**REVIEW ARTICLE** published: 17 January 2013 doi: 10.3389/fendo.2012.00184

# Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy

# *ZheruiWu1, Daniel Martinez-Fong2, Jean Trédaniel 1,3 and Patricia Forgez1\**

1 INSERM-UPMC UMR\_S938, Hôpital Saint-Antoine, Paris, France

2 Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional,

Mexico City, Mexico

3 Unité de Cancérologie Thoracique, Groupe Hospitalier Paris Saint-Joseph/Université Paris Descartes, Paris, France

#### *Edited by:*

Jae Young Seong, Korea University, South Korea

#### *Reviewed by:*

Jean Mazella, Centre National de la Recherche Scientifique, France Aixa R. Bello, University of La Laguna, Spain

#### *\*Correspondence:*

Patricia Forgez, INSERM-UPMC UMR\_S938, Hôpital Saint-Antoine, Bâtiment Raoul Kourilsky, 184 rue du Faubourg St-Antoine, 75571 Paris Cedex 12, France. e-mail<EMAIL_ADDRESS>
Cancer is a worldwide health problem. Personalized treatment represents a future advancement for cancer treatment, in part due to the development of targeted therapeutic drugs. These molecules are expected to be more effective than current treatments and less harmful to normal cells. The discovery and validation of new targets are the foundation and the source of these new therapies. The neurotensinergic system has been shown to enhance cancer progression in various cancers such as pancreatic, prostate, lung, breast, and colon cancer. It also triggers multiple oncogenic signaling pathways, such as the PKC/ERK and AKT pathways. In this review, we discuss the contribution of the neurotensinergic system to cancer progression, as well as the regulation and mechanisms of the system in order to highlight its potential as a therapeutic target, and its prospect for its use as a treatment in certain cancers.

**Keywords: cancer therapy, cancer progression, carcinogenesis, neurotensin, neurotensin receptor**

#### **INTRODUCTION**

Cancer is one of the first, if not the first cause, of death in the world. Conventional treatment methods include surgery, radiotherapy, and chemotherapy. In many cases, only a supportive treatment can be offered to the patient. The last few decades have been marked by the accumulation of knowledge about the inner workings of the normal and cancer cell. Thus arose the therapeutic arsenal against cancer, and the so-called targeted biological therapies. Due to these new drugs, great progress has recently been achieved in the treatment of cancers considered refractory to previous therapies, including cancers of the liver, kidney, and melanoma skin. But other tumors, such as breast and lung cancer, have also greatly benefited from these advances. However, remissions are often transient and do not yet provide definitive cures of the patient. In this context the neurotensin system is proposed to be a candidate for therapeutical development.

Neurotensin (NTS) is a tridecapeptide originally isolated from calf hypothalamus (Carraway and Leeman, 1973). It acts as neurotransmitter or neuromodulator in the central nervous system and as a local hormone in the periphery, mainly the gastrointestinal tract (Vincent et al., 1999). In the brain, NTS modulates dopaminergic transmission in the nigrostriatal and mesocorticolimbic pathways as well as hormone secretion from the anterior pituitary (Vincent et al., 1999). It also exerts potent hypothermic and analgesic effects when injected into the central nervous system (Popp et al., 2007). NTS has also been related to central nervous system pathology such as Parkinson disease and schizophrenia (St-Gelais et al., 2006; Mustain et al., 2011). In the periphery, NTS is released by endocrine-like N cells predominantly in the small intestine (Reinecke, 1985). NTS has a dual function, as a paracrine and endocrine modulator of the digestive tract and as a growth factor on a variety of normal or cancer cells (Vincent et al., 1999; Carraway and Plona, 2006). The NTS/NTSR1 complex has been proposed to contribute to cancer progression because of the various oncogenic effects induced by NTS in tumors and in cancer cells from diverse origins (Thomas et al., 2003; Dupouy et al., 2011). In this review, we elaborate on how the NTS/NTSR1 complex could be developed as a possible target for cancer therapy.

