Table 1).
Direct modulation of PLC activity
There is no evidence for Gα i binding to PLCβ, and it is well
established that Ca2+ signalling by many Gα i -coupled receptors
is via Gβγ -mediated activation of PLCβ [11–15]. The four
identified PLCβ isoforms can be stimulated by Gβγ subunits
[16,17], but there are marked differences in their sensitivities [16].
Thus the expression profile of PLCβ isoforms within cells
may dictate the ability of Gβγ to mediate phosphoinositide and
Ca2+ signalling. The more extensively studied PLCβ isoforms
(β1–β3) have distinct binding sites for Gα q and Gβγ [18,19],
and the effects of simultaneous occupation of these sites can be
synergistic [20], providing a potential mechanism for receptor
cross-talk at the level of PLC. For example, binding of Gα q
to a PLCβ isoform (possibly PLCβ3) may prime the enzyme to
subsequent activation by Gβγ subunits derived from activated
δ-opioid receptors in NG108-15 cells [21]. Exactly how Gα q/11
might sensitize PLCβ to Gβγ subunits is not clear, but one
possibility is through a conformational change in PLCβ that
relieves a steric hindrance to Gβγ binding (Figure 2, A → B).
Alternatively, binding of Gα q/11 and Gβγ to discrete sites [18,19]
may simply result in a conformation more favourable to enzyme
activity. Such an interaction could explain a number of examples
of cross-talk in which Ca2+ responses to Gα s - and Gα i -coupled
receptors are enhanced by prior or concomitant stimulation of a
co-expressed Gα q/11 -coupled GPCR (see above and Table 1) or
by the expression of constitutively active Gα q subunits [22].
Another mechanism for the modification of the sensitivity of
PLCβ to Gβγ subunits is the removal of its C-terminus (Figure 2,
A → C and B → C). The C-terminal tail of PLCβ is crucial to
the enzyme, co-ordinating diverse functions such as homodimerization, Gα q/11 binding and GAP (GTPase-activating protein) activity [23–25]. Removal of the C-terminus experimentally,
either through proteolytic cleavage by a Ca2+ -activated intracellular protease such as calpain [26,27] or by deletion mutation
[19,28] abolishes sensitivity to Gα q and can markedly increase
PLC activation by Gβγ [26]. Purification of truncated, Gβγ sensitive PLCβ3 from platelets [26] or bovine brain cytosol
[29] indicates that C-terminal modification may occur in vivo.
Furthermore, calpain can be activated and have functional effects
G-protein-coupled receptor cross-talk and Ca2+ signalling
Table 1
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Interactions between GPCRs leading to potentiation of Ca2+ and/or phosphoinositide signalling
The Table shows combinations of receptors that have been shown to interact to facilitate Ca2+ release or to potentiate an upstream event, e.g. inositol phosphate production by PLC. Each row details
a receptor combination and its signalling consequence. InsP x , total inositol phosphates.
Gα i/o -coupled receptor
Gα s -coupled receptor
Adenosine A1
Gα q/11 -coupled receptor
Enhanced signalling observed
Refs.
Bradykinin
Ins(1,4,5)P 3 production/Ca2+ mobilization
PKC activation
InsP x accumulation
InsP x accumulation
Ca2+ mobilization
Ca2+ mobilization/responses
Ins(1,4,5)P 3 production
Ca2+ mobilization/responses
Ca2+ responses
InsP x accumulation
Ins(1,4,5)P 3 production
Ca2+ mobilization
InsP x production
Ca2+ mobilization
Ca2+ responses
[197]
[198]
[199,200]
[201]
CCKA
Histamine H1
mGluR
Muscarinic
Vasopressin
P2Y2
Adenosine A2B
Opioid
Thyrotropin
P2Y
Bradykinin
Bombesin
Muscarinic M1
P2Y2
Bradykinin
P2Y
Muscarinic
[188,202]
[34]
[34,203]
[34]
[200,204]
[205]
[173]
[33]
InsP x accumulation
[22]
Ins(1,4,5)P 3 production/Ca2+ mobilization
[196,206,207]
2+
Ca
mobilization
2+
[61,149]
Somatostatin
Bradykinin
Muscarinic
Ca mobilization
Ca2+ mobilization/responses
[206]
[151,208]
Neuropeptide Y Y2
Muscarinic
Ca2+ mobilization
[151]
α 2 -Adrenoceptor
α 1 -Adrenoceptors
Bradykinin
Muscarinic
P2Y1/2
ATII receptor
Muscarinic M3
P2Y
P2Y1
InsP x accumulation
Ca2+ mobilization
Ca2+ mobilization
Ca2+ mobilization
Ca2+ mobilization
Ca2+ mobilization
[187]
[206]
[203,209]
[2,33]
[66]
[36]
Ca2+ mobilization
[192]
5-HT1B
P2Y
InsP x accumulation
[210]
Muscarinic
Bradykinin
Ca2+ mobilization
[206]
β 2 -Adrenoceptor
PTH
2+
CXCR2
P2Y2
Ca
CCR4
P2Y
Ca2+ responses
following stimulation of Gα q/11 -coupled GPCRs, including the
muscarinic M3 receptor [30]. However, the activation of calpain
and truncation of PLCβ have not yet been linked definitively.
Phosphorylation by PKA (cAMP-dependent protein kinase)
and/or PKC (protein kinase C) plays an important role in the
regulation of PLCβ isoforms [31], providing part of a wellrecognized negative-feedback loop. Phosphorylation and functional inhibition of PLCβ1 by PKCα is inhibited by Gβγ
subunits [32], providing another potential mechanism through
which activation of a Gα i - or Gα s -coupled receptor could enhance
phosphoinositide and Ca2+ signalling in the presence of Gα q/11 coupled receptor activation.
The mechanism by which Gβγ and Gα q/11 interact to enhance
the activity of PLCβ may dictate the experimental and physiological conditions under which potentiation of Ca2+ signalling
would be observed. For example, a change in the conformational
or phosphorylation state of PLCβ may be reversible and would
be appropriate for explaining examples of cross-talk that require
continued Gα q/11 activation [2,33,34], whereas an irreversible
cleavage would be appropriate for explaining examples of crosstalk that require priming but do not require ongoing Gα q/11 activ-
mobilization
[2]
[211]
ation [35,36]. As a general point, it is currently unclear whether
dependence on ongoing activation of Gα q/11 is common, as
many studies have used protocols that do not allow this to be
determined (e.g. simultaneous addition of agonists or the persistent presence of the priming agonist).
Cross-talk interactions involving Gα s -coupled GPCRs are
common (see Table 1), and this naturally implies a role for PKA.
In general, PKA actions at PLCβ are inhibitory [37–40], but
there are instances where the actions of PKA have been shown
to increase the activity of PLCβ [41,42]. Thus activation of
adenylate cyclase by Gα s -coupled receptors has the potential to
increase an ongoing Gα q/11 -mediated activation of PLC. There are
several other loci at which PKA could potentially act to enhance
intracellular Ca2+ signalling. These are discussed later in this
review.
