Seven transmembrane receptors, also termed G protein-coupled receptors (GPCR), form the largest class of the cell surface membrane receptors, involving 850 members in the human genome. GPCR are generally expressed in several different tissues in the same individual and involved in numerous physiological processes, including in the neuroendocrine system by playing a pivotal role in the control of feeding behavior, reproduction, growth, hydromineral homeostasis and stress response. At the cellular level, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways which are dependent or independent of G protein coupling (Rajagopal et al., 2010a).

Near 30% of therapeutic agents on the pharmaceutical market target GPCR (Hopkins and Groom, 2002). Whereas the majority of the GPCR-targeting drugs have proved their therapeutic benefit, some of them were associated with undesired effects (Table 1). Around 70% of the drugs which target GPCR are derived from the natural ligand and the use of agonist mimetics in clinical indication can act on the different tissues expressing the targeted GPCR and potentially induce undesired effects. Notably, prolonged treatment with GPCR-targeting agonist analogs was shown to induce preneoplastic and tumoral side effects. Here, we develop two examples of the use of GPCR-targeting drugs whose therapeutic benefits are tarnished by carcinogenesis risks. As a first example, glucagon-like peptide-1 receptor (GLP-1R) agonists used as anti-diabetic treatment were shown to induce preneoplastic lesions and/or cancers in the pancreas and the thyroid. The second example of ligands that we chose to develop does not initially target a GPCR, but the unexpected undesired effect is associated with a new GPCR target. Indeed, nuclear estrogen receptor antagonists such as tamoxifen are a breakthrough in the therapy and the prevention of breast cancer; however, long-term treatment was shown to be associated with an increased risk in endometrial cancer which was explained by the tamoxifen-induced activation of a GPCR, named G protein-coupled estrogen receptor (GPER).

We point out and discuss the need of more pharmacological studies to understand and overcome the undesired effects associated with the chronic administration of ligands which target GPCR. Deciphering the signaling networks engaged following GPCR activation appears to be primordial to unveil their contribution in the physiological and physiopathological processes.

One of the main physiological roles of GLP-1 is to enhance insulin secretion in a glucose-dependent manner. Thus, GLP-1 is an incretin hormone released after meals by L cells in the intestine (Figure 1) (Mojsov et al., 1987). GLP-1 exerts its physiological effects through binding to its specific G protein-coupled receptor, GLP-1R, which is primarily and positively coupled to adenylate cyclase, through Gαs-containing heterotrimeric G proteins, leading to the activation of second messenger pathways such as protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factor II (cAMP-GEFII, also known as Epac2) signaling pathways (Figure 2) (Thorens, 1992; Kashima et al., 2001; Mayo et al., 2003; Holz, 2004; Seino and Shibasaki, 2005; Doyle and Egan, 2007; Holst, 2007). In addition to its stimulatory effect on insulin secretion, GLP-1 suppresses the secretion of glucagon, a counter-hormone to insulin, thus maintaining glucose homeostasis following a meal (Nauck et al., 1993). GLP-1 plays also a key role in the homeostasis of β-cell mass by inducing β-cell proliferation and protecting against apoptosis which favor an expansion of β-cell mass (Figure 2) (Doyle and Egan, 2007). These functions are mediated via the activation of the cAMP/PKA/CREB (cAMP-responsive element binding protein) and the transactivation of the EGF-R (epidermal growth factor receptor) leading to the activation of phosphatidylinositol-3 kinase (PI3K), Protein Kinase Cζ (PKCζ), Akt-protein kinase B, Extracellular Regulated Kinase (ERK1/2) signaling pathways and to the up-regulation of the expression of the cell cycle regulator cyclin D1 (Buteau et al., 2003; Drucker, 2003; Trumper et al., 2005; Park et al., 2006; Doyle and Egan, 2007). The antiapoptotic effect of GLP-1 in β-cells also involves β-arrestin1 recruitment by GLP-1R which mediates the ERK1/2 activation leading to the phosphorylation and inactivation of the pro-apoptotic protein Bad (Quoyer et al., 2010). The properties of GLP-1 on insulin secretion and β-cell proliferation make GLP-1 one of the most promising therapeutic agent to treat type-2 diabetes. Moreover, GLP-1 analogs offer the advantage of improved glycemic control of type-2 diabetic patients, without inducing severe hypoglycemia (Phillips and Prins, 2011).

