G protein-coupled receptor-induced Akt activity in cellular proliferation and apoptosis

Authors

  • David C. New,

    1. 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
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  • Kelvin Wu,

    1. 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
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  • Alice W. S. Kwok,

    1. 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
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  • Yung H. Wong

    1. 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
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Y. H. Wong, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Fax: +852 2358 1552
Tel: +852 2358 7328
E-mail: boyung@ust.hk

Abstract

Akt (also known as protein kinase B) plays an integral role in many intracellular signaling pathways activated by a diverse array of extracellular signals that target several different classes of membrane-bound receptors. Akt plays a particularly prominent part in signaling networks that result in the modulation of cellular proliferation, apoptosis and survival. Thus, the overexpression of Akt subtypes has been measured in a number of cancer types, and dominant-negative forms of Akt can trigger apoptosis and reduce the survival of cancer cells. G protein-coupled receptors act as cell-surface detectors for a diverse spectrum of biological signals and are able to activate or inhibit Akt via several direct and indirect means. In this review, we shall document how G protein-coupled receptors are able to control Akt activity and examine the resulting biochemical and physiological changes, with particular emphasis on cellular proliferation, apoptosis and survival.

Abbreviations
CDK

cyclin-dependent kinase

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

ERK

extracellular signal-regulated kinase

FH

forkhead

GPCR

G protein-coupled receptor

LPA

lysophosphatidic acid

MAPK

mitogen-activated protein kinase

mTOR

mammalian target of rapamycin

NF-κB

nuclear factor κB

p70S6K

p70 ribosomal protein S6 kinase

PAR-2

protease-activated receptor-2

PDGFRα

platelet-derived growth factor receptor α

PI3K

phosphoinositide 3-kinase

PKB

protein kinase B

PTEN

phosphatase and tensin homologue

RTK

receptor tyrosine kinase

SHIP

SH2-domain-containing inositol polyphosphate 5-phosphatase

TNF

tumour necrosis factor

TSC

tuberous sclerosis complex

TSH

thyroid-stimulating hormone receptor.

Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinase that plays a pivotal role in many physiological processes, including metabolism, development, cell cycle progression, migration and survival [1–4]. The Akt subfamily of protein kinases consists of three isoforms – Akt1, Akt2 and Akt3 (also termed PKBα, PKBβ and PKBγ) – which are the products of distinct genes. All three proteins share a conserved tertiary structure of an N-terminal pleckstrin homology domain, a kinase domain and a C-terminal regulatory domain containing the hydrophobic motif phosphorylation site [5]. While the homology between the three isoforms allows for a degree of functional redundancy [1], there also seems to be considerable scope for isoform-specific activation and substrate specificity [3,6].

Akt plays an integral role in the phosphoinositide 3-kinase (PI3K) signaling pathways. PI3K pathways are activated in response to extracellular signals mediated by cell-surface receptors of the G protein-coupled receptor (GPCR), integrin and growth factor/receptor tyrosine kinase (RTK) superfamilies. Receptor-mediated activation of PI3K results in the generation of phosphatidylinositol (3,4,5)-trisphosphate from phosphatidylinositol (4,5)-bisphosphate, a reaction that is reversed by the enzymes phosphatase and tensin homologue (PTEN) and SH2-domain-containing inositol polyphosphate 5-phosphatase (SHIP). Both Akt and phosphoinositide-dependent kinase are recruited to the plasma membrane by phosphatidylinositol (3,4,5)-trisphosphate through their pleckstrin homology domains, where phosphoinositide-dependent kinase phosphorylates Akt1 on residue Thr308 in its kinase domain [7]. A second phosphorylation takes place at Ser473 in the hydrophobic motif region of Akt1. This phosphorylation event seems to be catalyzed by a number of different kinases, which are probably stimulus- and/or cell type-specific. This stabilizes the active conformation of Akt and allows it to translocate to the cytoplasm or nucleus to search for its many target proteins [8].

Akt's role in physiology suggests that aberrant Akt signaling may be a factor in disease states. Most notably, amplification of Akt isoform genes and Akt mRNA overexpression has been observed in many human cancers [9]. Akt activity in cancer cells may also be enhanced by the amplification of genes encoding PI3K or by a reduction in the activity of PTEN or SHIP [9]. It is therefore to be expected that the inhibition of PI3K, Akt and their downstream effectors has been targeted in the development of cancer therapies [10]. The involvement of Akt in cancer is not surprising given the ability of Akt to promote cellular proliferation through the direct and indirect phosphorylation of a number of cell cycle regulatory proteins [11], and its ability to inactivate pro-apoptotic factors, such as Bad, caspase-9 and forkhead (FH) transcription factors [12]. In contrast, it is thought that a reduction in Akt signaling may contribute to diabetes by reducing the survival of pancreatic β cells [13].