Regulation of PtdIns(4,5)P 2 supply
In addition to enhancing activity by direct modification of PLC,
it is possible that the same effect could be achieved by increasing
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284
Figure 2
T. D. Werry, G. F. Wilkinson and G. B. Willars
Proposed interactions between PLCβ, calpain and the Gα q/11 and Gβγ subunits of GPCRs
In the unstimulated PLCβ enzyme (A), the Gβγ -subunit-binding region is inaccessible to its ‘ligand’. However, in (B), binding of Gα q/11 causes both PLCβ activation and alteration of protein
conformation, such that the Gβγ -binding site becomes accessible, allowing Gβγ also to stimulate PLCβ activity. It is not clear whether ‘unfolding’ is required for PLCβ activation by Gα q/11 . The
Gβγ -binding site can be revealed by C-terminal truncation of either the folded or the unfolded form (C), possibly by calpain activated downstream of a GPCR.
Figure 3
Possible mechanisms for regulating the synthesis of PtdIns(4,5)P 2
The availability of PtdIns(4,5)P 2 may act as a controlling feature of phosphoinositide signalling via PLC. There is evidence that the activities of key enzymes in the synthesis of PtdIns(4,5)P 2 ,
namely PtdIns 4-kinase and/or PtdIns4P 5-kinase, are regulated under agonist stimulation independently of changes in the levels of substrate and product. Thus enhanced supply of PtdIns(4,5)P 2
could potentiate phosphoinositide and Ca2+ signalling. The precise mechanisms that regulate the activity of PtdIns 4-kinase and/or PtdIns4P 5-kinase are unclear, but may be a consequence of
the activation of either Gα q/11 - or Gα i -coupled receptors. R1 and R2 represent Gα q/11 - and Gα i -coupled receptors respectively. Lines shown represent stimulatory pathways; broken lines indicate
indirect or unknown routes of regulation. Further details are given within the text.
the supply of its substrate, PtdIns(4,5)P2 . This phospholipid represents a minor fraction of the total cellular phospholipids, and
its resynthesis may be required for both acute and sustained
phosphoinositide signalling [43]. It is conceivable that, in some
circumstances, the rate of PtdIns(4,5)P2 supply may limit PLC
activity, thereby limiting both Ins(1,4,5)P3 generation and Ca2+
signalling. Hence an increase in the activity of PtdIns 4-kinase
and/or PtdIns4P 5-kinase (Figure 3) could enhance both phosphoinositide hydrolysis and Ca2+ signalling. There is evidence that
c 2003 Biochemical Society
these kinases are regulated by agonist stimulation independently
of altered substrate and product concentrations [44,45]. The
potential mechanisms for such activation are diverse [45], and
include dependence on PKC [46], GTP [45,47], phosphatidic acid
[48] and Rho proteins [49,50].
Activation of Gα q/11 -coupled M3 muscarinic receptors in HEK
cells has been reported to enhance PtdIns(4,5)P2 levels by approx.
50 %, and this may allow for a long-lasting (> 1 h) enhanced
PLC activity on subsequent stimulation of the same receptor
G-protein-coupled receptor cross-talk and Ca2+ signalling
or different receptors coupling to Gα q/11 [35]. The potentiation
of signalling and enhanced levels of PtdIns(4,5)P2 were shown
to be sensitive to PTX (pertussis toxin), implying that a Gα i
coupling of the muscarinic M3 receptor is responsible. Subsequent
studies demonstrated that Gi -derived βγ subunits, Ca2+ and PKC
are required for the potentiation of PLC-mediated signalling
between a variety of receptor combinations [51,52]. Coupled with
the observations that Rho GTPases activate PtdIns4P 5-kinase
[49] and enhance the generation of PtdIns(4,5)P2 [53,54], that
inhibition of Rho can attenuate PLC-mediated signalling [50],
and that Gα q can stimulate RhoA directly [55], these data suggest
a mechanism for cross-talk involving Rho and an increased supply
of PtdIns(4,5)P2 (see Figure 3).
EF-hand-containing Ca2+ -binding proteins such as the NCS
(neuronal Ca2+ sensor) family are also able to regulate levels of
PtdIns(4,5)P2 , through interaction with, and activation of, PtdIns
4-kinase [56]. Thus expression of recombinant NCS-1 in PC12
cells enhances cellular levels of both PtdIns4P and PtdIns(4,5)P2 .
Furthermore, this was associated with an increased generation
of Ins(1,4,5)P3 and enhanced Ca2+ mobilization in response to
activation of endogenous Gα q/11 -coupled P2Y2 nucleotide receptors. Activation of PtdIns 4-kinase by proteins such as NCS-1
following activation of Gα q/11 -coupled receptors could regulate
levels of PtdIns(4,5)P2 and enhance the generation of Ins(1,4,5)P3
to a subsequent activation of PLC either by a second Gα q/11 coupled GPCR or by Gβγ subunits.
The activation of tyrosine kinases by GPCRs has also been
implicated in the regulation of Ca2+ release. For instance, Ca2+
responses that are independent of extracellular Ca2+ are decreased
in some cells by tyrosine kinase inhibitors [57,58], as are inositol
phosphate responses [58]. The mechanisms through which
tyrosine kinase activity potentiates phosphoinositide and Ca2+
signalling are unclear but, interestingly, levels of PtdIns(4,5)P2
are reduced by inhibition of Src [54,59]. The involvement of
Src in Ca2+ signalling mediated by 5-HT (5-hydroxytryptamine;
serotonin) in airway smooth muscle is via the regulation of
PtdIns(4,5)P2 levels [59], and this suggests that Src may be
another mediator through which a GPCR could enhance substrate
supply, thereby promoting cross-talk. Src-family proteins can
be activated by Gβγ subunits [60], and as such may be considered
good candidates for potentiating the signalling of a co-stimulated,
Gα q/11 -mediated pathway.
Sensitization of Ins(1,4,5)P 3 receptors
If enhanced PLC activity underlies cross-talk, then inositol
phosphate generation should also be enhanced. Despite such
findings (see Table 1), this is certainly not a consistent feature
[36,61]. One explanation is that local and/or very transient
enhancement of Ins(1,4,5)P3 generation may be responsible for
cross-talk, and this may not be detectable by the methods used.
Alternatively, cross-talk could simply occur in the absence of
enhanced PLC activity. One such possibility would be an increase
in the sensitivity of the Ins(1,4,5)P3 receptor, allowing further
Ca2+ release without any additional increase in Ins(1,4,5)P3 levels.
PKA can sensitize the Ins(1,4,5)P3 receptor through phosphorylation [62–65], and this provides a mechanism through
which receptors that elevate cAMP levels could participate in
cross-talk [65,66]. The sensitization of Ins(1,4,5)P3 receptors by
cAMP/PKA to basal levels of Ins(1,4,5)P3 could also explain
some examples of Ca2+ responses seen following stimulation of
cAMP-coupled receptors. Potentiation of Gα q/11 -mediated Ca2+
responses through either concomitant stimulation of Gα s -coupled
GPCRs or the use of forskolin has been well documented in nu-
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merous cell types, including brown fat cells [67], articular
chondrocytes [68], hepatocytes [63,66], pancreatic β-cells [69]
and parotid cells [70,71]. PKA is either strongly implicated, or
has been shown to be involved directly, in all these examples of
potentiation, although the precise mechanism has not been determined. However, there is some opposition to the idea that the
Ins(1,4,5)P3 receptor is the target for PKA, since there are conflicting reports on the functional consequences of such phosphorylation [62,72]. One alternative is that a PKA-mediated
phosphorylation event could enhance the Ca2+ loading of intracellular stores. Indeed, increased Ca2+ loading reduces the EC50
for Ins(1,4,5)P3 -mediated Ca2+ release [73], and some studies
have demonstrated a PKA-induced increase in the Ins(1,4,5)P3 releasable Ca2+ pool [63,74]. In a further example of cross-talk
between Gα q/11 - and Gα s -coupled receptors, recombinant PTH
(parathyroid hormone) receptors potentiate the Ca2+ signalling by
endogenous muscarinic receptors in HEK-293 cells [36,75]. This
interaction appears to be through a PTH-mediated sensitization
of Ins(1,4,5)P3 receptors, but is independent of cAMP [75].
