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ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(9):779–788 CN 31-1300/Q

Mini Review

CC Chemokine Receptor-coupled Signalling Pathways

( Department of Biochemistry, the Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China )

Abstract The isolation and characterization of multiple CC chemokine receptors (CCRs) in a wide range of tissues and cells signifies the functional diversity of CC chemokines. The realization that multiple chemokines activate individual receptors and that some chemokines are functional at several different CCRs, indicates that interplay between a complex network of intracellular pathways is required for the full expression of the physiological function of each ligand. In different cellular environments, chemokines can regulate distinct second messengers or even positively or negatively regulate the same signal transduction pathway. The specific interactions between many signalling molecules have been discerned in an increasing number of cellular systems and this information is being used to explain the physiological actions of chemokines. This review will attempt to summarize recent research by many groups that has revealed numerous subtleties of the CC chemokine-coupled signalling pathways.

Key words CC chemokine receptor (CCR); chemokines; chemotaxis; G protein; G protein-coupled receptor (GPCR)

The chemokines are a family of proinflammatory cytokines that act through cell surface receptors to regulate numerous routine physiological and pathophysiological processes, including hematopoiesis, T-cell activation, angiogenesis, inflammatory diseases as well as HIV-1 infection[1,2]. These small peptides are typically composed of 70－130 amino acids and are characterized by the presence of two disulphide bonds formed between four conserved cysteine residues. Chemokines are classified into four subfamilies according to the pattern of conserved cysteines in their amino acid sequences. They include at least twenty-six CC chemokines, seventeen CXC chemokines, two C chemokines and one CX3C chemokine[3－5].

Chemokines exert their effects through at least nineteen G protein-coupled receptors (GPCRs). The nomenclature of the chemokine receptors follows the notation used for the chemokine subfamilies and they are termed CCR1－10 (CC chemokine receptor 1－10), CXCR1－6, XCR1 and CX3CR1[4,6]. A remarkable feature of the chemokine receptors is their relative lack of selectivity in ligand binding, with many chemokine receptors binding more than one chemokine with high affinity. For example, eleven chemokines are reported to bind to the CCR1 receptor, including MIP-1α ( macrophage inflammatory protein 1α), MIP-1β, MIP-1δ, RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemotactic peptide 1), MCP-2, MCP-3, MCP-4, Lkn-1 (leukotactin-1), MPIF-1 (myeloid progenitor inhibitory factor 1) and HCC-1 (hemofiltrate CC chemokine 1), with varying affinities and acting with different degrees of agonism. Similarly, individual chemokines act as ligands for different receptors. For example, MCP-3 acts as a ligand for CCR1, CCR2, CCR3 and CCR5[4,7]. This promiscuity and the apparent redundancy of signalling that may arise poses many questions as to the control of chemokine signalling in different tissues expressing different combinations of chemokines, receptors and effectors.

Despite the apparent complexities of the chemokine signalling systems, the importance of individual chemokine receptors is gradually emerging from detailed studies on knockout mice, targeted gene disruption and the application of specific chemokine antagonists[6]. As an example, CCR1 knockout mice have been reported to have disordered trafficking and proliferation of myeloid progenitor cells and to display impaired inflammatory responses to a variety of stimuli. Control of the CCR1 signalling system was demonstrated to have clinical significance as CCR1 knockout mice display significantly reduced rejection responses to cardiac allografts[8]. This suggests that a strategy of blocking CCR1 signalling pathways may be useful in preventing rejection of transplanted tissues.

CCR5 has generated widespread interest because of its role as a co-receptor for HIV[9]. The identification of a naturally occurring mutant of this receptor, CCR5Δ32, and observations that homo and heterozygotes for this mutant have increased resistance to HIV infection and the development of AIDS[10] has highlighted the potential benefits to human health that could accrue from controlling the ability of CCR5 to bind ligands.
The CC chemokine receptors (CCRs) are thought to predominantly signal via Gi/o-coupled heterotrimeric G proteins to inhibit the activity of adenylyl cyclase (AC) and to regulate Ca²⁺ flux, however there is increasing evidence that CCRs are able to couple to a wider spectrum of G proteins to potentially influence the activity of numerous intracellular signalling pathways and gene transcription events. If we wish to exert a degree of control over chemokine signalling it will be necessary to unravel the complexities of promiscuity and apparent redundancy in chemokine expression, receptor binding and signalling. In this review we will outline recent efforts to delineate the intracellular pathways that define the CCR system.
The reader is directed to Table 1 for an overview of the signalling events activated by the individual receptors. In addition, Fig.1 graphically integrates the information presented. Throughout the text we shall identify chemokine ligands by their common name but upon their first use we will also include the official nomenclature[4].