GPCRs act as cell-surface detectors for a diverse spectrum of biological signals

To date, over 200 GPCRs have been matched with a ligand that activates the receptor to promote a wide variety of intracellular biochemical changes [14], even though it is estimated that the human genome encodes between 800 and 1000 GPCR subtypes [15,16]. Their pervasive influence, coupled with their cell-surface accessibility, has resulted in GPCRs becoming the targets of as many as 45% of modern medicines [17], which are used to treat conditions as diverse as inflammation, incontinence, hypertension, depression and pain [18].

GPCRs preferentially couple to heterotrimeric G proteins (consisting of α, β and γ subunits) that are grouped into four classes, known as Gαq/11, Gαi/o, Gαs and Gα12/13, based on the effector with which the α-subunit primarily interacts. The activated G proteins in turn promote the activation or inhibition of a variety of intracellular events, including the activation of phospholipases, mitogen-activated protein kinases (MAPKs), activation/inhibition of adenylyl and guanylyl cyclases, and the opening and closing of ion channels.

In this review, we shall investigate the ability of GPCRs to activate Akt signaling pathways both directly, through the interaction of Gβγ subunits with PI3K, and indirectly, through the GPCR transactivation of RTKs and integrins. We shall also examine the downstream signaling and physiological consequences of GPCR-induced Akt activation, paying particular attention to the consequences for cellular proliferation, survival and apoptosis.

GPCR activation of PI3K/Akt signaling pathways

GPCRs promote intracellular signaling through both Gα and Gβγ subunits, which can activate distinct, complementary or antagonistic pathways. As we will demonstrate, a large number of GPCRs, coupled to all four classes of G protein, activate PI3K/Akt pathways through either Gα or Gβγ subunits (see below, Table 1 and Fig. 1). Gβγ subunits are able to bind directly to and activate PI3K heterodimeric proteins containing either the p110β or the p110γ subunits [19]. Furthermore, it has been demonstrated that muscarinic and lysophosphatidic acid (LPA) GPCRs are only able to activate Akt in cells expressing the p110β or p110γ subunits, and that this activation is mediated by Gβγ subunits, but not by Gα subunits [20]. Direct activation of PI3K by Gα subunits has not been specifically measured but they can activate PI3K/Akt pathways by transactivating integrins, RTKs and other growth factor receptors.