On the other hand, GLP-1 receptor activation directly promotes cell proliferation and enhances cell survival in several tissues including neurons, fibroblasts, and cardiomyocytes (Brubaker and Drucker, 2004).

Estrogen hormone regulates the growth and the differentiation of many tissues playing a critical role in the development of the reproductive system but also in the nervous, immune, vascular, muscular, skeletal, and endocrine systems. The binding of 17β-estradiol, the natural endogenous estrogen, to the estrogen receptors ERα and ERβ (ER) is the main mechanism responsible for the diverse biological effects of the hormone (Pedram et al., 2006; Meyer and Barton, 2009; Meyer et al., 2009). These highly homologous receptors can shuttle between the cytoplasm and the nucleus and function as ligand-activated nuclear transcription factors that bind cis-acting estrogen response elements in the promoter and enhancer regions of hormonally regulated genes (genomic effects of estrogen) (Figure 3) (Ring and Dowsett, 2004; Edwards, 2005; Carroll and Brown, 2006). Estrogen also induces some rapid biochemical responses to estrogen stimulation which occur in seconds to minutes, such as the increase in intracellular free calcium and the activation of multiple intracellular kinases including ERK, PI3K, PKA, and PKC (non-genomic effects of estrogen) (Chen et al., 2008).

Estrogen is the one of the risk factors for breast tumors, which accounts for 40% of cancer among the women and approximately 50% of all breast cancers demonstrated elevated levels of ER expression (Pike et al., 2004). Consequently, anti-estrogen therapy has been extended such as the gold standard tamoxifen (Figure 3) (Deroo and Korach, 2006; Lorand et al., 2010). Unfortunately, long-term treatment with tamoxifen is associated with adverse effects such as an increased incidence of endometrial cancer and with breast cancer resistance to hormonotherapy. Moreover, these events were shown to be associated with G protein signaling- or growth factor-mediated pathways which were not blocked by tamoxifen antagonist, leading to the prediction that an alternative membrane-bound estrogen receptor exists (Wehling, 1997; Hammes and Levin, 2007; Meyer and Barton, 2009). In fact, an orphan GPCR was identified as an estrogen-binding membrane GPCR from vascular and cancer cells and is now included in the official GPCR nomenclature and was designated GPER or GPR30 by the International Union of Pharmacology (Revankar et al., 2005; Thomas et al., 2005; Prossnitz et al., 2008a; Alexander et al., 2011). Its localization seems to be predominantly intracellular due to the constitutive internalization of plasma membrane GPER (Figure 3) (Revankar et al., 2005; Otto et al., 2008; Cheng et al., 2011; Sanden et al., 2011). GPER is widely expressed in cancer cell lines and primary tumors of the breast (Carmeci et al., 1997; Filardo et al., 2000; Revankar et al., 2005; Albanito et al., 2008a), endometrium (Vivacqua et al., 2006a; Leblanc et al., 2007; He et al., 2009), ovaries (Albanito et al., 2007, 2008b; Henic et al., 2009), thyroid (Vivacqua et al., 2006b), lung (Siegfried et al., 2009), prostate (Chan et al., 2010), and testicular germ cells (Franco et al., 2011). GPER does not only bind estrogens but also other substances such as tamoxifen which displays estrogenic agonist activity on GPER notably in the reproductive systems (Figure 3) (McDonnell, 1999; Filardo et al., 2000; Thomas and Dong, 2006; Jordan, 2007; Albanito et al., 2008b; Orlando et al., 2010; Chevalier et al., 2012). Indeed, tamoxifen stimulates the cell proliferation and growth of cell lines of thyroid, ovarian, endometrial, and breast cancers (Filardo et al., 2000; Thomas et al., 2005; Vivacqua et al., 2006a; Albanito et al., 2007; Prossnitz et al., 2008b; Pandey et al., 2009). The discovery of GPER-selective agents and the elaboration of GPER knockout mice helped to examine GPER signaling pathways and strongly supported that GPER is associated with cancer proliferation, migration, invasion, metastasis, differentiation, prognosis, and drug resistance (Prossnitz et al., 2008b; Wang et al., 2010).

