GPCRs predominantly, although not exclusively 8, exert their effects by activating heterotrimeric G proteins. This promotes the release of free Gα and Gβγ subunits, which then initiate intracellular signal transduction. GPCRs preferentially couple to heterotrimeric G proteins that are grouped into four classes, known as Gα_q/11, Gα_i/o, Gα_s and Gα_12/13 9. Members of all four classes of Gα subunit have been shown to be involved in the regulation of cell growth and proliferation by virtue of the fact that constitutively active Gα mutants have been found in numerous tumours. The gsp oncogene (for

G_s

p

G_i

p

Numerous GPCRs utilize heterotrimeric G proteins to modulate cellular proliferation. Direct evidence of the involvement of G_i/o proteins has been obtained by the use of pertussis toxin (PTX) to block G_i/o-mediated signalling. For example, melatonin acting on G_i/o-coupled MT₁ receptors expressed in MCF-7 breast cancer cells suppresses estrogen and glucocorticoid-induced cell proliferation 12, possibly by inhibiting the steroid receptor-induced transcription of the cyclin D1 gene 1314. These effects of melatonin are entirely blocked by PTX. The use of PTX has also indicated that G_i/o proteins mediate the promotion of DNA synthesis by α₁-adrenergic receptors in osteoblasts 15, κ-opioid receptors in C6 glioma cells 16 and lysophosphatidic acid (LPA) receptors in human fibroblasts 17. Further examples of GPCR utilization of G_i/o proteins in proliferative responses can be found in Table 1.

The involvement of G_s proteins in a few GPCR-initiated responses has been determined using cholera toxin (CTX), which constitutively activates Gα_s subunits, preventing further activation by GPCRs. Glucagon-like peptide 2 (GLP-2) acts as a potent mitogen at Caco-2 intestinal epithelial cells but pretreatment of cells with CTX significantly reduces GLP-2-induced DNA synthesis 18. Likewise, CTX blocks the LPA-induced proliferation of retinal pigment epithelial cells 19, although the relative contribution of LPA receptor activation of G_i/o and G_s proteins in these responses was not determined. Other G_s-coupled GPCRs also play significant roles in promoting or inhibiting cell cycle progression, as witnessed by their effects on downstream effectors (see Table 1 and below).

While there is much compelling evidence that proves the involvement of G_q/11 and G_12/13-activated signalling pathways in cell cycle control (discussed in more detail below), direct experimental evidence of the GPCR activation of these G proteins for the purposes of cell cycle control is generally absent. A notable exception, however, is a study of NIH3T3 fibroblasts transfected with Gα₁₂. In the presence of LPA, these cells synthesize DNA and proliferate much more rapidly than untransfected cells, indicating that the LPA effects are mediated by the LPA receptor coupled to Gα₁₂ 20.

Cyclic AMP (cAMP) is generated from ATP by the AC family of enzymes. ACs are activated by Gα_s subunits while most isoforms are inhibited by Gα_i/o subunits. Gβγ dimers can either negatively or positively regulate AC isoforms. cAMP activates protein kinase A (PKA), which not only phosphorylates transcription factors, including the cAMP response element binding protein (CREB) and AP1 family members, but also modulates the activity of other signalling pathways (Fig. 1 and 21).

Parathyroid hormone (PTH) receptor activation in UMR-106 osteoblast cells inhibits the progression of cells into S phase. This blockage is accompanied by increases in p27^Kip1, an inhibitor of the cyclin-CDK complexes necessary for the G1 to S phase transition 7. As PTH is a G_s-coupled receptor, a cell permeable cAMP analogue mimicked the effects of PTH while a PKA inhibitor abolished the increases in p27^Kip1 levels 22. In complete contrast, activation of the thyroid stimulating hormone (TSH) receptor, also G_s-coupled, induced G1 to S phase progression in rat thyroid cells. The TSH-induced progression and increased DNA synthesis was associated with increases in the levels of c-Fos 23, a binding site for which is found in the promoter region of cyclin D1 14, as well as increases in the levels of two G1 cyclins, D1 and E 24. These effects were mimicked by a cAMP analogue 24 and cells containing a dominant negative mutant of CREB, which also activates the cyclin D1 promoter, had reduced levels of TSH-induced DNA synthesis and an increased cell cycle length 25. Similarly, estrogen and 17β-estradiol (E2) are thought to act, in part, as ligands for the orphan GPCR GPR30 26. The E2-induced proliferation of keratinocytes is accompanied by increases in the levels of cyclin D2, a key mediator of G1 to S phase progression in skin cells 27, and increases in the activity of cyclin D2-CDK4 or 6 complexes 28. E2 increased the amount of active CREB, a transcriptional activator of the cyclin D2 gene, and this, as well as the increased levels of cyclin D2 and proliferation, were reversed by a PKA inhibitor 28.