Table 1 CCR receptor activation of intracellular signalling pathways

Receptor	Tissue/ cell type	G protein	Activity	Reference
CCR1	Transfected HEK-293	Gi/o	↓AC	[13]
	Transfected COS-7	G14,βγ	↑IP	[38]
	HL-36		↑IP3/Ca²⁺	[40]
	HOS	Gi/o	↑PKC/NF-κB/↑ERK	[41,73]
	Monocytes	ND	↑AA/cPLA2	[40]
CCR2a	Transfected HEK-293	Gi/o	↓AC	[13]
	Transfected COS-7/HEK-293	βγ	↑IP3/Ca²⁺	[38,13]
	Monocytes	Gi/o	↑Ca²⁺ flux	[68]
	Monocytes	ND	↑cPLA2	[83]
CCR2b	Transfected HEK-293	Gi/o	↓AC	[14]
	Transfected COS-7	G14/16, βγ	↑IP3/Ca²⁺	[38]
	Monocytes/macrophages/Fibroblasts/ transfected COS-7	Gi/o, G14/16	↑ERK	[74－76]; unpublished data
CCR3	T lymphocytes	ND	↑PKA	[19]
	AML/Purkinje cells	Gi/o	↑Ca²⁺	[16,20]
	Transfected COS-7	G14/16	↑IP3	unpublished data
	Eosinophils/rat mast cell	ND	↑ERK/p38	[77,78]
CCR4	Hippocampal neurons/cerebellum	ND	↑CREB	[19,20]
	Platelets/glomerular podocytes/T lymphocytes/IANK	In part Gi/o	↑Ca²⁺	[18,44－46,20]
	Transfected G1A1/dorsal root ganglia/neurons	Gi/o	↓VDCC	[22]
	Hippocampal neurons	ND	↑ERK	[19]
CCR5	Transfected CHO/NG108	Gi/o	↓AC	[23,24]
	Transfected COS-7/CHO	Gi/o	↑IP3/Ca²⁺	[47,24]
	Transfected G1A1/dorsal root ganglia/neurons	Gi/o	↓VDCC	[22]
	Macrophages/T cells	ND	↑KCa/Kir	[70,71]
	T cells/glioma cells	ND	↑ERK/p38/↑JNK	[79]
CCR6	IANK	Gi/o/z/q	↑GTPγS	[25]
	Lymphocytes/dendrites/T cells	ND	↑Ca²⁺	[51－53]
	Purkinje cells	Gi/o	↑Ca²⁺	[20]
CCR7	IANK	Gi/q	↑GTPγS	[25]
	Lymphocytes/dendrites	ND	↑Ca²⁺	[54,55]
	Purkinje cells	In part Gi/o	↑Ca²⁺
CCR8	NK cells/purkinje cells/transfected HeLa cells	In part Gi/o	↑Ca²⁺/↑NF-κB	[20,46,57]
	Mouse thymic lymphoma cells	Gi/o	↑ERK	[80]
CCR9	Thymocytes	ND	↑Ca²⁺	[56]
CCR10	Podocytes	ND	↑Ca²⁺	[44]
US28	Transfected HEK-293	ND	↑CREB	[31]
	HCMV-infected fibroblasts/transfected HEK-293	Gi/16	↑Ca²⁺	[58]
UL33	Transfected HEK-293		↑CREB	[31]
UL12	Transfected erythroleukemia cells	ND	↑Ca²⁺	[60]
E1	Transfected HEK-293	ND	↑Ca²⁺	[61]

The activities induced or inhibited by individual CCRs are presented along with the G protein mediators and the relevant tissues or cell types. Activites identified with non-selective chemokines are not included. ND, no data available. KCa, Ca²⁺-dependent K⁺ channels; Kir, G protein-gated inwardly rectifying potassium channels.

Fig.1 Intracellular signalling pathways activated by CC chemokine receptors

The CC chemokine receptors are grouped together according to their G protein-coupling specificity. For descriptions of pathways modulated by individual CCRs, the reader should consult the text and Table 1. The diagram identifies the heterotrimeric G protein subunits that have been shown to couple with CCRs, as well as the signalling pathways that these interactions modulate. Dashed lines indicate that the role of intermediate proteins has not yet been elucidated. The experimental evidence identifying individual pathways and the interactions between their intermediates is described in the text.