Table 1.   GPCR-induced Akt activity and the consequences for cellular proliferation and apoptosis. A selection of examples is presented here demonstrating the signaling components employed in connecting GPCR-initiated signals to downstream events regulated by Akt. ↑, indicates an increase in protein levels or activity; ↓, indicates a decrease in protein levels or activity. AC, adenylyl cyclase; AFX, FOX04; ALXR, lipoxin A4 receptor; CDK, cyclin-dependent kinase; CREB, cAMP-response element binding; cyt c, cytochrome c; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FKHR, forkhead in rhabdomyosarcoma; FSH, follicle stimulating hormone; IGF-1, insulin growth factor-1; IGF-IRβ, insulin-like growth factor receptor; IRS-1, insulin receptor substrate 1; GnRH, gonadotropin-releasing hormone; GSK3β, glycogen synthase kinase 3β; LHRH, luteinizing hormone-releasing hormone; LPA, lysophophatidic acid; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor-κB; p70S6k, p70 ribosomal protein S6 kinase; PAR-2, protease-activated receptor-2; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PP2A, protein phosphatase 2A; PTP, protein tyrosine phosphatase, ROCK, Rho-associated kinase; ROS, reactive oxygen species; SHP, Src homology 2-containing phosphatase; TSC2, tuberous sclerosis complex 2; TSH, thyroid stimulating hormone; VPAC, vasoactive intestinal peptide receptor.
GPCRIntracellular pathwayFunctional consequencesReferences
Gi/o pathways
Adenosine A2↑PI3K/↑Akt/↓TSC2Survival[41]
Adenosine A3↑PLC/↑PI3K/↑Akt/↓ERKAnti-proliferation[56]
ALXR↓PDGF/↑EGF/↑PI3K/↑Akt/↑p27Kip1/↑p21Cip1/ ↓CDK2/↓cyclin EAnti-inflammatory effects, antiproliferation[47]
CXCR4↑Ca2+/↑Pyk2/↑PI3K/↑ERK/↑Akt↑DNA synthesis, proliferation[34]
δ-opioid↑PI3K/↑Akt/↓TSC2Survival[50]
↑PI3K/↑Akt/↓GSK3β/↓AFX/↓FKHRSurvival, proliferation[81]
Dopamine D2↑PP2A/↓Akt/↑GSK-3Dopamine-induced neurological responses[37]
κ-opioid↑PI3K/↑Akt/↓TSC2Survival[50]
LPA↑MMP/↑EGFR/↑PI3K/↑Akt↑Cyclin D1,↑DNA synthesis, proliferation, survival[24,25]
↑PI3K/↑Akt/↑p70S6kSurvival[70]
↑PI3K/↑Akt/↑NF-κBSurvival[80]
µ-opioid↑PI3K/↑Akt/↓TSC2Survival[50]
Muscarinic M2↑PI3K/↑AktSurvival[63]
Muscarinic M4↑PI3K/↑Akt/↓TSC2Survival[40]
Pheromone V2R↑ERK/↑Akt/↑CREBSurvival[64]
Somatostatin SST2↓PI3K/↓Akt/↓NF-κBAnti-proliferative, apoptotic, antiangiogenic and anti-invasive[36]
↑Src/↑SHP-1/↑SHP-2/↑PI3K/↑Ras/↑ERK/ ↑p27Kip1G1 cell cycle arrest[57]
Somatostatin SST2b↑PI3K/↑Akt/↑p70S6K↑DNA synthesis, survival, proliferation[45]
Gq/11 pathways
α-thrombin↑PI3Kβ/↑Akt/↑cyclin D/↑CDK4↑G1-S phase transition[46]
Angiotensin II type 1↑MMP/↑EGFR/↑PI3K/↑AktSurvival, proliferation[25]
↑EGFR/↑PI3K/↑Akt/↑mTOR/↑p70S6K↑G1 cyclins, ↓p27Kip1, ↓p21Cip1, proliferation[52]
Apelin APJ↑PI3K/↑Akt/↓caspase 8/↓cyt c/↓caspase 9/ ↓caspase 3Survival[68]
Bradykinin↑EGFR/↑PI3K/↑Akt↑Cyclin D1, ↑Cyclin E, ↑DNA synthesis, proliferation[26]
Bombesin↑EGFR/↑PI3K/↑Akt↑DNA synthesis, proliferation[26]
↑IGF-IRβ/↑Src/↑PI3K/↑AktSurvival[29]
Endothelin-1↑IGF-IRβ/↑Src/↑PI3K/↑AktSurvival[29]
↑Src/↑PI3K/↑AktGlut4 translocation[38]
GnRH↑PKC/↑Src/↑Pyk2/↑MMP/↑EGFR/↑PI3K/↑AktSurvival, proliferation[25]
LPA↑PI3K/↑Akt/↑NF-κBSurvival[80]
LHRH↑PKCα/↓Akt/↑GSK3/↑Bad/↑caspase 3Apoptosis[75]
Muscarinic M1↑PI3K/↑AktSurvival[63]
↑PTP/↓phosphorylated-IRS-1/↓PI3K/↓Akt/↑RhoA/ ↓ROCK-IApoptosis[73]
Muscarinic subtypes↑Src/↑PI3K/↑Akt/↑ERK/↓GSK3β/↓caspase 3/ ↑CREBSurvival, proliferation[67]
PAR-2↑Ca2+/↑PKC/↑Pyk2/↑Src/↑PI3K/↑AktActin reorganization and cell migration[33]
Serotonin↑PI3K/↑ROS/↑Akt/↑mTOR/↑p70S6KProliferation[53]
Vasopressin V1↑Ca2+/↑PKC/↑Pyk2/↑Src/↑EGFR/↑PI3K/↑Akt/ ↑mTOR/↑p70S6KCell growth, proliferation[51]
Gs pathways
Adenosine A2A↑Ca2+/↑Src/↑Trk/↑AktSurvival[65]
β-adrenergic↑AC/↑PKA/↑Src/↑EGFR/↑PI3K/↑AktMucin secretion[31]
FSH↑PI3K/↑Akt/↓FOXO1aFollicular survival and development[69]
TSH↑cAMP/↑PKA/↑PI3K/↑PDK1/↑mTOR/↑p70S6KProliferation, thyroid follicle activity[54]
↑cAMP/↑PKA/↑PI3K-Ras complex/↓ERKDNA synthesis, mitogenesis[55]
VPAC-1↑TrkA/↑PI3K/↑AktSurvival, development, differentiation[28]
G12/13 pathways
LPA↑Rho/↑p160ROCK/↑PDGFRα/↑PI3K/↑Akt/↓FKHR↓Transcription of apoptotic and   antiproliferative genes[30]
Thrombin↑Rho/↑p160ROCK/↑PDGFRα/↑PI3K/↑Akt/↓FKHR↓Transcription of apoptotic and   antiproliferative genes[30]
Figure 1.