G protein-coupled receptors are generally expressed in several different tissues and involved in numerous physiological processes. Many natural ligands can bind and activate several subtypes of GPCR. This is illustrated, for example, with cholecystokinin and somatostatin receptors (Guillermet-Guibert et al., 2005; Dufresne et al., 2006). Ligands can also activate different classes of receptors as illustrated before with estrogen (Prossnitz et al., 2008a). Such a diversity of receptors activation following agonist administration can engage multiple intracellular signaling pathways and be responsible for adverse effects in treated patients. Furthermore, biological effects triggered by the same GPCR result from the activation of G protein-dependent and -independent intracellular signaling pathways. Recently, signaling engaged after GPCR recruitment of β-arrestin proteins have emerged as new G protein-independent intracellular signaling pathways (Luttrell and Gesty-Palmer, 2010; Rajagopal et al., 2010a). To increase the complexity, a single GPCR has pleiotropic signaling properties and each signal can crosstalk at different levels with the transactivation of cell surface receptor having tyrosine kinase activity (EGF-R, PDGF-R, FGF-R, for examples) or serine/threonine kinase activity (TGF-β, for example) or with the formation of multimers, thus potentially influencing the signaling pathways of the different receptors (Burch et al., 2012; Wang and Lewis, 2013). Indeed, numerous biochemical and biophysical studies supports that GPCRs can form physiologically relevant homo-, hetero-, or oligo-mers (Angers et al., 2002). Homodimerization of the GLP-1R was shown to be critical for selective coupling of the receptor to physiologically relevant signaling pathways (Harikumar et al., 2012). Indeed, disruption of GLP-1R homodimerization completely abrogated the intracellular calcium mobilization response whereas it slightly reduced cAMP formation and phosphorylation of ERK. Furthermore, GLP-1R dimerization can discriminate between peptide and non-peptide-mediated receptor activation. In the chemokine receptors family, antibodies against the CCR2b promoted the receptor dimerization and second messenger production (Rodriguez-Frade et al., 1999), whereas an antibody directed against CCR5, that induces receptor dimerization, inhibits its function (Vila-Coro et al., 2000). Many studies was also conducted to analyze the role of GPCR heterodimerization and supported that heterodimerization could be the source of additional pharmacological properties which are different from those of the individual receptors. As a first example, the co-expression of the δ- and κ-opioid receptors in the same cell leads to an almost complete loss of binding to selective δ- and κ-ligands while preserving binding to non-selective ligands (Jordan and Devi, 1999). As a second example, somatostatin receptor SSTR1 displays internalization in cells when it is co-expressed with SSTR5, whereas monomeric SSTR1 is resistant to internalization in contrast to monomeric SSTR5, suggesting that the SSTR1 trafficking is modified by its heterodimerization with SSTR5 (Rocheville et al., 2000). Thus, homo- and heterodimerization between GPCR cause complexity in the receptor pharmacological properties that can be responsible of synergistic or antagonistic signaling cross-talks. This GPCR pharmacological and signaling complexity could account for unexpected pharmacological effects and have dramatic impacts on drug development. All together, these hallmarks indicate that undesired adverse effects can be expected with a prolonged agonist administration that targets a GPCR (Table 1). Moreover, many GPCR have already been shown to present proliferative and pro-tumoral properties (Table 2), suggesting that an increase of preneoplastic lesions and cancer incidence can potentially occur following chronic activation of GPCR. Thus, deciphering which signaling networks are engaged and orchestrated following GPCR receptor activation appears to be primordial to unveil their contribution in the cell fate.

One strategy to overcome these limitations would be to examine the initial steps following receptor activation. The release of X-ray structures of agonist/GPCR complexes (Chung et al., 2011; Lebon et al., 2011; Warne et al., 2011; Xu et al., 2011; Audet and Bouvier, 2012), the numerous biophysical and biochemical studies (Granier et al., 2007; Kahsai et al., 2011; Liu et al., 2012; Rahmeh et al., 2012) have enabled to show that different and selective ligands, named biased ligands, can induce or stabilize distinct receptor conformations and activate one (or several) signaling pathway(s) in contrast to non-biased agonists which activate all the signaling pathways (Vaidehi and Kenakin, 2010). Thus an understanding of the structure and dynamics of the ensemble of receptor conformations would greatly help the design of small molecules with functional selectivity or “biased signaling” properties and would provide more specific and efficient new drugs. Receptor structure/activity relationship studies, structure- and docking-based virtual screening are now widely applied in drug discovery and must take in account the existence of different receptor conformations activating specific signaling pathways.