1 Regulation of adenylyl cyclase

Adenylyl cyclase (AC) is an enzyme which catalyses the synthesis of cyclic adenosine monophosphate (cAMP) from ATP. All nine AC isozymes are activated by interaction with Gαs subunits. However, each type of AC isozyme exhibits different sensitivity to Gαi, Gαz and Gαo. Type 1－6 ACs can be inhibited by Gαi1－3 and Gαz. However, GαoA/B only inhibit type 1 AC but not types 2, 4－6 AC. In addition to Gα subunits, Gβγ subunits are also known to play important roles in the regulation of AC activity. Type 1 AC is inhibited by Gβγ. On the other hand, Gβγ stimulates types 2, 4 and 7 AC in the presence of activated Gαs subunits[11] or following phosphorylation by protein kinase C (PKC)[12]. Thus activation of Gi/o-family heterotrimeric proteins by CCRs may inhibit or activate AC depending on the tissue-specific pattern of expression of receptors, G proteins and AC isozymes, as well as the relative strength of the Gα and Gβγ-mediated signals.

In human embryonic kidney 293 cells (HEK-293) stably expressing CCR1, challenging cells with a variety of CCR1 ligands induces potent inhibition [EC50 values for MIP-1α (also known as CCL3) and RANTES (CCL5) approximately 0.1 nmol/L] of forskolin-stimulated cAMP accumulation[13]. CCR2a also inhibits AC activity with equal potencies when cells are challenged with MCP-1 (CCL2). Furthermore, the CCR2 mediated responses are sensitive to pretreatment of the cells with pertussis toxin (PTX), indicating that the CCR2 mediated inhibition of AC proceeds via a Gi/o-coupled pathway[13]. CCR2b similarly mediates inhibition of AC in response to MCP-1[14].

CCR3-mediated signalling events in induced eosinophil acute myelogenous leukaemia (AML) cell lines are known to be sensitive to PTX, implying that Gi/o-coupled pathways transduce CCR3 signals[15]. However, when the ability of eotaxin (CCL11), a selective CCR3 agonist, to inhibit forskolin-stimulate AC activity was tested, it was observed that CCR3 did not induce inhibition of AC in AML cells[16]. These cells do express CCR3 and respond to eotaxin in a number of physiological and biochemical assays[16], suggesting to the authors that CCR3 is coupled to a PTX-sensitive pathway that does not involve Gi proteins. However, considering the previously discussed sensitivity of AC isozymes to Gαi/o subunits, it is also possible that this cell type does not express AC isoforms that respond to Gαi and/or Gαo. There are currently no reports of the inhibition of AC by CCR3 in heterologous systems. Although, it has been observed that eotaxin promotes adhesion, aggregation and migration of interleukin-stimulated T lymphocytes, and these CCR3-mediated events were completely blocked by a specific protein kinase A (PKA) inhibitor, H89[17]. Even though cAMP levels were not directly measured in the study, this indicates that CCR3 may induce increases in cAMP levels in certain cell types and that the activity of the downstream pathways activated by cAMP is required for CCR3-mediated cellular events to occur. It is not known whether this occurs via activation of Types 2, 4 or 7 AC by Gβγ subunits released from Gi/o heterotrimeric complexes to potentiate signals from Gs-coupled receptors, or whether cAMP levels are increased by an as yet undocumented interaction of CCR3 and Gs.

A similar phenomenon may be operating in platelets, which express CCR4. In these cells, the CCR4 selective ligand macrophage-derived chemokine (MDC) (CCL22) induces Ca²⁺ mobilization and platelet aggregation but does not inhibit the prostaglandin-induced cAMP accumulation[18]. In hippocampal neurons, MDC promoted accumulation of the cAMP responsive element binding protein (CREB) to the nucleus[19], and in rat cerebellar slices MDC increased the cellular levels of phosphorylated CREB[20]. This suggests that activation of CCR4 is also able to promote increases in AC activity resulting in activation of downstream effectors. However, it has been reported that chemokine-induced phosphorylation of CREB is blocked by U0126, an inhibitor of the mitogen activated protein kinase (MAPK) pathway intermediate MAPK/ERK kinase (MEK)[21], so it is not at all clear whether CCR4 is able to modulate AC activity. It is known that CCR4 couples to Gi/o family proteins as the MDC-induced Ca²⁺ currents in G1A1 cells expressing CCR4 cells are PTX sensitive[22].

Expression of CCR5 in CHO or NG-108-15 cells results in cell sensitivity to the ligands RANTES or MIP-1β (CCL4) in a PTX-dependent manner[23,24]. This study also noted that overexpression of Gαi2 strongly potentiated the CCR5 mediated inhibition of AC activity, providing further evidence that subunits of the Gαi family are able to mediate CCR5 responses[23].