 Routes to Akt activation. G12/13-, Gi/o-, Gq-, and Gs-coupled receptors are all known to activate the phosphoinositide 3-kinase (PI3K)/Akt pathway through either Gα or Gβγ subunits. Gαq and Gαs subunits utilize secondary messenger systems [Ca2+, cAMP, reactive oxygen species (ROS)] to promote PI3K/Akt activation. Whilst Ca2+-induced PI3K activation is a feature of Gi/o-coupled GPCRs, Gβγ subunits released from these receptors are also able to directly bind to and activate PI3K. In addition to these mechanisms, all four classes of GPCRs are able to activate the PI3K/Akt pathway by transactivating RTK at the plasma membrane either through matrix metalloproteinases (Gi/o-, Gq-, and Gs-coupled receptors) or through Rho/Rho-associated kinase (Rock)-mediated expression of RTK ligands (G12/13-coupled receptors). The Rho/Rock pathway can also indirectly inhibit PI3K activity, although the signaling components involved have not yet been elucidated (indicated by a dashed line).

Numerous RTKs and integrins can independently activate PI3K/Akt pathways [21] but it has also been reported that they can be transactivated by GPCRs through Gα- or Gβγ-dependent pathways [22,23]. For example, ligands for the LPA, endothelin-1 and thrombin receptors all promote DNA synthesis in Rat1 fibroblasts by transactivating the epidermal growth factor receptor (EGFR, an RTK). Such transactivation requires the activation of matrix metalloproteases to release EGF from its membrane-bound form, which then stimulates the EGFR and downstream extracellular signal-regulated kinase (ERK) pathways [24]. PI3K/Akt pathways are also activated by a similar method of transactivation [25]. In Swiss 3T3 cells, bradykinin and bombesin promote cellular proliferation by an EGFR-dependent formation of a signaling complex that activates PI3K/Akt cascades [26]. A number of other RTKs are also transactivated by GPCRs [27–29], and it has been demonstrated that constitutively active Gα12 subunits activate PI3K/Akt signaling via the transactivation of the platelet-derived growth factor receptor α (PDGFRα) [30]. However, it is not clear whether transactivation of RTKs by GPCRs can also occur through the induced expression of RTK ligands. Alternatively, the RTK ligand requirement may be bypassed by the GPCR-induced Src family tyrosine kinase activation of RTKs [27], as evidenced by the Src kinase-dependent EGFR transactivation promoted by the β-adrenergic receptor in gastric mucosal cells [31].

Src-family kinases are firmly embedded in signal transduction pathways activated by diverse extracellular stimuli playing a significant role in the crosstalk between many pathways, including those that facilitate the GPCR activation of Akt [32]. The protease-activated receptor-2 (PAR-2) utilizes Gαq subunits to promote the activation of protein kinase C and the mobilization of intracellular Ca2+, leading to the formation of a complex containing Src-family kinases, the focal adhesion kinase, Pyk2, and PI3K [33]. Similar findings have been made for the Gi/o-coupled CXCR4 receptor, which promotes DNA synthesis via a Pyk2/PI3K/ERK pathway [34]. These complexes may form as part of larger integrin/paxillin signaling platforms that promote the phosphorylation and activation of PI3K subunits [35].