Interestingly, the potentiation of Ca2+ mobilization responses
to metabotropic P2Y nucleotide receptors by isoprenaline or
adenosine in rat cerebellar type I astrocytes was also independent
of the associated elevations of cAMP, but were dependent upon
activation of Gs proteins, leading the authors to suggest a possible
role for Gβγ subunits [33]. Indeed, other studies have argued
for a role of Gβγ subunits in the sensitization of Ins(1,4,5)P3
receptors [76], suggesting a mechanism through which Gα i/o - or
Gα s -coupled receptors could enhance Ca2+ release independently
of effects on levels of cAMP.
The ryanodine receptor can also be phosphorylated and activated by PKA [77] and by Ca2+ /calmodulin-dependent protein
kinase II [78]. Although this provides a mechanism to link
increases in cAMP and Ca2+ respectively to enhanced Ca2+
release, specifically in cardiac tissue expressing ryanodine receptor 2, a role in cross-talk between GPCRs has not been fully
explored.
In many cell types, the activation of PKG (protein kinase G;
G-kinase) by nitric oxide inhibits agonist-mediated Ca2+ release
from intracellular stores via reduced generation of Ins(1,4,5)P3
and/or phosphorylation of the Ins(1,4,5)P3 receptor [79]. This
provides a feedback loop in which the Ca2+ -mediated activation
of nitric oxide synthase inhibits further Ca2+ release. Although
this occurs in a wide variety of cell types, it is not the only effect
of nitric oxide on Ca2+ release. In hepatocytes, phosphorylation of
the Ins(1,4,5)P3 receptor by PKG increases its sensitivity to
Ins(1,4,5)P3 , resulting in the generation of oscillatory Ca2+ signals
or the potentiation of agonist-mediated Ca2+ responses [80,81].
The activation of GPCRs can increase the generation of nitric
oxide [82,83]. Although it is well established that PLC-coupled
GPCRs increase generation through a Ca2+ -mediated activation
of nitric oxide synthase, a cAMP-dependent activation has also
been demonstrated [84]. Therefore nitric oxide, through PKG
and increased sensitivity of the Ins(1,4,5)P3 receptor, provides a
further mediator that, under appropriate circumstances, is able to
enhance Ca2+ mobilization. Indeed, in the intact liver, nitric oxide
generation in endothelial cells acts as a paracrine regulator of the
spatial and temporal aspects of agonist-mediated Ca2+ signalling
in hepatocytes [85]. Although this illustrates signalling between
different cell types, a similar mechanism could operate in a single
type of cell, providing a mechanism for cross-talk at the level of
Ca2+ mobilization.
The Ins(1,4,5)P3 receptor therefore provides a target for
mediating cross-talk that results in enhanced Ca2+ release but does
not require enhanced PLC activity. It should be noted, however,
that demonstration of enhanced Ins(1,4,5)P3 generation does not
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T. D. Werry, G. F. Wilkinson and G. B. Willars
rule out the Ins(1,4,5)P3 receptor (or several other sites) as the
primary focus for cross-talk, as enhanced [Ca2+ ]i could itself
provide positive feedback to increase the activation of PLC.
A role for Ins(1,3,4,5)P 4 ?
The metabolism of Ins(1,4,5)P3 by Ins(1,4,5)P3 3-kinase results
in the generation of Ins(1,3,4,5)P4 , which has a considerable
history as a potential intracellular second messenger. There are,
however, a number of mechanisms through which Ins(1,3,4,5)P4
could participate in cross-talk to enhance the mobilization of
Ca2+ . First, in particular circumstances, Ins(1,3,4,5)P4 enhances
the agonist-mediated mobilization of Ca2+ via the Ins(1,4,5)P3
receptor [86,87]. It has been suggested that the most likely
explanation is that Ins(1,3,4,5)P4 is able to enhance the size of the
accessible Ca2+ store (see also below) by regulating the location
and structure of the endoplasmic reticulum (ER), possibly via
GAP1IP4BP and GAP1m [87,88]. Hence, priming of the cell by
Ins(1,3,4,5)P4 could allow subsequent agonists access to Ca2+
stores that were previously hidden.
Ins(1,3,4,5)P4 has a 10-fold higher affinity for inositol
phosphate 5-phosphatase than does Ins(1,4,5)P3 , and this accounts
for the ability of Ins(1,3,4,5)P4 to protect Ins(1,4,5)P3 from
metabolism both in vitro [89] and in cells [90]. The inositol
phosphate 5-phosphatase also has a 100-fold lower V max for
Ins(1,3,4,5)P4 than for Ins(1,4,5)P3 [91], meaning that, following
the removal of an Ins(1,4,5)P3 -generating agonist, Ins(1,3,4,5)P4
will be removed with much slower kinetics than Ins(1,4,5)P3 .
The generation of Ins(1,3,4,5)P4 by a priming agonist could
therefore protect the Ins(1,4,5)P3 generated in response to a
second agonist from inactivation by the inositol phosphate 5phosphatase. It has been demonstrated that such protection is
responsible for the ability of a low concentration of carbachol
to activate the Ca2+ -release activated current on a second, but
not on a first, challenge in RBL-2H3 cells with stable expression
of the muscarinic M1 receptor [90]. Although it was argued that
the ‘protected’ Ins(1,4,5)P3 caused Ca2+ release from a store
specifically associated with the Ca2+ -release activated current and
which did not contribute to global Ca2+ signals, these data clearly
demonstrate the ability of Ins(1,3,4,5)P4 produced in response to
an initial challenge to reveal Ins(1,4,5)P3 -mediated Ca2+ release
to a subsequent challenge.
Interactions between second messengers accessing distinct
Ca2+ stores
There is a growing interest in the novel Ca2+ -releasing agent
NAADP (nicotinic acid–adenine dinucleotide phosphate). Initially studied in sea urchin eggs [92], this mediator accesses
thapsigargin-insensitive stores [93], setting it apart from most
other rapidly releasable Ca2+ stores. This store has recently
been identified as the reserve granule in sea urchin eggs,
which is functionally equivalent to the lysosome [94]. NAADP
synthesis appears to be potentiated by elevations in cAMP [95],
and may prime Ins(1,4,5)P3 -sensitive Ca2+ stores for release
by Ins(1,4,5)P3 [96], providing a theoretical mechanism for
cross-talk. In some cell types, there is considerable interplay
between Ins(1,4,5)P3 , NAADP and cADPR (cyclic ADP-ribose)
in regulating the strength of Ca2+ signalling. In sea urchin eggs, the
NAADP-stimulated Ca2+ spike initiates a series of CICR (Ca2+ induced Ca2+ release) oscillations that depend entirely upon Ca2+
release from Ins(1,4,5)P3 - and cADPR-sensitive Ca2+ stores [96].