There are currently no reports demonstrating the coupling of CCR6-10 to changes in cellular cAMP levels either in endogenous or heterologous systems. However, GTPγS incorporation assays combined with antibody capture techniques demonstrated that liver and activation-regulated chemokine (LARC) (CCL20) activation of CCR6 promotes guanine nucleotide exchange on Gαi, Gαo and Gαz subunits and that Epstein-Barr virus-induced receptor ligand chemokine (ELC) (CCL19) activation of CCR7 promotes exchange on Gαi subunits in interleukin-2-activated natural killer (IANK) cells[25]. This coupling might be expected to allow CCR6 and CCR7 to regulate AC activity, although as was observed for CCR3 and CCR4 this may depend on the co-expression of these receptors in cells expressing appropriate AC isozymes.
In cells endogenously expressing CC chemokine receptors, there are reports that CC chemokines are able to both positively and negatively regulate the AC/cAMP/PKA/CREB pathway. Unfortunately, many of the chemokines are specific for several receptors and thus it is often not possible to be precise about which chemokine receptor has been activated. For example, MIP-1α increases cAMP levels in the human growth factor-dependent cell line M07e, whereas RANTES is without effect[26]. Addition of a membrane soluble form of cAMP to the cell growth medium was shown to mimic the growth suppressive effects of MIP-1α on M07e cells. MIP-1α is a potent agonist for CCR1 and CCR5 and RANTES is a ligand at CCRs 1, 3 and 5[4]. Coupled with a lack of information on the PTX responses of the cells, it is unclear which receptor is acting through which G proteins to modulate cAMP levels. Indeed, it is possible that the cAMP levels measured arise from competing responses elicited by different chemokine receptors. Supporting a role for CC chemokine-induced increases in cAMP is evidence that the cell polarization and adhesion effects on T lymphoblasts induced by RANTES and MIP-1α are mimicked by cAMP agonists and inhibited by PKA inhibitors[27]. The chemokine-induced effects are abolished by PTX pre-treatment of the cells, establishing that Gi/o-coupled proteins, probably Gβγ subunits, are able to promote cAMP formation, which results in physiological activation of the cells.

In contrast, MIP-1α and RANTES both inhibit forskolin-stimulated cAMP formation in human hepatoma and endothelial cells[28], and RANTES decreases cAMP levels and PKA activity in human astrocytes[29]. In astrocytes, RANTES is a powerful stimulator of the production of proinflammatory mediators and this property is mimicked by the PKA inhibitor H89[29]. In human monocytes, the chemoattractant properties of MIP-1α and RANTES are significantly inhibited by treatment with forskolin and IBMX, both of which raise intracellular cAMP levels[30].

There are also a number of virally-encoded proteins that have homology to chemokine receptors and bind CC chemokine ligands. US28 is a GPCR encoded by human cytomegalovirus (HCMV) with 30% amino acid identity to CCR1 and recognises several CCR1 ligands[4]. HCMV infection can cause vascular disease and this is dependent on the expression of US28, which promotes the activation of a number of signal transduction pathways, including increasing the transcriptional activity of CREB[31]. US28 and the related HCMV-encoded CCR homologue UL33 also demonstrate constitutive CREB activity in the absence of chemokines[32]. Expression and signalling by HCMV-encoded chemokine receptors leads to smooth muscle cell migration in response to CC chemokines[33]. Therefore, these receptors are potential therapeutic targets for the treatment of HCMV-accelerated vascular diseases.

2 Regulation of phospholipase C-coupled pathways

In mammalian cells, the enzyme phosphoinositide-specific phospholipase C (PLC) catalyses the hydrolysis of phosphatidylinositol to inositol-trisphosphates (IP3) and diacylglycerol (DAG). These second messengers trigger numerous downstream events, including the mobilization of Ca²⁺ from intracellular stores and the activation of PKC. Thus far, three subfamilies of PLC isozyme, designated as PLC-β, -δ, and -γ, have been identified. Of these, only PLC-β(1－4) isozymes are known to be activated by Gαq, Gα11, Gα14, Gα15, and Gα16 subunits. In addition PLC-β2 and PLC-β3 can be activated by Gβγ subunits[34]. This sensitivity to Gβγ subunits allows PLC-β to be stimulated by activation of Gi/o heterotrimeric complexes. In the complex signal transduction networks involving GPCRs there are numerous examples where Gi-linked receptors activate PLC via Gβγ subunits (for example, reference [35]). In addition, the augmentation of Gq/11-dependent signals by Gi/o-linked receptors has been described as a means of PLC-β activation. This requires preactivation of PLC-β by a Gq/11-coupled receptor along with Gβγ stimulation arising from Gi/o-coupled receptor activation[36].