GPCR activation may also lead to the inactivation of Akt. It has been reported that somatostatin SST2 receptors directly form a complex with the p85 regulatory subunit of PI3K. Agonist activation induced the dissociation of this complex, preventing PI3K activation [36]. Following agonist-induced activation, dopamine D2 receptors are internalized and form a multiprotein complex that includes β-arrestin, protein phosphatase 2A and Akt. Protein phosphatase 2A inactivates Akt, thereby relieving Akt's inhibition of glycogen synthase kinase 3β and allowing it to mediate dopamine-induced neurological responses [37]. An alternative mode of β-arrestin-mediated PI3K/Akt inhibition is proposed to occur upon activation of the Gαq-coupled PAR-2 receptor. Upon recruitment to the PAR-2 receptor, β-arrestin forms a complex with PI3K and spatially restricts its enzyme activity, thereby modulating the PAR-2 receptor activation of PI3K/Akt mediated by Pyk2 and Src-family kinases (see the preceding discussion) [33]. In contrast, another study has indicated that β-arrestins mediate the endothelin A receptor activation of Akt by recruiting Src-family kinases that phosphorylate and activate Gαq, ultimately leading to PI3K/Akt pathway activation [38]. Nevertheless, the vital role of β-arrestins in modulating the apoptotic events following the activation of some GPCRs was highlighted by a study which showed that in mouse embryonic fibroblasts devoid of β-arrestins the N-formyl peptide receptor, vasopressin V2, chemokine CXCR2 and the angiotensin II AT1A receptors all promote apoptosis through the activation of PI3K, MAPKs and Src kinases, leading to the activation of caspase pathways [39]. Reconstituting the β-arrestins prevented the GPCR-induced apoptosis, suggesting that for some GPCRs β-arrestins constrain their apoptotic abilities. The same study also demonstrated the GPCR selectivity of these events because in the absence of β-arrestins the CXCR4 and β2-adrenergic receptors were unable to activate apoptosis.

Recent studies have indicated that constitutively active Gα subunits of the Gαq/11 and Gα12/13 subfamilies may actually inhibit the EGFR-mediated activation of Akt in transfected HEK-293 cells [40]. This contradicts the previously noted ability of constitutively active Gα12 subunits to potentiate PDGFRα-mediated PI3K/Akt signaling [30]. It is not immediately apparent whether these studies relate to GPCR signaling because RTKs are able to utilize heterotrimeric G protein pathways independently of GPCR activation [41].

Akt mediation of GPCR-induced cell cycle control

GPCRs have been widely reported to mediate mitogenic signals leading to cellular proliferation [2,42], and the overexpression or mutation of many GPCR subtypes in numerous cell types is thought to contribute to deregulated growth and tumour development [43,44]. The transmembrane and intracellular pathways mediating the GPCR control of cell cycle progression are extensive [2], with all pathways converging in the nucleus to regulate the expression, localization, activity or stability of a small number of cell cycle proteins that are critical for the orderly progression from the G1 to S phases of the cell cycle. Akt, in response to GPCR activation, directly interacts with some of these cell cycle proteins or exerts its effects through its downstream partners (Fig. 2).

Figure 2.

 Targets of Akt phosphorylation. Activated (phosphorylated) Akt isoforms are able to regulate key cellular and physiological processes by phosphorylating a wide range of substrates involved in cellular survival (blue), glucose metabolism (orange), cell cycle progression (green), and protein synthesis (pink). Dashed lines indicate a translocation event.

Evidence suggests that the GPCR activation of Akt pathways can be either proliferative or antiproliferative, depending on the nature of the stimulus and the cell type observed. Competing effects on cell cycle progression generated simultaneously by the same extracellular signal have been observed, suggesting that the final outcome of a signaling event relies on the balance of several competing mechanisms. For example, activation of the SST2a receptor in Chinese hamster ovary cells promotes the sustained activation of the MAPK family member p38 and the up-regulation of the cell cycle inhibitory protein p21Cip1. Conversely, activation of the SST2b receptor resulted in the activation by PI3K of both Akt and the p70 ribosomal protein S6 kinase (p70S6K), which led to cell cycle progression [45], probably through induction of the expression of cyclins (key proteins for the G1 to S phase transition) [2]. Both somatostatin receptor subtypes were shown to be activating the same Gαi subtypes but it was postulated that the Gβγ subunit pairings may have been receptor subtype selective [45]. Although we now know that GPCR interactions with β-arrestins may also control PI3K/Akt activation (as discussed above), a study on α-thrombin receptor signaling demonstrated that this GPCR activated Akt in β-arrestin-dependent and -independent ways. β-arrestin-independent activation of Akt was more prolonged than β-arrestin-dependent activation and led to cyclin D1 accumulation, cyclin D1-cyclin-dependent kinase (CDK) 4 activity and cell cycle progression [46]. The intermediaries between Akt and cyclin D1 accumulation were not determined but it is known that the cyclin D1 protein is stabilized by the Akt-mediated inactivation of glycogen synthase kinase 3β, which normally phosphorylates and promotes the degradation of cyclin D1. In addition, Akt also phosphorylates and inactivates FH transcription factors, which bind to and activate the p27Kip1 promoter (another cell cycle inhibitory protein). Akt may also reduce the stability of p27Kip1, and Akt phosphorylation of p27Kip1 adversely affects its nuclear localization [11]. Indeed, the anti-inflammatory lipoxins act through GPCRs to inhibit the PDGFR-mediated activation of Akt and the subsequent decrease in the levels of p21Cip1 and p27Kip1, as well as inhibiting the PDGFR-mediated cyclin E–CDK2 complex formation and cell cycle progression [47].