This phenomenon is also observed in starfish eggs [97]. However,
the only demonstration in mammalian cells is in pancreatic acinar
c 2003 Biochemical Society
cells, where muscarinic receptor-stimulated local Ca2+ responses
(spikes) are converted into global responses (i.e. potentiated) by
the presence of NAADP and/or cADPR, but not Ins(1,4,5)P3
[98,99]. Furthermore, responses stimulated by cholecystokinin
are potentiated by Ins(1,4,5)P3 , but not by either NAADP or
cADPR [99]. The priming of CICR seen in sea urchin eggs appears
to be due to overloading of these Ins(1,4,5)P3 - and cADPRsensitive stores [96]. As discussed above, store overloading
decreases the EC50 for Ca2+ release [73], in this instance to a point
where basal levels of Ins(1,4,5)P3 are able to contribute crucially
to the perpetuation of NAADP-induced Ca2+ oscillations. It is
unclear how much effect NAADP-mediated priming would have
on receptor-induced Ca2+ elevations [particularly those induced
by Gα q/11 -stimulating agonists that will raise Ins(1,4,5)P3 levels
substantially above basal]. Whether interplay between these
messengers is a more widespread phenomenon than has currently
been established, or if it is a valid mechanism for GPCR crosstalk, remains to be seen, but these studies provide an elegant
demonstration of cross-talk at the level of second messengers.
Increased availability of intracellular Ca2+
The idea that a cell has a finite amount of Ca2+ in a single,
large reservoir has long been contested, and it is possible that
the ER may contain stores of Ca2+ that are functionally, if not
physically, discrete [100]. This concept has been supported by
work showing that, during the elevation of Ins(1,4,5)P3 , the
ER Ca2+ concentration can become heterogeneous, suggesting
that Ca2+ in discrete cisternae does not automatically equilibrate
across the entire store when Ca2+ is lost from adjacent regions
[101,102]. It has also been shown that even Ca2+ stores accessed
by the same mediator [i.e. Ins(1,4,5)P3 ] produced in response
to different agonists can, at least partially, be discrete from
one another [103]. Furthermore, the size of the Ins(1,4,5)P3 sensitive store may be subject to regulation by, for example,
Ins(1,3,4,5)P4 (see above). GTP also increases the size of
the store in hepatocytes in a manner that is dependent upon the
cytoskeleton [104]. These events may be directly linked, as GTP is
required for tubulin polymerization and also controls cytoskeletal
organization through Rho proteins. As has been noted [104], these
aspects can be influenced by agonists, which suggests that the
accessibility of Ca2+ stores may be regulated. Aligned to this,
it has been suggested that movement of Ca2+ between distinct
stores may be GPCR-driven, and that this may allow cross-talk
between receptors, resulting in enhanced Ca2+ mobilization [36].
Despite the lack of effect of cytoskeleton-disrupting agents on
cross-talk between Gα s -coupled PTH receptors and Gα q -coupled
muscarinic receptors [75], this may not be sufficient to dismiss
completely the hypothesis that Ca2+ transfer occurs between
discrete stores. The lack of involvement of the cytoskeleton may
simply indicate a different mechanism for mediating Ca2+ transfer,
or that the two stores (donor and acceptor) are in close proximity
and do not require cytoskeletal rearrangements to allow exchange
between discrete Ca2+ pools.
In isolated guinea pig hepatocytes, PKA is responsible for the
ability of angiotensin II to reveal Ca2+ mobilization to isoprenaline
[66]. Although part of this effect may be mediated through
the sensitization of Ins(1,4,5)P3 receptors (see above), PKA also
increases the size of the relevant Ca2+ pool, as demonstrated by
enhanced thapsigargin-mediated release. Whether this is mediated
through store-shifting or an alternative mechanism such as
enhanced activity of the sarco/endoplasmic reticular Ca2+ -ATPase
(SERCA) is unclear. There is a precedent for PKA-dependent
regulation of SERCA activity, as shown by its effects in the
G-protein-coupled receptor cross-talk and Ca2+ signalling
heart. Thus cardiac muscle expresses the regulatory protein
phospholamban, an endogenous inhibitor of SERCA1a and
SERCA2a, but not SERCA3 [105]. Phospholamban can be
phosphorylated by PKA, decreasing its ability to inhibit SERCA
activity. This may be the major mechanism through which βadrenoceptor agonists enhance Ca2+ removal and relaxation in
the heart.
Regulation of Ca2+ handling
Although mitochondria have relatively low-affinity Ca2+ -uptake
mechanisms, they are able to participate in Ca2+ signalling as a
consequence of large local increases in Ca2+ concentration that
may not be reflected by global measurements of [Ca2+ ]i . Mitochondria are able to remove and later release cytosolic Ca2+ ,
thereby influencing the magnitude and duration of Ca2+ signalling events [101,106–108]. Although there is some evidence that
mitochondria can participate in CICR following Ins(1,4,5)P3 mediated release of Ca2+ from the ER [109], there is no evidence that mitochondria release Ca2+ directly in response to
GPCR stimulation. Therefore GPCR-mediated Ca2+ release from
mitochondria is unlikely to account for cross-talk, but inhibition
of mitochondrial Ca2+ uptake could reduce the buffering action of
the mitochondria and effectively enhance [Ca2+ ]i . Interestingly,
compounds that uncouple mitochondria, for example FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone], acutely
increase the number of HeLa cells responding to Ca2+ -releasing
hormones and prolong the time to recovery of basal [Ca2+ ]i [110].
Furthermore, in brown adipocytes, stimulation of β-adrenoceptors
limits Ca2+ buffering, possibly through lowered mitochondrial
membrane potential and therefore decreased activity of the Ca2+
uniporter [111]. Reduced mitochondrial Ca2+ uptake may account
for comparatively uninhibited Ca2+ signalling [111,112], and
could underlie certain examples of cross-talk.
In addition to altered Ca2+ handling by mitochondria, it is also
possible that changes in either Ca2+ uptake within the cell or Ca2+
extrusion at the plasma membrane could result in enhanced
[Ca2+ ]i , but these aspects have not been investigated thoroughly
in relation to cross-talk between GPCRs.
GPCR oligomerization
There is accumulating evidence that GPCRs are components of
multi-protein signalling complexes and that these regulate the
localization and function of receptors. GPCRs form complexes
with a wide variety of signalling and regulatory proteins, and
the advantages of this co-localization are immediately apparent.
A number of different types of GPCRs also form homo- and/or
hetero-multimeric complexes (often referred to as dimers), and
the impact of this on receptor physiology and pharmacology has
attracted great interest (for review, see [113]). These complexes
exist constitutively [113], but, although far from clear, there
is some evidence to suggest that they may also be regulated
dynamically by agonist activation [114–119].