The activity of chemokine receptors is most commonly assayed by measuring changes in intracellular Ca²⁺ levels following application of agonists. Mobilization of Ca²⁺ from intracellular stores indicates that PLC-β has been activated, although it does not define which G protein family has transduced the chemokine message from receptor to effector. However, increases in intracellular Ca²⁺ levels do not necessarily prove that the PLC/IP3 pathway has triggered Ca²⁺ release from intracellular stores. An alternative explanation would be the chemokine-mediated opening of channels in the outer cell membrane allowing Ca²⁺ flux into the cell. This possibility has been demonstrated experimentally and will be explored in this and subsequent sections. In addition, CC chemokines are able to promote Ca²⁺ fluxes via the cyclic adenosine diphosphate-ribose (cADPR)/ryanodine receptor pathway and the sphingosine kinase/sphingosine 1-phosphate cascade[37].

CCR1 receptors transiently expressed in COS-7 cells do not induce IP3 formation following challenge with MIP-1α, suggesting that CCR1 does not activate PLC-β pathways via endogenous Gq/11 family proteins, and that endogenous PLC-β1 is not activated by Gβγ subunit released from Gi/o proteins. Upon co-transfection with Gα14, but not Gα16, MIP-1α is able to induce IP3 formation. When COS-7 cells were co-transfected with CCR1 and PLC-β2, IP3 formation was induced by MIP-1α in a PTX sensitive manner[38], demonstrating that in the presence of appropriate Gα subunits and effector isozymes CCR1 can activate PLC-β via different pathways. In an HL-60 cell line endogenously expressing several CCRs, the application of different chemokines with overlapping but not identical selectivities identified a CCR1-mediated increase in intracellular Ca²⁺ levels[39]. The nature of the transducing G protein was not identified. Interestingly, it has been observed that the rise in intracellular Ca²⁺ levels following activation of CCR1 expressed in HEK-293 cells is completely inhibited by the PLC inhibitor U73122, indicating that the Ca²⁺ is released from intracellular pools. However, it was noticed that in the absence of extracellular Ca²⁺, the intracellular levels induced by CCR1-mediated events were considerably reduced[40]. This implies that CCR1 is able to promote the influx of Ca²⁺ into a cell by activating Ca²⁺ channels (see below) as well as by promoting the release of Ca²⁺ from IP3-sensitive intracellular pools. Other studies have confirmed the CCR1-mediated activation of a PLC/IP/DAG/PKC pathway in human osteogenic sarcoma (HOS) cells stably expressing CCR1. In this cell line, the chemokine Lkn-1 (CCL15) promotes IP3 formation and redistribution of PKCδ from the cytosol to the membrane. Furthermore, Lkn-1 increases the binding activity of the transcriptional regulator NF-κB, and this activation is dependent on Gi/o signalling, PLC and PKC activity[41]. In HOS cells, Lkn-1 is a powerful chemoattractant, whose activity is dependent on the synthesis of new proteins, as demonstrated by the inhibition of chemotaxis by cycloheximide and actinomycin D[41]. This indicates that the PLC/PKC pathway activated by Lkn-1 ultimately leads to gene transcription and translation, possibly by activation of NF-κB.
Similarly, in COS-7 cells CCR2a and CCR2b promote IP3 formation through Gβγ-subunit release from Gi/o proteins in the presence of PLC-β2[38]. In HEK-293 cells, PTX-sensitive Ca²⁺ mobilization by CCR2a activation does not require co-transfection of PLC-β2[13], presumably because Gβγ subunit sensitive PLC-β isozymes are endogenously expressed in this cell type. CCR2b, but not CCR2a, is also able to promote IP3 formation via G14 and G16 proteins[38].
Activation of CCR3 receptors by the specific ligand eotaxin induces calcium transients in AML cells and cultured Purkinje cells. The calcium release in both of these cell types is completely inhibited by pre-treatment of the cells with PTX[16,20]. This indicates that in these cell types CCR3 does not functionally couple to Gq/11-family proteins, however we have recently observed that in heterologous systems CCR3 can promote IP3 formation when co-expressed with either Gα14 or Gα16 (unpublished data). It is predicted that CCR3 and Gα14 and Gα16 will be co-expressed in lymphoid cell lines[4,42,43] but PTX-resistant activation of PLC-β-coupled pathways has not yet been demonstrated.

Activation of CCR4 leads to Ca²⁺ mobilization in many cell types, including platelets[18], glomerular podocytes[44], CD4+ T lymphocytes[45] and IANK cells[46]. The G protein involvement in these Ca²⁺ responses has not been investigated. In IANK cells the CCR4-mediated chemotaxis is PTX-sensitive, but a direct link between Ca²⁺ levels and cell motility was not demonstrated[46]. In cultured cerebellar Purkinje cells, MDC-induced Ca²⁺ levels are reduced by approximately 50% following PTX treatment[20]. Surprisingly, in the same study the response to thymus- and activation-related chemokine (TARC) (CCL17), another fairly selective CCR4 agonist, was completely insensitive to PTX treatment. This data suggests that CCR4 is linked to PLC-β activation by more than one pathway and that CCR4 ligands do not necessarily induce all pathways with equal potency and efficacy.