Due to the differential expression patterns and levels of AC isoforms, the multiplicity of phosphodiesterases that can degrade cAMP and the regulation of ACs by Ca²⁺/calmodulin and a variety of kinases 21, it is perhaps not surprising that activation of G_s-coupled receptors can lead to contradictory effects on cell cycle progression depending on the cell type and GPCR studied (Table 1 and 14). It has been suggested that the differences may be the result of different cAMP concentrations, with lower levels inducing cyclin D expression whereas higher levels induce p27^Kip1 expression 28. In addition, elevated levels of cAMP and the activation of PKA results in cell type specific modulation of MAPK pathways 29, while it is probable that Gβγ subunits released from GPCR-activated G_s proteins can activate MAPKs (Fig. 1 and 30).

It is not yet clear whether G_i/o-coupled GPCR-induced reductions in basal cAMP levels can independently affect cell cycle progression but it is likely that intracellular cAMP levels are the product of competing signals from G_s and G_i/o proteins. There are examples of G_i/o-coupled receptors modulating cell cycle progression, e.g. the melatonin MT₁ receptor-mediated inhibition of proliferation in rat uterine cells 31, however these effects are likely to be mediated by a variety of other intracellular pathways (see following sections) rather than by the inhibition of AC activity.

Mammalian cells express three major classes of MAPKs, the extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 kinases, the activation of which results in the stimulation of transcription factors and the regulation of the expression of cell cycle proteins 3233. GPCRs activate MAPKs via several distinct mechanisms, i.e. by using β-arrestin/endocytotic pathways, transactivating RTKs or by second messenger activation. The β-arrestin pathway generally results in the retention of MAPKs in the cytoplasm and transient MAPK activity, limiting their role in the activation of nuclear substrates and proliferation (discussed in 34). However, GPCR activation of β-arrestin dependent pathways does not exclude the possibility of sustained ERK activation 35 or of nuclear translocation of ERK activity and the promotion of proliferation, as demonstrated for the neurokinin NK-1 receptor 36. In contrast, RTK-mediated and second messenger activation of MAPK pathways generate the sustained MAPK activity that is often thought critical to the GPCR regulation of cell cycle progression 32.

As well as its documented role in activating Raf-1 (see above), PKC also acts as a key mediator of a number of other GPCR-induced proliferative pathways. PKC isoforms, as well as DAG, are able to activate the protein kinase D (PKD) family of serine/threonine kinases 57. Indeed, the proliferation of Swiss 3T3 cells in response to the activation of G_q-coupled bombesin or vasopressin receptors is greatly potentiated by the overexpression of PKD 58. The pathways connecting GPCR activation to the control of cell cycle progression have not yet been outlined but it is known that PKD can activate ERK pathways and phosphorylate c-Jun (Fig. 4 and 57).

PKC also activates the NF-κB transcription factors by initiating a series of phosphorylation and degradation events 59. In mouse embryonic cell lines expressing both dopamine D₁ (G_s-coupled) and D₂ (G_i/o-coupled) receptors, the administration of dopamine resulted in a PKC-dependent increase in NF-κB DNA binding activity, along with increases in the levels of p21^Cip1 and p27^Kip1 and an inhibition of DNA synthesis 60. However, in an embryonic fibroblast model NF-κB binds to and activates the cyclin D1 promoter region, leading to G1 to S phase progression (Fig. 4 and 61). Other GPCRs, including the G_i/o-coupled μ-opioid receptor 62, the somatostatin SST₂ receptor acting via Gα₁₄ 63 and the adenosine A₁ receptor acting via Gα₁₆ 64 also promote NF-κB activation. This activity appears to be mediated by numerous intracellular pathways, including those dependent on PKC, ERK, Src, PI3K, JNK, and PLCβ, although the role of G_i/o-coupled receptor activation of these pathways in NF-κB mediated cell cycle progression is yet to be investigated.