The potential importance of GPCR dimerization has been
emphasized by the observation that GABAB receptor 1 and
GABAB receptor 2 (where GABA is γ -aminobutyric acid) are
inactive as monomers, but form functional heterodimers [120–
122]. In addition, both homo- and hetero-dimerization (between
receptors of the same or different families) can alter the
functional properties of receptors within the complex [113–
115,117]. A variety of receptor combinations that form dimeric
assemblies have also been shown to enhance Ca2+ signalling (not
necessarily dependent on any existing Ca2+ signalling capability
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in the monomers). For example, heterodimers of angiotensin II
AT1 receptors with bradykinin B2 receptors enhance G-protein
coupling, inositol phosphate responses and Ca2+ signalling in
response to agonists of either receptor [123]. Although that
study demonstrated that heterodimerization can influence receptor
coupling, the heterodimerization was not agonist-dependent and
the potentiated signalling required only co-expression of the
receptors and not co-stimulation. However, it has been argued
strongly that heterodimers of mGluR1α (metabotropic glutamate
receptor 1α) and the adenosine A1 receptor provide the molecular
basis for the ability of agonists of either receptor to enhance the
Ca2+ signalling mediated by the other receptor type [124]. In
another example of dimer formation, heterodimerization between
the chemokine receptors CCR2 (C-C chemokine receptor 2) and
CCR5 was agonist-dependent, and co-stimulation also resulted in
a synergistic increase in [Ca2+ ]i [119]. Evidence that mixed complexes of receptors enhance coupling to Gα q/11 has also been
obtained in studies demonstrating that antagonism of either
bradykinin B1 or B2 receptors reciprocally inhibits agonistmediated inositol phosphate production in prostate cancer PC3
cells [125]. Despite convincing evidence of GPCR dimerization
and an association of this with altered receptor function, including
enhanced Ca2+ signalling, the precise molecular mechanisms are
unknown. In the case of enhanced Ca2+ signalling, it is indeed
unclear in some instances if this is mediated directly as a consequence of dimerization, or whether it arises simply from
the co-expression of the receptors. There are, however, several
consequences of dimerization that could result in enhanced
mobilization of intracellular Ca2+ , including: (i) altered ligandbinding properties; (ii) surrogate transduction/transactivation; and
(iii) changes in the specificity of G-protein coupling.
(i) Alteration of binding characteristics
There is accumulating evidence that oligomerization of GPCRs
can alter the selectivity and affinity of ligand binding. This has
been documented for opioid receptor heterodimers [126,127] and
for adenosine receptor heterodimers with P2Y1 or P2Y2 nucleotide
receptors [128]. An alteration in binding properties could sensitize
receptors to ligands, thereby allowing responses to lower agonist
concentrations. This has been demonstrated for heterodimers
between somatostatin sst5 and dopamine D1 receptors, in which
the affinity of each receptor was increased synergistically by the
presence of the other receptor, although ligand selectivity was
unaltered [129]. A similar effect occurs when 5-HT receptors
and cannabinoid CB1 receptors are co-expressed [130]. Receptor
dimerization can also result in the formation of novel binding
sites. For example, heterodimers of adenosine A1 receptors and
P2Y1 nucleotide receptors display a novel pocket that binds P2Y1
nucleotide receptor agonists, but signals in a manner typical of
adenosine A1 receptors (PTX-sensitive inhibition of adenylate
cyclase) and is blocked by adenosine A1 receptor antagonists
[128]. Such studies provide evidence that altered binding as
a consequence of dimerization (or an alternative mechanism)
can regulate receptor signalling, although it is currently unclear
whether there are examples that enhance Ca2+ mobilization.
(ii) Surrogate transduction
Surrogate transduction describes a model in which receptors
‘borrow’ the transduction machinery of a dimeric partner by, for
example, ‘domain swapping’ (Figure 4) [131]. This may result in a
response typical of one receptor partner, but occurring in response
to a ligand at the other. An elegant study using somatostatin
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288
Figure 4
T. D. Werry, G. F. Wilkinson and G. B. Willars
Models for surrogate transduction/domain swapping
(1) Monomeric signalling: receptor A and receptor B have distinct binding and signalling characteristics. (2) Surrogate transduction/domain swapping. (i) The two receptors bind in a dimeric
conformation. This complex binds receptor B-specific agonists, but transduces a receptor A-type signalling event. This model describes the temporary ‘hijack’ of transduction machinery by receptor
B from nearby receptor A. (ii) Dimerization allows receptor A to present its signalling machinery (e.g. Gα subunit) to receptor B, allowing receptor B to signal in a receptor A-type manner. Whether
these models (2i and 2ii) are experimentally or even conceptually separable remains to be determined.
receptors illustrates this principle. Thus the sst1 somatostatin receptor (which does not internalize) forms a dimer with a Cterminally truncated mutant of sst5 (318-sst5) that is unable
to couple to adenylate cyclase, but retains its agonist binding and
internalization characteristics [132]. This dimer internalizes in
response to an sst1 agonist (i.e. an sst5-like event). In addition,
the coupling of 318-sst5 to adenylate cyclase can be restored
in the dimer (i.e. an sst1-like signalling event in response to sst5
stimulation). Dopamine D1 receptors also rescue the coupling
of 318-sst5 to adenylate cyclase without controlling agonist
selectivity [129]. Domain sharing or swapping has also been
demonstrated in chimaeras of the α 2 -adrenoceptor and muscarinic
M3 receptor in which transmembrane domains VI and VII of one
receptor were fused to transmembrane domains I–V of the other
[133]. Phosphoinositide hydrolysis by the muscarinic M3 receptor
was seen only in cells expressing both constructs, indicating
that the functional coupling had been reconstituted. Domain
swapping may also be involved in functional interactions between
dopamine D2 and D3 receptors [134]. Hence a GPCR heterodimer
may ‘mix-and-match’ its binding and signalling components to
form a novel signalling profile, including positive alterations in
Ca2+ mobilization. A variant of this model may also account for
potentiation of Ca2+ signalling in cells co-expressing angiotensin
II AT1 and bradykinin B2 receptors, where the bradykinin B2
receptors are thought to ‘present’ G-proteins to the AT1 receptors
within a dimeric complex to facilitate Ca2+ signalling in response
to AT1 receptor agonists [123].
(iii) Alteration of G-protein specificity
Dimerization may also influence G-protein coupling. For example, heterodimerization of Gα i/o -coupled µ- and δ-opioid receptors results in a switching to Gα q -mediated responses [135].
In addition, CCR2 and CCR5 chemokine receptors, both of
which couple preferentially to Gα i/o , are able to stimulate Gα q/11
when heterodimerized [119]. Although CCR2 and CCR5 are
both able to elevate [Ca2+ ]i in their monomeric forms via a
PTX-sensitive Gβγ -mediated stimulation of PLCβ, dimerization
converts this Ca2+ signal into a PTX-resistant type. Since the
mechanism by which G-protein switching occurs is not yet fully
understood, the same effect could also theoretically be achieved
without dimerization in cells co-expressing interacting GPCRs.
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These examples represent true gain-of-function interactions, since
the interaction between the two receptors induces downstream
signalling that is not a property of either receptor alone. In
examples of cross-talk resulting in enhanced release of Ca2+
where activation of a ‘priming’ receptor is required, this would
imply that the event resulting in altered G-protein coupling (e.g.
dimerization or altered relationships within a dimer) would be
agonist-induced.
G-protein coupling infidelity
There is accumulating evidence that GPCRs are able to couple
to G-proteins from more than one family, independent of any
physical interaction between receptors (i.e. oligomerization; see
above), and that the nature of this coupling may be regulated by
factors downstream of other GPCRs. The coupling of a single
receptor type to more than one class of G-protein has been
demonstrated for a wide variety of receptors. Although such
‘promiscuity’ can clearly occur as a result of receptor overexpression [136–139], there is evidence to suggest that promiscuity is
a physiological reality, at least for some receptors [140–142].
One possible mechanism underlying such promiscuity is that
different states of the same receptor are able to couple to different
G-proteins. Indeed, the chemokine receptor, CXCR2, has highand low-affinity binding forms, and these conformations appear
to have distinct coupling specificities in response to the same
ligand [143]. The low-affinity form mediates elevation of [Ca2+ ]i ,
whereas the high-affinity form mediates neutrophil chemotaxis
[143], suggesting that stabilization of the low-affinity form could
result in potentiation of Ca2+ signalling.