In COS-7 cells transiently expressing CCR5, a variety of known agonists promote IP3 accumulation[47]. In CHO-K1 cells stably expressing CCR5, MIP-1β stimulates intracellular Ca²⁺ release, as measured in an aequorin-based functional assay. PTX pretreatment of the cells completely abolished the response, indicating the involvement of Gi/o proteins in CCR5-mediated activation of PLC-β-coupled pathways[24]. Interestingly, several HIV strains are known to induce intracellular inositol phosphate formation in neurons[48]. While it is known that CCR5 activation of Gi/o-coupled pathways is not require for HIV infection[49], these authors hypothesize that viral induction of chemokine signalling pathways may play a role in the development and progression of HIV-associated dementia.
CCR6 expressed in HEK-293 cells respond to their selective ligand LARC with mobilization of intracellular Ca²⁺[50]. LARC also induces Ca²⁺ mobilization in peripheral blood lymphocytes[51], dendritic cells[52], memory T cells[53]. In cultured cerebellar Purkinje cells, the LARC-induced signalling is abolished by PTX pretreatment of the cells[20]. The ability of CCR6 to couple to Gq/11 family subunits has not been thoroughly investigated although LARC is known to induce binding of GTPγS to Gαq in IANK cells[25]. In the same study, the selective CCR7 ligand ELC also activated nucleotide exchange on Gαq subunits, and in lymophocytes and dendritic cells, ELC induces Ca²⁺ mobilization[54,55]. In cultured cerebellar Purkinje cells, CCR7-induced Ca²⁺ fluxes are partially reduced by PTX pretreatment[20] and this is in agreement with the previously observed Gαq activation by CCR7. However, we have recently observed that CCR7 is unable to promote IP3 formation through activation of endogenous Gq/11 proteins in COS-7 cells and furthermore, is unable to functionally couple to G14 or G16 proteins (unpublished data).

CCRs8, 9 and 10 also promote increases in intracellular Ca²⁺ levels[44,46,56] in cells endogenously expressing these receptors. In cultured cerebellar Purkinje cells, CCR8-induced Ca²⁺ fluxes were not affected by PTX treatment of the cells indicating the lack of involvement of Gi/o-coupled pathways[20]. This data correlates well with results that demonstrate that CCR8 is able to functionally couple to Gα16 in transfected HeLa cells to enhance the activity of NF-κB, a transcriptional regulator[57].

The viral CC chemokine receptor homologue US28 activates Ca²⁺ flux when expressed in HEK-293 cells and challenged with the chemokines RANTES or MCP-3. It was determined that US28 is able to trigger Ca²⁺ flux via Gαi or Gα16 subunits[58]. Furthermore, human fibroblasts infected with HCMV respond to RANTES with increased Ca²⁺ flux, while HCMV with a disrupted US28 open reading frame does not induce the same response[59]. When the human herpesvirus 6 protein UL12, a CC chemokine GPCR homologue, is expressed in human cells it responds to a number of CC chemokines with increased Ca²⁺ flux[60], as does the E1 protein encoded by the equine herpesvirus 2[61].

3 Ion channel regulation

Two groups of Ca²⁺ channels, voltage-dependent Ca²⁺ channels (VDCCs) and second messenger-operated Ca²⁺ channels (SMOCs), are regulated by GPCR-coupled pathways. Five types of VDCCs have been cloned or identified, designated as L-, N-, P-, Q-, and R-types. L-, N- and P/Q-type VDCCs are inhibited by Gi/o family proteins while N- and P/Q-type VDCCs can be stimulated by Gs proteins[62] but Go appears to be more efficient than Gi[63]. Moreover, Gβγ subunits have been suggested to inhibit N-type channels by a PLC-β stimulated PKC pathway[64]. Thus, it might be expected that stimulation of CC chemokine receptors may lead to the inhibition of N-, L-, and P/Q-type VDCCs via Gαi/o- and Gβγ-mediated pathways. In addition, it has been shown that intracellular inositol phosphates can trigger the opening of SMOCs[65], so it is possible that CCRs induce the opening of SMOCs via a PLC-dependent pathway.

Indeed, CCR4 and CCR5 are able to mediate the PTX-sensitive inhibition of N-type VDCCs in transiently transfected cells as well as in subpopulations of neuronal cells[22]. The physiological relevance of this inhibition is not clear but may provide a link between chemokines released in the periphery and the central nervous system.