#### **NEUROTENSIN/NEUROTENSIN RECEPTOR COMPLEX NEUROTENSINERGIC SYSTEM**

The biological effects of NTS are known to be mediated through three receptors, two G protein-coupled receptors, NTSR1 and NTSR2, and a single transmembrane domain sorting receptor, the NTSR3 (Vincent et al., 1999; Mazella and Vincent, 2006). NTSR3 is a member of the receptor family related to the yeast sorting receptor Vps10p (Petersen et al., 1997).

#### *Neurotensin receptor 1, NTSR1*

"fendo-03-00184" — 2013/1/12 — 17:45 — page 1 — #1

The effects of NTS are primarily transmitted through its high affinity receptor, the NTSR1 which has a sub-nanomolar affinity for NTS. This 424 amino acid receptor has been identified in the brain and in various cancer cells (Tanaka et al., 1990). The signaling pathways induced by the NTS/NTSR1 complex have been studied in different cellular types, such as N1E-115, HT-29, and NTSR1-transfected CHO overexpressing NTSR1. The stimulation of NTSR1 by its ligand (NTS) leads to activation of phospholipase C (PLC) via its coupling to the prime Gαq/11 subunit (Wang and Wu, 1996; Najimi et al., 2002). The activation of PLC leads to the production of inositol triphosphate (IP3) and diacylglycerol from membrane phospholipids (PIP2). These two second messengers induce the activation of PKC and the mobilization of intracellular calcium which are key oncogenic effectors (Bozou et al., 1986; Snider et al., 1986; Turner et al., 1990).

Several signaling pathways potentially involved in cell proliferation, survival, migration, and invasion are described after NTSR1 stimulation. The signaling mechanisms mediating the effects of neurotensinergic system involve multiple pathways and are cell-dependent. The NTS-induced PKC/ERK signaling pathway is the most well studied. The PKC activation stimulated by NTS/NTSR1 was demonstrated by using broad isotype inhibitors, such as Gö6976 which specifically inhibit the conventional PKCs α and β1 in CHO-NTSR1-transfected cells and in endogenously NTSR1-expressing cells from colon, lung, and pancreatic cells (Poinot-Chazel et al., 1996; Seufferlein and Rozengurt, 1996; Ehlers et al., 2000; Muller et al., 2011). The NTSR1 contribution in this activation was confirmed by its inhibition by a specific NTSR1 antagonist, the SR 489692 (Gully et al., 1993). PKC activation induce mitogen-activated protein kinase (MAPK) via direct stimulation of Raf-1, independently of Ras, or by transactivation of the epidermal growth factor receptor (EGFR) (Guha et al., 2003). EGFR transactivation by NTS/NTSR1 complex has been observed in several cell lines. In prostatic cancer PC3 cell line, NTS activates mitogenesis through EGFR transactivation in a PKC-dependent pathway, and the stimulation of the Raf–MEK– ERK. This effect was also PI3 kinase (PI3K)-dependent (Hassan et al., 2004). NTS also induces a time-dependent increase in Tyr845 EGFR phosphorylation, c-Src phosphorylation and signal transducer. NTS is also an activator of transcription 5b (Stat5b), a downstream effector of Tyr845 EGFR phosphorylated (Amorino et al., 2007). In colonic HT-29 cells, the EGFR tyrosine kinase inhibitor, gefitinib, blocks NTS-stimulated phosphorylation of both MAPK and Akt, indicating the transactivation of EGFR independently of PKC activation. However, in the colonic HCT116 cells, NTS/NTSR1 induces a PKC-dependent MAPK phosphorylation and an EGFR metalloproteinase-mediated transactivation, that is associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt is only partly inhibited by gefitinib, suggesting an additional mechanism to EGFR transactivation (Muller et al., 2011). The mechanism of NTS-induced EGFR transactivation is still not clearly elucidated. The release of EGFR ligands-like (TGF-α, Hb-EGF, or amphiregulin), pre-existing at the plasma membrane, as proligand, by NTS has been proposed. These ligands are released by proteolytic cleavage involving enzymes of the metalloproteinase family, including ADAMS (disintegrin and metalloprotease) and MMP (matrix metalloproteinase; Sanderson et al., 2006; Zhao et al., 2007; Kataoka, 2009). Once released, these ligands bind to EGFR and activate the downstream signaling cascades of EGFR activation (Hassan et al., 2004). These results suggest a cooperative relationship between the neurotensinergic system and EGFR pathway.