Akt-induced phosphorylation of the tumour suppressor tuberous sclerosis complex (TSC)2 (also known as tuberin) causes the dissociation of TSC2 and TSC1 (also known as hamartin), relieving their inhibition of the mammalian target of rapamycin (mTOR) kinase [48]. Increased mTOR activity reduces the stability of p27Kip1, releasing its restrictions on cell cycle progression. In addition, mTOR activates the proliferative kinase p70S6K[11]. Some GPCRs have now been shown to couple to this PI3K/Akt/tuberin/mTOR system. In PC-12 and other neuronal cells, the Gi/o-coupled α2-adrenergic receptors, muscarinic M4 receptors, as well as the δ-, κ- and µ-opioid receptors, all promote TSC2 phosphorylation via a PI3K/Akt-dependent pathway [41,49,50]. Despite such evidence, a direct role for a Gi-coupled GPCR/Akt/mTOR signaling axis in cellular proliferation has not been demonstrated, as it has been for anti-apoptotic, pro-survival pathways (see below). However, activation of the Gq-coupled vasopressin V1 receptor in mesangial cells potently stimulates cell growth and proliferation by a Pyk2/Src-dependent transactivation of EGFR followed by an mTOR-dependent activation of p70S6K and cell cycle progression [51]. A very similar proliferative EGFR/PI3K/Akt/mTOR/p70S6K pathway is activated by Gq-coupled angiotensin II type 1 receptors in mouse embryonic stem cells, leading to increases in the expression levels of G1 cyclins and their CDK partners, along with decreases in the levels of p21Cip1 and p27Kip1[52]. Mitogenic responses through these pathways have been reported for a number of other Gq-coupled receptors, including those for serotonin [53].

The proliferative actions of Gs-coupled GPCRs mediated by these pathways have not been reported. Nevertheless, activation of the Gs-coupled thyroid-stimulating hormone receptor (TSH) in thyrocytes results in proliferation via an Akt-independent pathway activated by the TSH receptor interaction with PI3K, leading to the activation of p70S6K and mTOR [54]. A separate study has indicated that the TSH receptor promotes PI3K pathway activation and DNA synthesis by stimulating the association of PI3K with Ras [55]. Ras is known to bind to and activate several PI3K subtypes [19], and itself is a major target of GPCR activity [2].

In relation to GPCR control of proliferation, Akt control of ERK has also been recorded. For example, agonist stimulation of the Gi-coupled adenosine A3 receptor expressed in human melanoma cells triggers PI3K phosphorylation of Akt, leading to a reduction in the levels of active, phosphorylated ERK1/2 and an inhibition of cellular proliferation [56]. ERKs are known to regulate the transcriptional activity of several transcription factors that control the expression of G1 cyclins and CDK inhibitors [2]. A seemingly similar PI3K/ERK-dependent pathway is activated by SST2 receptors, leading to the induction of p27Kip1[57].

Akt mediation of GPCR-induced survival and anti-apoptotic pathways

A key role of Akt is to facilitate cell survival and to prevent apoptotic cell death. In fact, dominant negative alleles of Akt reduce the ability of growth factors, extracellular matrix and other stimuli to support cell survival. Conversely, the overexpression of Akt can rescue cells from apoptosis [9]. This is achieved by the phosphorylation and inactivation of pro-apoptotic factors such as Bad, caspase-9 and FH transcription factors.