A far more common observation is the chemokine-induced activation of Ca²⁺ channels, whose activities have been shown in a number of studies to account for the majority of the intracellular Ca²⁺[40,66]. When the kinetics of the changes in Ca²⁺ levels are closely monitored there often appears to be a biphasic mobilization. In T cells, RANTES produces a biphasic mobilization of Ca²⁺ levels with the initial transient rise due to intracellular release followed by a sustained influx of Ca²⁺ from the extracellular environment[67]. In monocytes, MCP-1, a CCR2 agonist, induces increases in Ca²⁺ levels, which are dependent on extracellular Ca²⁺ levels and are partially inhibited by the Ca²⁺ channel blocker Ni²⁺. Thapsigargin, which empties intracellular Ca²⁺ stores, also partially blocks Ca²⁺ mobilization, but when combined with Ni²⁺ completely blocks the actions of MCP-1[68]. Furthermore, the MCP-1-induced Ca²⁺ levels are sensitive to PTX but not cholera toxin (CTX), suggesting that the effects were predominantly Gi/o-mediated. However, the Ca²⁺ level increases induced by MCP-2, a broad spectrum CC chemokine, were sensitive to CTX but not PTX, implicating a role for Gs proteins[68].

Neurotransmitters and hormones are known to regulate many kinds of potassium channels directly via Gα and Gβγ subunits or indirectly via second messengers[69]. Chemokine activation of CCR5 on macrophages opens calcium-activated potassium channels[70] and on T cells opens voltage-gated potassium channels[71].

4 Regulation of MAPK pathways

GPCRs regulate cell proliferation and differentiation through a family of MAPKs. These serine/threonine protein kinases are capable of phosphorylating several transcription factors, thereby regulating subsequent transcriptional events. There are at least three subtypes of MAPK, the extracellular signal-regulated kinases (ERKs) are mainly stimulated by growth factors, whereas c-Jun NH2-terminal kinases (JNKs, also referred to as the stress activated protein kinases, SAPKs) and p38 MAPK are more responsive to cellular stress[72].

In astrocytes, RANTES induces the phosphorylation of MEK, ERK1/2 as well as p90 ribosomal S6 kinases (RSK) and CREB. This cascade of events is inhibited by U0126, a MEK inhibitor, and dominant negative mutants of RSK and CREB blocked the transcriptional activation of chemokine synthesis[21]. RANTES also induces Raf-1 kinase activity and this is sufficient to induce the transcription of proinflammatory chemokines. The PKA inhibitor H-89 is also able to promote chemokine synthesis[29]. This suggested to the authors that RANTES signalling induces the activation of a Raf/MEK/ERK pathway resulting in increased synthesis of chemokines, which may prolong the inflammatory response, and that activation of this MAPK kinase pathway works in concert with RANTES-induced inhibition of AC. As RANTES is a potent ligand for CCRs1, 3 and 5 the receptor mediating these events was not identified. In HOS cells expressing CCR1, Lkn-1 activation of ERK1/2 is PTX-sensitive and required the activity of PLC, PKCδ and Ras. Activation of this cascade by Lkn-1 induces the immediate early genes c-fos and c-myc, which may regulate the Lkn-1-mediated cell cycle progression[73].

Activation of CCR2b, but not CCR2a, induces rapid induction of ERK activity in a PKC/MEK dependent manner. In human monocytes and transiently transfected cells, this effect is partially PTX-sensitive[74] and may involve an element of mediation by G14 and G16 proteins (unpublished data). Conversely, in murine peritoneal macrophages and skin fibroblasts the MCP-1-induced ERK phosphorylation is completely PTX-sensitive[75,76]. MCP-1 challenge of murine peritoneal macrophages also demonstrated the MCP-1-mediated phosphorylation of JNK and activation of the c-jun transcription factor[75].

CCR3-mediated phosphorylation and activation of ERK1/2 and p38, but not JNK, in eosinophils is required for cellular chemotaxis[77]. In rat basophilic leukemia (RBL-2H3) mast cells CCR3-mediated activation of MAPK pathways is dependent on Rac activity, which correlates with CCR3-mediated actin reorganization known to be necessary for cell motility[78].

There are also reports that CCR4-mediated activation of ERK1/2 in hippocampal neurons partially protects the cells against the neurotoxicity of the HIV-1 envelope protein gp120[19]. In contrast CCR5 is thought to potentiate the neurodegenerative effects of HIV infection. When CCR5 was activated by its ligand MIP-1β along with co-stimulation of T cells and glioma cells by gp120, both p38 and JNK pathways were activated but stimulation by MIP-1β alone resulted in activation of ERK1/2. Selective inhibition of p38 prevented the gp120 induction of MMP-9, a protein thought to be involved in the development of HIV-induced dementia, suggesting that a strategy of targeting p38 may have clinical benefits[79]. CCR8 may also provide an element of protection against external challenges that induce apoptosis by activating ERK1/2 in mouse thymic lymphoma cell lines via Gi/o proteins[80].