Activation of MAPK pathway by NTS results in gene transcription stimulation, due to transcription factor activation, such as the induction of the early growth response gene-1 (Egr-1), the Ets family factors ELK1, and the AP-1 transcription factor family (Poinot-Chazel et al., 1996; Portier et al., 1998; Ehlers et al., 2000; Zhao et al., 2007). In colonic HCT166 cells, inhibition of PKC was shown to block NTS-induced DNA synthesis (Muller et al., 2011). In a tumor-initiating cell line derived from hepatocellular carcinoma (HCC) which is characterized by membrane expression of

CD133, addition of exogenous NTS resulted in concomitant upregulation of IL-8 and CXCL1 with simultaneous activation of MAPK and Raf-1, and promotion of angiogenesis, tumorigenesis, and self-renewal (Tang et al., 2012). The activation of MAPK via NTSR1 is mainly associated with uncontrolled cell growth, which can aggravate the growth of tumors (Harikumar et al., 2010; Kisfalvi et al., 2010).

Neurotensin also induces RhoGTPases and the non-receptor kinases focal adhesion kinase (FAK) and Src. Neurotensinergic system stimulation can modulate the activity of small RhoGT-Pases Rac1, Cdc42, and RhoA, which are partly responsible for the dynamics of the cytoskeleton. This modulation has an effect on cell migration. In the cell line U373 glioblastoma, the NTS has been associated with the stimulation of the activity of protein Rac1, RhoA, and Cdc42 (Zhao et al., 2003; Servotte et al., 2006). In addition, in small cell lung and prostate cancer cell lines, it has been shown that NTS can enhance the activity of focal adhesion kinase (FAK) (Tallett et al., 1996; Lee et al., 2001).

# *Neurotensin receptor 2, NTSR2*

The low affinity receptor of NTS, NTSR2, is a protein of 410 amino acids, with a high homology to NTSR1 (64%) (Chalon et al., 1996). NTSR2 exhibits a low binding property for NTS and this binding can be inhibited by levocabastine, a non-peptide histamine H1 receptor antagonist (Schotte et al., 1986). SR48692, which has a lower affinity for NTSR2 than for NTSR1, can stimulate the activity of this receptor (Yamada et al., 1998). When the cloned mouse NTSR2 coding sequence is expressed in *Xenopus laevis* ovocytes, NTS, neuromedin N, levocabastine, and SR48692, are capable of triggering an inward current which is calcium-dependent (Mazella et al., 1996). Using CHO cells transfected with the cloned rat or human NTSR2 cDNA, levocabastine and SR 48692 can mobilize intracellular Ca2+ more intensively than NTS agonists and phosphorylate Erk1/2, suggesting that NTSR1 and NTSR2 receptors present distinct functional characteristics (Botto et al., 1997; Yamada et al., 1998; Gendron et al., 2004). In CHO cells transfected with human NTSR2 cDNA, both NTSR1 antagonists, SR48692 and SR142948A, enhance inositol phosphate (IP) formation with subsequent [Ca2+] immobilization, induce arachidonic acid release, and stimulate MAPK activity. Interestingly, these activities were inhibited by NTS and levocabastine in a dose-dependent manner. In summary, the signaling pathway triggered by NTSR2 is cell-dependent, and mainly based on its overexpression. This response is far different from that of the physiological endogenous expression.