Members of this family of kinases are firmly embedded in signal transduction pathways activated by diverse extracellular stimuli 65. They also play a significant role in the crosstalk between many pathways. We have already seen that Src kinases play a part in the GPCR-induced transactivation of RTKs (see preceeding discussion and Fig. 2). The GPCR/Src/RTK sequence of events is poorly understood, involving either Gα or Gβγ subunit stimulation of Src or Src-activating pathways 46. GPCRs can also transactivate focal adhesion complexes consisting of integrin heterodimers that act as extracellular matrix receptors. The transactivation is Src-dependent and leads to the formation of a signalling platform that includes Src, the focal adhesion kinase p125FAK or its homologue Pyk2, paxillin, as well as the adaptor proteins required to promote Ras family-dependent signalling pathways, particularly those that use MAPKs and PI3Ks as intermediates (reviewed in 66). Angiotensin II utilizes just such a p125FAK/Rac1/JNK pathway to promote the proliferation of vascular smooth muscle cells 67. Gastrin and other neuropeptides, through their agonistic effect on G_q- and G_12/13-coupled GPCRs, are also thought to promote G1 to S phase transition, in part, via their activation of similar Rho/integrin/p125FAK/paxillin signalling complexes 6869. This would include the endothelin receptors, which promote DNA synthesis in primary astrocytes using a combination of an adhesion dependent Src/Rho/p125FAK/paxillin and an apparently Rho/adhesion-independent Pyk2/ERK pathway 70. The G_i/o-coupled CXCR4 receptor promotes DNA synthesis via a Pyk2/PI3K/ERK pathway (Fig. 5 and 71).

In the absence of RTK transactivation, Src activity is required for GPCR-induced proliferation of a number of alternative pathways. The G_q-coupled substance P receptor (NK-1) promotes the proliferation of human glioblastoma cells in a Src-dependent manner. Inhibition of Src activity prevents the phosphorylation and activation of PKCδ and ERK in these cells 72. ERKs are known substrates of PKCδ 73. The mitogenic G_q-coupled endothelin receptors activate ERKs via a Src-dependent pathway that requires the Gα_q-subunit activation of PLCβ and Ca²⁺ release 74. A similar pathway was identified in CHO cells expressing the gastrin-activated CCK2 receptor, where proliferation was mediated by a PKC/Src activation of p38 MAPK 75. In contrast, the anti-proliferative effects of the α_1B-adrenergic receptor in HEK293 cells are Src family kinase dependent. Such activity stimulates a Rho family GEF, Dbs, and cdc42, a Rho family member, activating a MAPK kinase, MKK4, and JNK (Fig. 5 and 76).

Other studies have shed light on the G_i/o-coupled GPCR activation of Src-mediated proliferation. Serotonin 5HT_1E, dopamine D₂ and α_2C-adrenergic receptors all promote the proliferation of NIH3T3 cells via the Gα_i-subunit activation of Src, which activates C3G, a RapGEF. As was discussed above, RapGEFs, including Epac, activate Rap-1/B-Raf/ERK pathways leading to proliferation (Fig. 5 and 77). Alternatively, the dopamine D₄ receptor promotes DNA synthesis via Src/Src homology 2-containing protein (SHC)/Ras/ERK pathway 78. The precise mechanism of Gα_i activation of Src is still under investigation but both Gα_i and Gα_s directly bind to and activate Src family kinases 79.