5 Regulation of phospholiase A2 activity

Arachidonic acid (AA) acts as a second messenger for receptors in the superfamily of GPCR and receptor tyrosine kinases. AA hydrolysis from phospholipids is regulated by the cytosolic form of phospholipase A2 (cPLA2) in response to hormones[81]. AA is a precursor of proinflammatory eicosanoids[82] and is therefore of considerable relevance to the study of chemokine action.

The selective CCR1 ligand MPIF-1 (CCL23) promotes rapid AA release from monocytes and the activity of PLA2 is required for the formation of filamentous actin formation, an essential step for cellular motility[40]. Monocyte chemotaxis induced by several CC chemokines is also inhibited following antisense inhibition of cPLA2 expression[83]. In this study, the chemotaxis induced by MCP-1 was completely inhibited by antisense knockout of cPLA2, suggesting that AA formation is an absolute requirement for CCR2-induced monocyte chemotaxis.

6 Concluding remarks

The G protein-coupled CC chemokine receptors are known to modulate a series of effectors through G proteins and second messengers. CCR-induced regulation of some of these effectors including AC, PLC-β, and MAPK pathways has been confirmed in both heterologous systems and cells endogenously expressing CC chemokine receptors. There is also considerable evidence for the involvement of Ca²⁺, K+, phosphoinositide 3-kinase[84] and small GTPases[37] in the transduction of chemokine signalling but the role of individual G protein subunits has often not been clearly identified. Since many of the components of these pathways have been cloned, the putative signalling pathways linking CCRs to their effectors can be reconstituted and examined in a defined environment. This has undoubtedly enabled the elucidation of many of the subtleties of CCR signalling and will allow further refinement of our understanding of many of the puzzling aspects of these signalling events. This approach must always be complemented by examination of signalling pathways in endogenous tissues as overexpression of various signalling components may allow measurement of activities that do not occur naturally, due to different levels of expression or spatial and temporal separation. For example, in this review we have presented examples where CCRs have differing effects on AC and Ca²⁺ channels, depending on the subtype of effector isozyme present in particular tissues.

A number of CCR-coupled transduction pathways are yet to be described adequately. For example, the ability of some CCRs to couple to G16 proteins opens up a number of new signalling possibilities, including activation of signal transducers and activators of transcription (STAT)-dependent pathways[85] and NF-κB activation[57].

Given the number of signalling pathways that have been demonstrated to mediate CC chemokine-induced events, it will be particularly important to establish which pathway or, more likely, which combination of pathways are actually required to regulate a physiological event in a given cell type. As an example of the current uncertainty in the relevance of individual pathways, the presented data on human monocytes is instructive. In this cell type, the chemoattractant properties of MIP-1α and RANTES are significantly inhibited by treatment with forskolin and IBMX, implying a vital role for CCR regulation of AC-coupled pathways[30]. In addition, antisense knockout of cPLA2 reduces MIP-1α and RANTES-induced chemotaxis by approximately 75%[83]. This suggests that either there is a degree of overlap between these two pathways or that they operate independently and both are required to trigger the biochemical mechanisms that ultimately regulate the multitude of cellular events required for cell motility.

The studies described suggest that controlling CC chemokine signalling activity may be beneficial to the treatment of numerous pathophysiological states, and indeed CCR ligands are being actively investigated for their therapeutic applications. For example, a number of non-peptide CCR1 antagonists have been described with applications in the treatment of multiple sclerosis and improving the success of organ transplants[6]. The increasing use of the CCRs in high-throughput drug screening protocols and the availability of highly promiscuous chimeric G protein adaptors[86] (Wong et al., submitted for review; Hazari et al., submitted for review), will certainly facilitate the discovery of new ligands that may increase our ability to control CC chemokine signal transduction cascades.

CCRs clearly activate a network of signalling pathways many of which have been shown to play vital roles in the regulation of physiological and pathophysiological events. Unravelling the interplay between these interwoven pathways is undoubtedly a daunting task but one which will result in a much fuller understanding of how we may be able to regulate many physiological processes

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Received: June 30, 2003 Accepted: July 2, 2003
This work was supported in part by the Hong Kong Jockey Club and grants from the Research Grants Council of Hong Kong (HKUST 2/99C and DAG98/99.SC05) and the University Grants Committee (AoE/B-15/01) to WONG Yung H.. WONG Yung H. was a recipient of the Croucher Senior Research Fellowship

*Corresponding author: Tel, 852 2358 7328; Fax, 852 2358 1552; e-mail, [email protected]