# *Neurotensin receptor 3, gp85/sortilin, NTSR3*

"fendo-03-00184" — 2013/1/12 — 17:45 — page 2 — #2

NTSR3 functions as a modulator of neurotensinergic signaling when it is co-expressed with another receptor of NTS, and as a functional receptor involved in the migration when expressed alone. This receptor is not NTS-specific. It can bind other ligands such as lipoprotein lipase, proneurotrophins, protein RAP (receptor-associated protein), or protein SAP (sphingolipid activator protein) (Nielsen et al., 1999; Lefrancois et al., 2003). NTSR3 may act as a co-receptor to participate in true NTS/NTSR1 signaling. The study by immunoprecipitation using the adenocarcinoma cell line HT29, demonstrated that the NTSR3 forms heterodimers with the NTSR1. Additionally, upon NTS stimulation, the NTSR1/NTSR3 complex is internalized and the interaction between the two receptors modulates both the NTSinduced phosphorylation of MAPK and the phosphoinositide (PI) turnover mediated by NTSR1 (Martin et al., 2002).

In the human microglial cell line C13NJ, NTSR3 is the only known endogenous NTS receptor. In these cells, NTS elicited cell migration by a mechanism dependent on both PI3K and MAPK pathways (Martin et al., 2003). The NTS/NTSR3 complex has been shown to phosphorylate both Erk1/2 and Akt kinases in a murine microglial cell line (Dicou, 2008).

# **NEUROTENSIN/NEUROTENSIN RECEPTOR COMPLEX AND CANCER BIOLOGY**

Few years after its discovery, high-level expression of NTS was found in the plasma of pancreatic tumor patients (Gutniak et al., 1980). This discovery inspired investigations on the relationship between NTS and cancer. Many studies have since been performed to clarify the role of NTS in carcinogenesis in diverse cancer cells.

# **PANCREATIC CANCER**

Pancreatic cancer is the eighth leading cause of cancer death in the world (Yabushita et al., 2012). It has the poorest prognosis amongst all human malignant solid tumors, mainly due to its high rate of metastasis (Cheng et al., 2012). The growth promoting action of NTS has been observed in pancreatic cancer cell lines both *in vitro* and *in vivo*. In both cases, the NTSR1 antagonist, SR48692, inhibited the NTS-induced effects (Sumi et al., 1993; Iwase et al., 1997). Recently, NTS was shown to protect insulin producing cells (b-TC3, INS-1E) against apoptosis induced by IL-1b and staurosporine (STS) (Coppola et al., 2008). NTSR2 and NTSR3 have been shown to be essential for the NTS mediated survival of these cells (Beraud-Dufour et al., 2009). NTS also influenced the migratory ability of pancreatic cancer cells, while NTS significantly reduced the migration levels of collectively migrating cells on vitronectin, NTS significantly increased the levels of individually migrating cells (Mijatovic et al., 2007). Thus, NTS-induced migration is dependent on the extracellular matrix environment and their respective migratory mode.

# **COLORECTAL CANCER**

Colorectal cancer is the third most common cancer worldwide and the fourth most common cause of death (Jemal et al., 2011). NTS stimulates the growth of mouse and human colon cancer cell lines in tissue culture and after being xenografted into nude mice (Maoret et al., 1999). *In vivo*, systemic NTS administration stimulates tumor size and weight, DNA, RNA, and the protein contents of the murine colon cancer, MC26 (Yoshinaga et al., 1992). Recently, the expression of NTSR1 was significantly correlated to an increase in the number of tumors when sporadic cancer was generated in mouse models by inflammation. However, no effect of NTSR1 expression was noticed on the number of aberrant crypt foci or tumor size, suggesting that the NTS/NTSR1 signaling complex has a major role in tumor progression (Bugni et al., 2012). NTS is also known to enhance colon cancer cell migration by increasing IL-8 expression and secretion. These effects are blocked by NTSR1 antagonists and curcumin, a diet-derived chemopreventive and/or chemotherapeutic agent which blocks AP-1 and NF-κB induction (Wang et al., 2006a). The role of IL-8 identified as an integral part of the metastasis process, was shown due to its triggering of the release of enzymes [MMPs, and uroplasminogen activator (uPA)], responsible for extracellular matrix degradation (Xie, 2001).