Bad belongs to the Bcl2 family of apoptotic proteins. In some cell types, unphosphorylated Bad forms a complex with pro-survival members of the Bcl2 family at the mitochondrial membrane, inducing the release of cytochrome c from the mitochondria and triggering caspase-mediated apoptosis. Akt phosphorylation of Bad leads to its sequestration in the cytosol by 14-3-3 proteins, preventing it from binding to its partners at the mitochondrial membrane [9]. Likewise, Akt also phosphorylates and inactivates caspase-9, thereby inhibiting the terminal execution phase of apoptosis [12,58]. In the absence of Akt activity, FH family members are found in the nucleus where they initiate apoptosis through the enhanced expression of specific pro-apoptotic Bcl2 family members. Additionally, FH transcription factors promote the expression of the tumour necrosis factor (TNF) receptor-associated death domain and of the TNF-related apoptosis-inducing ligand, leading to the activation of death-receptor signaling and caspase-mediated apoptosis [59]. Activated Akt phosphorylates FH family members, which are then exported from the nucleus and sequestered in the cytoplasm by their interaction with 14-3-3 proteins [12]. Akt-dependent cell survival may also be achieved by the activation of the nuclear factor-κB (NF-κB) transcription factor and the direct phosphorylation and activation of the cAMP-response element binding protein. These two transcription factors have been implicated in the promotion of the expression of genes encoding survival proteins, such as c-myc, inhibitor-of-apoptosis proteins 1/2 and Bcl2 [9,60].

GPCR-mediated inhibition of apoptosis was observed many years ago when, for example, the activation of muscarinic M3 receptors endogenously expressed in rat cerebellar granule neurons protected the cells against K+-induced apoptosis [61]. In neuronal PC12 cells, agonist activation of exogenously expressed muscarinic M1 receptors protected against apoptosis induced by growth factor withdrawal [62]. The intracellular pathways responsible for mediating these effects are gradually being revealed and it is now clear that Akt-dependent signaling is a vital avenue for the transmission of pro-survival, anti-apoptotic signals emanating from GPCRs. For example, in transfected COS-7 cells both Gq-coupled M1 and Gi-coupled M2 muscarinic GPCRs are able to activate Akt and prevent UV-induced apoptosis [63], while the Go-coupled V2R pheromone receptor promotes the survival of vomeronasal stem cells via a pathway dependent on Akt and cAMP-response element binding protein activation [64]. Gs-coupled receptors have also been noted to utilize Akt-dependent mechanisms to promote cell survival. Adenosine acting through the A2A receptor transactivates the Trk neurotrophin RTKs, which in turn activate Akt and cell survival [65], while an uncharacterized Gs-coupled receptor for the peptide hormone ghrelin protects pancreatic β-cells against induced apoptosis via both Akt and MAPK pathways [66].

The GPCR-mediated signaling events downstream of Akt have also begun to be characterized. In oligodendrocytes, carbachol (a nonselective muscarinic receptor agonist) significantly reduces caspase-mediated apoptosis by stimulating PI3K/Akt pathways [67]. The role of caspases in GPCR-induced cell survival is further confirmed by the ability of the peptide hormone apelin to decrease the activation of caspase 9, as well as caspases 3 and 8. In mouse osteoblasts, this inhibition of caspase activity and the apoptotic activity induced by serum deprivation, steroids or TNF-α were blocked by inhibitors of PI3K and Akt [68].

It is apparent that GPCRs also modify the expression and activity of members of the Bcl2 family of proteins in order to regulate cell survival and apoptosis. As discussed above, the Akt-mediated phosphorylation of FH transcription factors removes them from the nucleus, preventing them from promoting the expression of pro-apoptotic Bcl2 proteins. The initiation of these pro-survival responses by GPCRs is little studied, but there are indications that GPCR/Akt/FH transcription factor/Bcl2 protein pathways are relevant to cell survival. For example, follicle stimulating hormone is thought to play a role in follicular survival and development in the ovary. When expressed in HEK-293 cells, the follicle stimulating hormone receptor rapidly promoted the phosphorylation and inactivation of the FOXO1a FH transcription factor, probably by Akt [69]. The consequences of FOXO1a inactivation were not examined in this study but other work has described the ability of GPCRs to inhibit the expression of Bcl2 proteins. In multiple cell types, the Gi-coupled LPA receptors activate PI3K/Akt/p70S6K pathways as part of their cell survival mechanism [70]. It is suspected that the activation of these pathways by LPA and sphingosine 1-phosphate receptors results in the suppression of the cellular levels of the pro-apoptotic Bcl2 family member Bax [71]. In PC12 cells, it was determined that M4 receptors induced a Gβγ subunit-dependent activation of Akt and were able to augment nerve growth factor (NGF)-mediated cell survival [40]. This Akt activation was accompanied by the degradation of TSC2. While not directly measured in this study, removal of TSC2 would be expected to promote mTOR activity. mTOR is thought to affect apoptosis and cell survival in several different ways, including by regulating the expression levels of the anti-apoptotic Bcl2 family member Bcl-XL[72].