Although GPCRs can modulate a large variety of distinct signaling pathways, classification of biased ligands are actually restricted to two groups depending on their ability to activate two main transduction pathways (Whalen et al., 2011): (1) G protein-biased ligands which promote G protein activation without β-arrestin recruitment and (2) β-arrestin-biased ligands which recruit β-arrestin to the receptor and initiate consecutive signaling pathways in the absence of G protein activation.

The vast majority of biased ligands identified so far exhibits exclusive β-arrestin activity for a number of receptors (Rajagopal et al., 2010a; Whalen et al., 2011), including the AT1 angiotensin II receptor, β1- and β2-adrenergic receptors, or the CXCR7 decoy receptor (Wei et al., 2003; Wisler et al., 2007; Kim et al., 2008; Rajagopal et al., 2010b). The parathyroid hormone (PTH) analog, D-Trp(12),Tyr(34)-PTH(7-34), binds the PTH receptor 1 (PTHR1) and activates β-arrestin-dependent but not classical G protein-dependent signaling (Gesty-Palmer et al., 2009; Gesty-Palmer and Luttrell, 2011). In mice, this PTH biased agonist induces anabolic bone formation without stimulating bone resorption, comparatively with the non-selective agonist PTH(1-34) which induces both functions. Thus, this PTHR1 biased ligand may present interesting properties for the treatment of metabolic bone diseases such as osteoporosis and is a proof of concept that the exploitation of β-arrestin biased agonism may offer therapeutic benefit.

Few ligands have been yet identified as perfect G protein-biased ligands, namely inducing G protein signal transduction without any β-arrestin recruitment (Whalen et al., 2011). GMME1 ligand binding to the CCR2 chemokine receptor leads to calcium mobilization, caspase-3 activation and consecutive cell death, but does not recruit β-arrestin2 (Rafei et al., 2009). Selective ligands that activate G protein-coupling by FSH-R (follicle-stimulating hormone receptor) and PTH-1R have been also reported (Bisello et al., 2002; Wehbi et al., 2010). Of note, some ligands classified as G protein-biased can induce a weak β-arrestin recruitment by the targeted GPCR (Whalen et al., 2011). For example, oxyntomodulin and glucagon are full agonists in GLP-1R-mediated cAMP accumulation but partial agonists in recruiting β-arrestins to this receptor, suggesting that oxyntomodulin and glucagon are biased ligands on the GLP-1R (Jorgensen et al., 2007).

Interestingly, some ligands are biased in regard to the different G protein families and can trigger opposite cellular responses (Reversi et al., 2005; Sensken et al., 2008). For example, oxytocin receptors (OTR) coupling to Gi inhibits cell proliferation, whereas its coupling to Gq stimulates cell proliferation. Atosiban, an oxytocin derivative, was shown to act as a competitive antagonist on OTR/Gq coupling, and to display agonistic properties on OTR/Gi-coupling, thereby leading to the selective inhibition of cell growth (Reversi et al., 2005; Busnelli et al., 2012). SOM230 which activates the somatostatin receptor sst2A behaves as agonist for Gi coupling and inhibition of adenylyl cyclase, but antagonizes somatostatin’s actions on intracellular calcium and ERK phosphorylation which can be activated by a Gi/Go independent process (Cescato et al., 2010).

Biased signaling can also exist with respect to other signaling proteins than G proteins and arrestins. The internalization of apelin receptor takes different signaling pathways depending of the apelin isoforms (Lee et al., 2010). Indeed, apelin-13-activated receptors dissociated rapidly from β-arrestin1 and were recycled to the cell surface through a Rab4-dependent mechanism, while the apelin-36-internalized receptors trafficked with β-arrestin1 to intracellular compartments and were targeted by Rab7 to lysosomes for degradation. CCL19 and CCL21 ligands both induce β-arrestin2 recruitment by the receptor CCR7, but activate different GRK (G protein receptor kinase) isoforms (Zidar et al., 2009). Indeed, CCL19 leads to robust CCR7 phosphorylation and β-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. The functional consequences are that only CCL19 leads to classical receptor desensitization whereas both agonists are capable of signaling through GRK6 and β-arrestin2 to ERK kinases.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.