# **PROSTATE CANCER**

Prostate cancer is the most common male malignancy in Western countries and the second most common cause of male cancer-related death in the UK and USA (Jemal et al., 2011). The androgen-dependent human prostate cancer LNCaP cell line has been shown to exhibit an autocrine growth response to NTS in androgen-deprived only conditions (Sehgal et al., 1994). Vias et al. (2007) found that long-term anti-androgen treatment of LNCaP cells produced a sub-line exhibiting upregulated expression of NTS and NTS receptors, which increased the proliferation rate, accelerated cell cycle progression, and increased invasiveness through Matrigel. These effects are sensitive to NTS siRNA. NTSR1 expression was found at very high levels in the human androgen-independent PC3 cell line, derived from prostate cancer metastasized to bone. The growth responses of these cells to NTS were found at concentrations close to human postprandial blood levels (Seethalakshmi et al., 1997). These studies proposed that NTS is a potential autocrine, paracrine, and endocrine regulator of prostate cancer growth in humans, after androgen ablation therapy and during the devastating final stages of the disease.

# **LUNG CANCER**

Lung cancer is the most common cause of cancer-related deaths throughout the world (Jemal et al., 2011). High concentration of NTS is present in and secreted from half classic small cell lung cancer cells (SCLC; Moody et al., 1985). NTS is one of the 73 genes overexpressed in the highly metastatic human lung cell line, H460-M, as compared to control cells (de Lange et al., 2003). In lung cancer cell lines, NCI-H209 and H345, SR48692 inhibited NTS-mediated calcium mobilization cells and c-fos mRNA induction and proliferation in a dose-dependent manner (Moody et al., 1985). SR48692 also inhibited tumor growth of NCI-H209 xenografts.

#### **BREAST CANCER**

"fendo-03-00184" — 2013/1/12 — 17:45 — page 3 — #3

Breast cancer is the most common cancer in women, and the second leading cause of cancer deaths in women worldwide (Jemal et al., 2011). NTS immunoreactivity has been observed in breast cancers *in vivo*.  The NTS anti-apoptotic effect has been described in the cell line MCF-7 originating from breast adenocarcinoma. Prolonged exposure to JMV449, a NTS-specific agonist, protected MCF-7 cells from serum deprivation-induced death, and reduced the number of apoptotic cells by two to three times. These effects have been predicted to be due, in part, to NTS-mediated induction of Bcl-2 mRNA and protein levels which depends on stimulation of MAPK (Somaï et al., 2002). By using NTSR1 Sh-RNA and SR 48692, tumor growth was significantly decreased when NTSR1 expression

was abolished or blocked in experimental tumors of the breast (Souaze et al., 2006a).

# **REGULATION OF THE NEUROTENSINERGIC SYSTEM REGULATION OF NTS**

Neurotensin deregulation has been observed in many cancers such as in colonic adenocarcinomas, small cell lung carcinomas, nonsmall cell lung adenocarcinomas, medullary thyroid carcinomas, and in fibrolamellar HCCs (Baca and Schmidt-Gayk, 1981; Ulich et al., 1983; Moody et al., 1985; Dammrich et al., 1988; Ehrenfried et al., 1994). DNA methylation has been shown to play a crucial role in the expression of the gut endocrine gene neurotensin/neuromedin N (NT/N). In the human hepatoma cell line Hep G2 cells, methylation of a NT/N promoter construct resulted in a severe reduction of the promoter activity, whereas treatment with the demethylating agent 5-azacytidine-induced NT/N expression (Dong et al., 1998). These observations have been confirmed in different human colon cancer cell lines, either expressing or not the gene coding for NTS (Dong et al., 2000). Interestingly, NTS gene can be regulated by Ras while expressed. Both wild-type and activated Ras enhances expression of NTS in the gut-derived CaCo-2 cell line, by activating the proximalAP-1/CRE motif (Evers et al., 1995). More recently, the PI3K catalytic subunit, p110alfa, was demonstrated to negatively regulate NTS secretion *in vitro* and *in vivo*. This process involves several regulatory proteins such as α-tubulin deacetylase, small GTPase, and kinase D-interacting substrate (Li et al., 2012).