It seems that given the correct conditions, GPCRs can actually promote apoptosis. In HeLa cells, this can be achieved by an M1 receptor-mediated inhibition of insulin receptor-stimulated Akt activation or by a direct activation of caspase/RhoA/Rho-associated kinase pathways [73], possibly by up-regulating the expression of the pro-apoptotic Bax [74], in contrast to the previously noted suppression of the cellular levels of Bax by the LPA and sphingosine 1-phosphate receptors [71]. An alternative approach to the induction of apoptosis has been adopted by the luteinizing hormone-releasing hormone, which inhibits the insulin growth factor-1 receptor-mediated activation of Akt. Inhibition by the LHRH receptor in pituitary cells results in a reduction in Bad phosphorylation and a reduction in the ability of insulin growth factor-1 to rescue cells from apoptosis [75].

It is clear that Akt-dependent survival pathways represent an attractive target for the development of anticancer agents. In fact, inhibitors of mTOR not only cause cell cycle arrest but also promote apoptosis directly by sensitizing cells to the effects of DNA-damaging agents [72,76]. The contribution that the regulation of GPCR activity may make to the modulation of these potentially therapeutic pathways is being intensively investigated [77,78]. One approach to cancer therapy has been to target nonselectively the activity of heterotrimeric G proteins using compound BIM-46174. A variety of biochemical assays indicate that BIM-46174 inhibits the formation and/or dissociation of the Gα/Gβγ heterotrimeric complex. Exposure of a variety of human cancer cells to BIM-46174 inhibits their growth by inducing caspase-dependent apoptosis. In mice, this drug seems to complement established chemotherapeutic regimes [79]. Individual GPCRs have also been targeted. For example, LPA receptors couple to several different G protein subfamilies to activate Akt and the transcriptional activity of NF-κB. In androgen-insensitive prostate cancer PC3 cells, this activation of Akt and NF-κB is required to escape cell death. Therefore, it has been suggested that as NF-κB is constitutively activated in prostate cancer, a strategy of targeted disruption of the LPA/Akt/NF-κB pathway may benefit androgen-insensitive prostate cancer treatment [80]. In small cell lung cancer cells with constitutively active Akt signaling pathways, the application of the δ-opioid receptor antagonist naltrindole promotes apoptosis. This correlated with reduced levels of phosphorylation and activity of PI3K, Akt, glycogen synthase kinase 3β and FH transcription factors, as well as the up-regulation of several pro-apoptotic gene products [81].

Conclusions

There is little doubt that Akt is a crucial intermediary in many intracellular signaling pathways initiated by diverse extracellular stimuli acting at several classes of membrane-bound receptors. Recent years have produced a growing body of evidence that clearly establishes GPCRs as key initiators of the modulation of Akt-dependent signaling events. Furthermore, the regulation of Akt-dependent cellular proliferation and cellular survival in human cancer cells by numerous GPCRs has opened up the possibility of controlling cellular events through the use of ligands for a variety of receptors. The investigation of the applicability of such an approach for therapeutic benefit is in its infancy but, as we have described, progress has been made with efforts to control growth and trigger apoptosis in human cancer cells by targeting heterotrimeric G proteins and individual GPCRs [79,80]. However, experimental data obtained from transgenic and knockout mice dictates that a cautious approach to targeting Akt activity will be necessary. Mice deficient in Akt2 display insulin resistance and type-II diabetes-like syndrome, while both Akt1 and Akt2 are required for platelet activation [8]. Encouragingly, expression of a dominant negative, kinase dead mutant of Akt using an adenoviral vector selectively induced apoptosis in tumor cells with elevated levels of Akt activity but not in normal cells [82]. This suggests that unlike normal cells, tumor cells are dependent on increased Akt activity for survival, indicating that short-term inhibition of Akt signaling may not be toxic to normal cells.

As with many aspects of cellular signaling, much remains to be uncovered in order to deepen our understanding of how individual GPCRs functionally interact with different G proteins to initiate a cascade of events leading to the activation of different Akt subtypes, which in turn trigger a multitude of downstream pathways. Many of these GPCR-initiated events are likely to be cell-type specific and modulated by the actions of a host of other extracellular and intracellular cues that must ultimately be integrated to achieve the required biochemical/physiological outcomes.

Acknowledgements

This work was supported, in part, by grants from the Research Grants Council of Hong Kong (HKUST 3/03C, HKUST 6443/06 m), the University Grants Committee (AoE/B-15/01), and the Hong Kong Jockey Club. YHW was a recipient of the Croucher Senior Research Fellowship.

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