Activation of MAPKs is not the only consequence of the GPCR-induced activation of Src family kinases. An increasing number of GPCRs activate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways as a means to modulate cell cycle progression. The gastrin-activated CCK2, muscarinic M₅ and α_1B-adrenergic G_q-coupled receptors, as well as the G_s-coupled melanocortin MC₅ receptor induce increases in cell proliferation by activating JAK and STAT family members 808182. The definitive involvement of Src in these pathways has not been established and it is possible that a combination of the direct activation by Src kinases and Ras-dependent MAPK pathways is required for full STAT transcriptional activation 83. Interestingly, the promiscuously coupled Gα₁₄ and Gα₁₆ subunits are similarly able to mediate the activation of Src and JAK/STAT pathways following activation of several GPCRs 848586, although whether this leads to the modulation of cell cycle progression is not yet known. The ability of Gα_i/o subunits to promote the Src-mediated activation of STATs is well documented 83. What is less clear is the role of G_i/o-coupled GPCRs in controlling cell cycle progression via these pathways. Intriguingly, in NIH3T3 cells, Gα_i2 mediates the Src activation of STAT3, and this may promote the expression of cyclin D1 (Fig. 5 and 87).

Extracellular signals transduced by both RTKs and GPCRs converge upon the activation of a family of PI3Ks. Activation of these lipid kinases by GPCRs is thought to be dependent on the direct binding of Gβγ subunits and Ras to PI3Ks 88. PI3K activation initiates a phosphorylation cascade leading to the activation of Akt (also termed protein kinase B) and its downstream kinases phosphoinositide-dependant kinase 1 (PDK1), glycogen synthase kinase 3 (GSK3), p70 ribosomal protein S6 kinase (p70^S6K), mammalian target of rapamycin (mTOR) and others 89. In addition, we have already seen how GPCRs can activate PI3K pathways via RTK or integrin transactivation 414266. Following direct or indirect GPCR-induced PI3K activation, cell cycle progression is regulated by the effect of PI3K-activated kinases on the expression and stability of cell cycle proteins, or by the modulation of the activity of other signal transduction pathways. For example, somatostatin SST₂ receptors expressed in Chinese hamster ovary cells (CHO) inhibit proliferation by activating a PI3K-dependent Ras-Rap1/B-Raf/ERK pathway, resulting in an increase in the levels of p27^Kip1 protein (Fig. 6 and 90). It has also been shown that sustained activation of p38 by activation of the SST_2a receptor subtype leads to upregulation of p21^Cip1 and cell cycle inhibition. However, this can be antagonized by activation of SST_2b receptor, which activates PI3K, p70^S6K, Akt and proliferation (Fig. 6 and 91). This suggests that the final outcome of a signalling event relies on the balance of several competing mechanisms.

Several studies have shed further light on the effect of the activation of GPCR/PI3K pathways on cell cycle proteins. For example, thrombin receptor activation in vascular smooth muscle cells leads to reduced levels of p27^Kip1 and increased cellular proliferation 92, while in embryonic fibroblasts the evidence suggests that thrombin receptor activation of PI3K/Akt pathways promotes cyclin D1 accumulation, cyclin D1-CDK4 activity and cell cycle progression 9394. Furthermore, it has been postulated that thrombin receptor activation of ERK activity ultimately leads to enhanced translocation of CDK2 into the nucleus and fibroblast proliferation 95. Moreover, sphingosine 1-phosphate activation of the EDG-1 receptor activates p70^S6K, promoting cyclin D1 expression and proliferation (Fig. 6 and 96). The reduction in p27^Kip1 levels and the upregulation of cyclin D protein are thought to be the primary cell cycle effects of PI3K activation by RTKs 89. The cyclin D1 protein is stabilized by the Akt-mediated inactivation of GSK3, which normally phosphorylates and promotes the degradation of cyclin D1. Akt also phosphorylates and inactivates forkhead (FH) transcription factors, which bind to and activate the p27^Kip1 promoter. PI3K pathways may also reduce the stability of p27^Kip1, and Akt phosphorylation of p27^Kip1 adversely affects its nuclear localization. Akt-induced phosphorylation of the tumour suppressor TSC2 (also known as tuberin) causes the dissociation of TSC2 and TSC1 (also known as hamartin), relieving their inhibition of mTOR kinase. Increased mTOR activity reduces the stability of p27^Kip1 (Fig. 6 and 89). Some GPCRs have now been shown to couple to this PI3K/tuberin system 97, although the significance for cellular proliferation has not been established.