#### **REGULATION OF NTSR1**

The mechanisms involved in NTSR1 deregulation in cancer cells have been studied in the context of colorectal carcinogenesis. These studies implicate an important role of the Wnt/β-catenin pathway deregulation. The mutation or loss of the protein APC (adenomatous polyposis coli) causes a dysfunction in the degradation of β-catenin. The accumulation of the latter in the cytoplasm, and its subsequent translocation to the nucleus induces NTSR1 gene expression via its association with transcription factors Tcf/Lef (T cell factor/lymphoid enhancing factor) (Souaze et al., 2006b). The NTSR1 promoter can be activated by the complex β-catenin/Tcf because it contains a consensus site for the transcription factors Tcf. In agreement with this result, it has been demonstrated that inhibitors of GSK-3β (protein kinase involved in the phosphorylation of β-catenin and its degradation) which cause the significant accumulation of β-catenin, upregulates the level of NTSR1 transcription (Wang et al., 2010). Similar results have been obtained in other cancers such as lung, prostate, and breast cancers (Chesire et al., 2002; Turashvili et al., 2006).

#### **REGULATION OF NTSR1 BY ITS OWN LIGAND**

Upon acute agonist exposure, and under physiological conditions, initiated by β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2), the NTS/NTSR1 complex is internalized and degraded in lysosomes through clathrin-coated vesicles. Cell resensitization occurs from *de novo* receptor synthesis a few hours after agonist removal (Souaze and Forgez, 2006; Law et al., 2012). However, some studies on cellular models such as the murine neuroblastoma cell line N1E-115 and human colon cancer cell line HT-29, showed a change in the traffic situation when the cell had a prolonged exposure to saturating doses of agonist (Souaze et al., 1997; Najimi et al., 1998). Instead of being degraded in the lysosome, NTSR1 accumulated transiently with NTS in the perinuclear recycling compartment (PNRC) where it was latter recycled to the plasma membrane (Toy-Miou-Leong et al., 2004). More recent research has shown the activity of endothelin-converting enzyme-1 (ECE-1) and βARRs being crucial for NTSR1 recycling and enhance NTS degradation (Law et al., 2012). Thus, NTS stimulation induces cellular adaptation by altering the degradation process of NTSR1. This phenomenon leads to permanently sensitizing cells to the neurotensinergic signal. The implementation of this mechanism could lead to deregulation of multiple signaling pathways involved in the cancer progression such as MAPK and its target genes.

#### **NEUROTENSIN/NEUROTENSIN RECEPTORS AND THERAPY**

The implications of the previous sections suggest a more direct role for NTS/NTSR1 in cancer growth and progression, than has been previously attributed. Nevertheless, the ability to develop therapeutic strategies, around this complex, remain a challenge. Yet, despite them, the characteristics and qualities associated with this system should provide new pharmaceutical approaches as the system becomes further studied.