Activation of PKA following stimulation of β 2 -adrenoceptors
results in phosphorylation of this receptor and its subsequent
switching from Gα s to Gα i [144,145]. A mechanism by which
Gα s - or Gα i/o -coupled receptors could be diverted towards Gα q/11
coupling is attractive as far as Ca2+ cross-talk is concerned, and
this has been demonstrated for the Gα s -coupled prostacyclin
receptor. Thus PKA-mediated phosphorylation at serine-357 of
the mouse prostacyclin receptor is responsible for switching from
Gα s to Gα i and Gα q [146]. Whether, for example, heterologous
receptor phosphorylation and subsequent G-protein switching
occurs to mediate cross-talk is unknown.
G-protein-coupled receptor cross-talk and Ca2+ signalling
Decrease in GPCR desensitization
Many GPCRs appear to desensitize, either fully or partially,
within seconds of agonist addition. The generally accepted
mechanism for this desensitization is receptor phosphorylation
following agonist activation. If these events are sufficiently rapid
to limit the initial response ([Ca2+ ]i elevation), then a reduction or
prevention of desensitization could potentiate signalling. Furthermore, reducing the desensitization of receptors coupled to Gα i/o
or Gα s could enhance Gβγ -mediated signalling to PLC or, alternatively, allow the receptor to couple more efficiently to Gα q/11 .
GRKs (G-protein-coupled receptor kinases) appear to be key in
the rapid desensitization of the majority of GPCRs, and it is
becoming apparent that the activities of these kinases are also
subject to acute regulation. For example, various GRKs are negatively regulated by Ca2+ sensor proteins or calmodulin [147],
providing a potential route for attenuating GPCR desensitization
during elevations of [Ca2+ ]i . Such a model has been partially explored using NCS-1 and the Gα i -coupled dopamine D2 receptor
[148]. Thus NCS-1 has been shown to interact with GRK2, in an
apparently Ca2+ -dependent manner, and this interaction is associated with reduced phosphorylation of the dopamine D2 receptor.
Additionally, receptor internalization is also decreased, while
the ability of receptor activation to inhibit forskolin-stimulated
cAMP accumulation is enhanced. It was argued that such an interaction serves to couple dopamine receptor activation with Ca2+
signalling, and this has clear implications for receptor cross-talk.
Acceleration of GPCR resensitization
An alternative to the inhibition of desensitization leading to crosstalk is to promote receptor resensitization. This could explain
cross-talk in which Ca2+ signalling by a Gα i/o - or Gα s -coupled
GPCR occurs only during the activation of a Gα q/11 -coupled receptor (Figure 1) [2,34,61,149–151]. Accordingly, activation of
the Gα i/o - or Gα s -coupled receptors could accelerate the rate
of resensitization of the Gα q/11 -coupled receptor and allow another
round of Ca2+ signalling. It has been shown for Gα q/11 -coupled
5-HT2A receptors expressed in HEK cells that inhibition of
internalization leads to increased resensitization of these receptors
[152]. However, it is not clear whether this effect could be
achieved by another GPCR. In terms of receptor cross-talk,
perhaps the closest example is the Ca2+ signalling that occurs
following the addition of an inhibitor of angiotensin-converting
enzyme in cells co-expressing this enzyme and the bradykinin
B2 receptor. This mechanism is proposed to be through the
dephosphorylation and resensitization of bradykinin B2 receptors
[153], although a recent study has argued against this [154]. To
date, the role of altered receptor phosphorylation status in crosstalk has not been fully defined, but it may well prove to be a fertile
area of investigation.
Enhanced receptor recycling or receptor recruitment
Initial studies with the β 2 -adrenoceptor demonstrated that, following agonist exposure and receptor desensitization, resensitization requires receptor internalization, followed by their
dephosphorylation and subsequent re-insertion into the plasma
membrane [155]. This has become the general paradigm for
resensitization of GPCRs. In terms of cross-talk resulting in
enhanced Ca2+ signalling, it is conceivable that activation of Gα i or Gα s -coupled receptors could enhance the recycling of Gα q/11 coupled receptors, thereby allowing for increased production of
Ins(1,4,5)P3 and further Ca2+ signalling. Alternatively or complementary to this, activation of one receptor type could recruit
289
to the plasma membrane an intracellular pool of a different receptor type and thereby enhance signalling. For example, in both a
renal epithelial cell line, LLCPK, and rat renal outer cortical cells,
activation of receptors for neuropeptide Y or atrial natriuretic peptide results in the rapid (<1 min) recruitment of α 1A -adrenoceptors
or dopamine D1 receptors respectively from the interior of the cell
to the plasma membrane [156]. Furthermore, the co-addition of an
α 1A -adrenoceptor agonist and neuropeptide Y blocks the usually
rapid internalization of the Gα q/11 -coupled α 1A -adrenoceptor.
Although not tested in that study, this phenomenon may be
expected to result in enhanced Ca2+ signalling downstream of the
Gα q/11 -coupled α 1A adrenoceptor. In another example of agonistmediated receptor insertion, activation of Gα q/11 -coupled P2Y1
nucleotide receptors in isolated murine dorsal root ganglion cells
recruits δ-opioid receptors to the plasma membrane in a Ca2+ dependent manner [157]. In these cells, the typically Gα i -coupled
δ-opioid receptor mediates the release of Ca2+ from Ins(1,4,5)P3 sensitive stores, and the insertion of more receptors is argued to
result in enhanced and sustained Ca2+ responses [157].
GAPs and signal termination
The intrinsic GTPase activity of Gα subunits acts as a timer to
limit the duration of G-protein activation [158,159]. Certain effector proteins are able to increase this intrinsic GTPase activity
(i.e. act as GAPs), a pertinent example being the activity of
PLCβ as a GAP for Gα q subunits [160–162]. In addition, a
family of proteins known as the RGS (regulators of G-protein
signalling) proteins have been identified as GAPs for Gα subunits
[158,159]. Such proteins are key regulators (generally inhibitory)
of signalling by GPCRs, and any reduction in their GAP activity
could account for enhanced signalling.
The ability of PLCβ to act as a GAP for Gα q [160–162] provides negative feedback, limiting PLC activity. This GAP activity
can be strongly inhibited by Gβγ subunits, as can the GAP
activity of certain RGS proteins [162]. Although signalling
properties have not been examined in detail, it could be that
decreased GAP activity enhances signalling by a Gα q/11 –PLCβ
complex and potentially, therefore, increases Ca2+ signalling. Our
understanding of RGS protein regulation is in its infancy, but there
is emerging evidence for dynamic regulation of their GAP activity.
For example, phosphorylation of RGS2 by PKC prevents GAP
activity towards Gα 11 and potentiates phosphoinositide signalling [163]. PtdIns(3,4,5)P3 also inhibits the activity of a number
of RGS proteins, and this can be relieved by Ca2+ /calmodulin
[164,165]. This interplay is thought to have major roles in the
characteristic ‘relaxation’ behaviour of G-protein-gated K+ channels in cardiac myocytes [166] and in determining the oscillatory
nature of Ca2+ signals in pancreatic acini [167]. Furthermore,
inhibition of RGS proteins results in the conversion of oscillatory
Ca2+ responses into more robust, sustained elevations in pancreatic acinar cells [167], demonstrating that RGS protein inhibition
can indeed enhance Ca2+ signalling. Phosphorylation of the
photoreceptor-specific RGS9-1 by PKA reduces its GAP activity
[168], suggesting that Gα s -coupled GPCRs could regulate signalling by inhibiting RGS proteins from the same family as RGS9-1
(e.g. RGS6/7/9/11). Therefore regulation of RGS proteins may
reduce their GAP activity and enhance PLC activation by either
Gα q/11 or Gβγ subunits.
