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281 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]). c 2003 Biochemical Society 282 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 1 283 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 c 2003 Biochemical Society 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- 285 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 c 2003 Biochemical Society 286 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 287 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 c 2003 Biochemical Society 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. c 2003 Biochemical Society 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 c 2003 Biochemical Society 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. 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