Pooling of liberated Gβγ subunits
Gβγ subunits appear to have an important role in several examples
of cross-talk between Gα i/o - and Gα q/11 -coupled receptors, given
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290
T. D. Werry, G. F. Wilkinson and G. B. Willars
the sensitivity of these phenomena to Gβγ scavengers such
as the C-terminal tail of GRK2 or the transducin Gα subunit, Gα t
[22,169]. This role is also highlighted by the positive effects
of Gβγ overexpression [170]. It has been suggested that the
concentration of free Gβγ subunits may need to reach a relatively
high level before they are able to stimulate effectors, particularly
Gβγ -sensitive isoforms of PLCβ. Indeed, many receptor types exhibit PTX-sensitive (i.e. Gi/o -protein-mediated) PLCβ responses
that are dependent upon receptor number [171]. This suggests that
gain-of-function cross-talk between two receptors could arise
from the pooling of Gβγ subunits from two receptor populations.
However, this view should be set against the observations that
increasing receptor number does not automatically increase the
likelihood of a Gβγ -mediated Ca2+ response, since Gα s -coupled
vasopressin V2 receptors massively overexpressed in L cells do not
mimic the Ca2+ signalling capability of Gα s -coupled luteinizing
hormone receptors expressed at 8-fold lower levels in the same
cells [140]. Furthermore, it has been argued that there may be
effector selectivity for each isoform of Gβγ [172], and although
this is not a consistent finding [170], it suggests that different
Gβγ dimers may not be able to unite to a common cause.
Gβγ subunits may contribute to cross-talk by other means. For
example, in the presence of excess GDP-bound Gα q/11 originating
from activation of thyroid-stimulating hormone receptors, it has
been suggested that Gβγ subunits derived from Gα i -coupled
adenosine A1 receptors form a heterotrimeric complex with
Gα q/11 –GDP that may again be activated by thyroid-stimulating
hormone receptors and lead to potentiated PLC activity [173].
Adenosine A1 receptors and α 2c -adrenoceptors have also been
argued to provide Gβγ subunits directly to Gα q -coupled
bradykinin B2 or P2Y nucleotide receptors, resulting in enhanced
binding of GTP to Gα q and enhanced signalling [170]. Therefore
Gβγ subunits can bring about enhanced Ca2+ signalling by
accelerating G-protein re-association and facilitating the activation of the Gα q/11 signalling pathway.
PHYSIOLOGICAL AND EXPERIMENTAL SIGNIFICANCE OF
CROSS-TALK
Early work on the integration of Ca2+ and cAMP signals in
guinea pig liver cells [174], pancreatic β-cells (reviewed in [175])
and airway smooth muscle (reviewed in [176]) demonstrated the
complexity of intracellular Ca2+ signalling in a physiological
setting. The possibility cannot, however, be excluded that the
use of model cell and receptor systems results in some examples
of cross-talk that are of little physiological relevance. Indeed,
the possibility that the overexpression of receptors may alter
their function must be considered. However, evidence exists to
suggest that cross-talk that results in enhanced Ca2+ signalling is
not simply a consequence of the overexpression of recombinant
receptors and/or the use of cell lines that do not represent their
normal cellular environment.
One recent, very convincing example of endogenously expressed receptors interacting to enhance Ca2+ mobilization is
the enhancement of mGluR1-mediated inward currents and Ca2+
signals in isolated Purkinje cells by concomitant activation of
GABAB receptors with baclofen [177]. The precise mechanism
underlying this interaction is unclear, but may involve a GABAB mediated stimulation of PLC and Ca2+ mobilization through
activation of Gα i/o . The release of endogenous GABA by electrical
stimulation in the cerebellar cortex also mimics the effect of baclofen in enhancing the mGluR1-mediated slow excitatory postsynaptic current elicited by stimulation of parallel fibres. This
suggests that the interaction is extremely relevant in a physio
c 2003 Biochemical Society
logical context. It has been suggested that mobilization of Ca2+
by mGluR1 in Purkinje cells may act as a co-incidence detector
for parallel- and climbing-fibre inputs, resulting in long-term
depression at parallel-fibre–Purkinje-cell synapses [178]. This
may be key to motor co-ordination within the cerebellar system
[179], and the enhancement of mGluR1 function by GABAB
receptors may therefore also contribute to this [177]. Activation
of GABAB receptors also potentiates the accumulation of inositol
phosphates in response to activation of α 1 -adrenoceptors in slices
of rat cerebral cortex [180]. Such potentiation and the subsequent
enhanced release of Ca2+ from intracellular stores has been linked
to the ability of GABA to induce long-term potentiation in the rat
visual cortex [181]. This long-term potentiation is also dependent
upon activation of 5-HT2 receptors, suggesting that GABAB
receptors may similarly potentiate post-synaptic phosphoinositide
and Ca2+ signalling by 5-HT2 receptors in the cerebral cortex.
There is considerable evidence for cross-talk between group I
mGluRs (that stimulate phosphoinositide turnover) and
group II mGluRs (that inhibit adenylate cyclase and modulate a
variety of ion channels). For example, in both adult and neonatal
rat hippocampus and in neonatal rat cerebral cortex, activation
of group II mGluRs (which alone do not affect phosphoinositide
turnover) markedly potentiates the effects of group I mGluRs
[182–186]. Synergy has also been demonstrated between
mGluR1α and adenosine A1 receptors at the level of Ca2+
signalling in transiently transfected HEK-293 cells [124]. Coimmunoprecipitation shows a close and subtype-specific interaction between these receptors, while immunocytochemistry
demonstrates co-localization in primary cultures of rat cortical
neurons. These data suggest that a direct physical interaction
may underlie the functional interplay between these receptors.
Stimulation with both adenosine and quisqualic acid also provides
significantly more protection against NMDA (N-methyl-Daspartate)-induced excitotoxicity in cultured cortical neurons than
either agonist alone. Whether this is associated with enhanced
Ca2+ mobilization was not investigated. Furthermore, although it
was not clear whether the neuroprotective effects of adenosine
and quisqualic acid are mediated through identical mechanisms,
or whether co-stimulation results in truly synergistic effects on
neuroprotection, it was argued that these receptor interactions
may be important for modulating the role of mGluR1 α in neurodegeneration and neuroprotection.
As discussed above, activation of P2Y1 nucleotide receptors
in isolated mouse dorsal root ganglion cells results in the
Ca2+ -dependent insertion of δ-opioid receptors into the plasma
membrane [157]. This is mediated through the fusion of large
dense-core vesicles containing δ-opioid receptors with the plasma
membrane. Activation of δ-opioid receptors similarly results in
the insertion of new δ-opioid receptors by a mechanism dependent
upon Ca2+ release from Ins(1,4,5)P3 -sensitive stores. It was
suggested that the spatio-temporal pattern of Ca2+ signalling
by δ-opioid receptor activation [as a result of mobilization
from Ins(1,4,5)P3 -sensitive stores and capacitative Ca2+ entry] is
favourable for the fusion of these large dense-core vesicles. Thus
the insertion of δ-opioid receptors in response to activation of
either P2Y1 nucleotide receptors or δ-opioid receptors themselves
results in enhanced and sustained Ca2+ signalling by δ-opioid
receptors. The consequence of this is enhanced release of the
neuropeptides substance P and CGRP (calcitonin gene-related
peptide). As these neuropeptides are pro-nociceptive, δ-opioid receptor antagonists could prove to be a useful adjunct to µ-opioid
receptor agonists in the treatment of inflammatory pain [157].
Several studies have also shown cross-talk resulting in enhanced
phosphoinositide and/or Ca2+ signalling in glial cells. For example, α 2 -adrenoceptor activation synergizes with activation of
G-protein-coupled receptor cross-talk and Ca2+ signalling
α 1 -adrenoceptors in the generation of phosphoinositides in primary cultures of rat glial cells [187]. Others have shown that,
in cultured rat cortical astrocytes, activation of adenosine A1 receptors elevates [Ca2+ ]i through a Gα i/o -dependent process only
in the presence of group I mGluR activation [188].
Other examples of cross-talk have been reported between receptors expressed on cells derived from peripheral tissues. For
example, in ciliary body epithelial cells from the rabbit, both Ca2+
and phosphoinositide responses were potentiated synergistically
by simultaneous activation of muscarinic receptors and α 2 -adrenoceptors [189]. Similarly, the release of Ca2+ from Ins(1,4,5)P3 sensitive stores in response to oxytocin via PTX-insensitive
G-proteins is augmented by ATP acting through PTX-sensitive Gproteins in mouse mammary myoepithelial cells [190]. This has
led to the suggestion that the contraction of myoepithelial cells
in milk-ejection responses may be enhanced by such cross-talk
[190].
In platelets, challenge with 5-HT results in little or no
aggregation on its own, but markedly enhances that occurring
in response to adrenaline [191]. This effect is dependent upon
a number of signalling pathways, including that involving PLC
and Ca2+ , suggesting that this heterologous receptor interaction
is important in regulating aggregation responses. In another
example of cross-talk between endogenously expressed receptors,
activation of Gα s -coupled PTH receptors potentiates the Ca2+
release mediated by activation of Gα q -coupled P2Y1 nucleotide
receptors in the clonal rat osteoblast line UMR-106, whereas PTH
alone is unable to elevate [Ca2+ ]i [192]. The mechanism through
which PTH receptor activation potentiates P2Y1 nucleotide
receptor-mediated Ca2+ mobilization was not defined, but does
not involve cAMP. The potentiation effect is also observed in
the phosphorylation of the transcription factor CREB (cAMP
response element-binding protein) and induction of c-fos. Activation of this proto-oncogene has been strongly implicated in the
regulation of osteoblast functions, including proliferation and
differentiation [193,194]. The local release of nucleotides may
therefore act to sensitize osteoblasts to systemic factors such as
PTH and result in the localized remodelling of bone [192].
The above examples illustrate that cross-talk between GPCRs
that results in enhanced Ca2+ signalling is not restricted to recombinant, overexpressed receptors, but is a phenomenon that is
present in both cell lines and isolated cells between endogenously
expressed receptors. In reality, linking this cross-talk to the regulation of physiological functions remains to be shown definitively
in many cases. Since GPCRs are expressed so ubiquitously, and
because most cell types express multiple types of GPCRs that
couple to different G-proteins, there is the potential for this type
of cross-talk in nearly every cell type. As discussed above, such
interactions are likely to have physiological relevance. From
a general perspective, enhanced [Ca2+ ]i responses may simply
increase the functional response (e.g. stronger contraction, increased secretion, etc.). Alternatively, the requirement for simultaneous activation of two receptors allows coincidence detection,
and could also provide some protection against inappropriate
signalling. Such an ability to tailor a response would serve to
increase the flexibility of function in certain cells and, in doing
so, refine and widen the array of actions performed. Further, it
has been suggested that, in cells in which opposing actions are
mediated by Gα i - and Gα q/11 -coupled receptors, potentiation of
Gα q/11 -mediated effects may be part of a feedback loop, lowering
the threshold for antagonism of Gα i -mediated effects [170].
Interactions between different endogenously expressed receptors or between a recombinant receptor and a different endogenously expressed one may also influence the outcome and
interpretation of experimental studies. For example, despite indi-
291
vidually expressed µ- and δ-opioid receptors coupling to the
inhibition of adenylate cyclase, µ-opioid receptor agonists elevate
[Ca2+ ]i in cells co-expressing the two receptor types, possibly
through the formation of a heterodimer with distinct signalling
properties [135]. Similarly, the generation of extracellular nucleotides, in response to either agonist stimulation or mechanical
disruption (e.g. stirred suspensions), is likely to be significant
if nucleotide receptors are present and able to participate in crosstalk [195]. Failure to recognize the impact of this endogenous
nucleotide may lead to the misinterpretation of data. Such
potentiation of Ca2+ responses downstream of Gα i/o -coupled
receptors due to the release of ATP has already been demonstrated
[2,61,196].
From an experimental perspective, this cross-talk also has
the potential to be exploited in high-throughput screening in the
search for novel therapeutic compounds. With the availability of
equipment such as the fluorescence imaging plate reader, one
of the most widely used high-throughput assays for GPCRs is the
measurement of receptor-mediated changes in [Ca2+ ]i . Cross-talk
that results in enhanced Ca2+ mobilization provides a mechanism
through which the activity of Gα i/o - or Gα s -coupled GPCRs could
be easily screened using the fluorescence imaging plate reader,
thereby accelerating the search for novel ligands at such receptors.
SUMMARY
The aim of this review has been to present an overview of
both the phenomenon of GPCR cross-talk that results in enhanced
Ca2+ signalling and the range of mechanisms that may be involved.
It should be clear that there is considerable diversity among these
mechanisms and that it is difficult to ascertain with any certainty
which are the major causes or contributors to cross-talk. To date,
most studies on cross-talk have been largely descriptive, and it
has been difficult to define the precise mechanism(s). Indeed,
even where evidence has been presented to support one model, it
is not difficult to suggest alternatives that explain the data equally
well. Currently favoured mechanisms, at least as indicated by
the volume of publications, are those involving convergence at
PLCβ, Gβγ pooling and sensitization of the Ins(1,4,5)P3 receptor
by phosphorylation. Receptor dimerization and protein–protein
interactions are also becoming more widely accepted, but there
are a number of mechanisms suggested here that have been
less thoroughly explored. As our knowledge of signal regulation
has increased, the number of possible molecular mechanisms
underlying GPCR cross-talk has also expanded. Possibilities in
addition to those discussed here may well exist, and indeed it
is likely that new models will continue to emerge. However, we
hope that this review reflects much of the current thinking in this
area and focuses attention on the need to verify experimentally
the mechanisms and functional relevance of cross-talk between
GPCRs that results in enhanced mobilization of Ca2+ .
Financial support from the BBSRC (ref. S211) and AstraZeneca is gratefully acknowledged.
We express many thanks to Dr John Challiss and Dr Stephen Tovey for helpful discussion
and constructive comments during the preparation of this review. Finally, many apologies
to those who have contributed to this area but whose work we have not cited.
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