In the periphery and in the central nervous system, NTS mainly modulates the action of other molecules which are the principal effectors. Support for this view was confirmed by experiments with NTS- or NTSRs-deficient mice. These mice do not present any physiological disorder, are viable, and show normal growth and overt behavior. The deficient mice were also useful to implicate the NTS system in body temperature control (Pettibone et al., 2002), feeding regulation, weight control, and locomotion (Remaury et al., 2002). Mice were also less responsive or sensitive to exogenous agents or conditions. NTSR1 ko mice were less sensitive to the anorectic effects caused by leptin (Kim et al., 2008), and less responsive to morphine-induced analgesia (Roussy et al., 2010). Experiments with NTSR1- and NTSR2-deficient mice indicate a role in the regulation of ethanol consumption. However, NTSR1 regulates ethanol intoxication while NTSR2 is involved in ethanolinduced hypnosis (Lee et al., 2010, 2011). This suggests that the disruption of NTS system in the periphery is unlikely to generate side effects superior to the benefits expected in cancer treatment. On the other hand, the quasi exclusive alteration of the NTS system in tumors would confer an advantage in its use as a therapeutic target.

#### **BIOLOGICAL TOOLS USED TO INTERACT WITH THE NTS/NSTR SYSTEMS**

Few biological tools have been developed to specifically target NTS system. The two most studied ones are the NTSR1 antagonists, SR 48692, and SR142948A (Gully et al., 1996, 1997). These molecules were developed to counteract NTS effects in central and peripheral nervous system. For this purpose, both molecules are specific and efficient. These compounds have been extensively used to successfully counteract exogenous NTS oncogenic actions. Nevertheless, these molecules remain weak and problematic to use to counteract intense autocrine regulation. For example, at high concentrations, SR48692 is toxic for the cells, and as no significant information exists on the biodegradability of these molecules, the dosage levels

"fendo-03-00184" — 2013/1/12 — 17:45 — page 4 — #4

are unknown. In addition, SR 48692 acts as a low affinity agonist with NTSR2 (Yamada et al., 1998). NTRS2 expression also occurs in the gastric mucosa, in neuroendocrine cells of the stomach, and the small and large intestine, and in cells of the exocrine pancreas. NTSR2 is rarely detected in human-derived tumors (Schulz et al., 2006). Recently, NTSR2 was found overexpressed in B cell leukemia patient's cells (Saada et al., 2012), and upregulated in prostate cancer cells luminal phenotype (Swift et al., 2010). It will be difficult to estimate NTSR2 impact on tumorigenesis until more information on its expression in human tumors becomes available.

In contrast, NTSR3 exhibits an ubiquitous expression in normal and tumoral cells. Its contribution on neoplastic progression is not known. The exact impact of SR48692 on NTSR3 remains complex. The most advanced hypothesis is that SR 48692 inhibits the oncogenic effects through NTSR1, when NTSR1 is expressed and in conjunction or not with NTSR3. In this context it was shown that SR 48692 inhibited the tumor growth of colon, breast, pancreas, and small lung cancer cells xenografted in mice (Iwase et al., 1997; Maoret et al., 1999; Moody et al., 2001; Souaze et al., 2006a). SR 48692 counteracts exogenously treated NTS in pancreas cells MIA PaCa-2 cells, bearing high and low affinity sites (Iwase et al., 1997), or circulating NTS as in the case of SW480, MDA-MB 231, and NCI-H209 cells (Maoret et al., 1999; Moody et al., 2001; Souaze et al., 2006a). It has to be noted that the use of SR 48692 does not stop tumor growth, but delays the growth rate by two to three times. The creation of the experimental tumor in mice results in tumors having an abnormal ratio (up to 10×) between the tumor size and body size, an observation uncommon and not seen in human tumors. Summarizing the effects induced by NTS, at the onset of cancer, NTS acts on the progression rather than on cellular transformation. Those actions, mediated by NTS include proliferation, survival, and metastatic effects. It is, therefore, not surprising to observe that the abolition of NTS/NTSR1 expression or signaling enhances the effect of several anti-tumoral treatments. For example, NTSR1 inhibition efficiently sensitized prostate regulation in orthotopic human tumor xenografts in mice to radiotherapy by significantly reducing tumor size, and in prostate cancer cells bearing NTS autocrine regulation (Valerie et al., 2011).

Another approach employed in the development of specific biological tools, uses Sh-RNA or Si RNA targeting NSTR1. The total or partial abolition of the target results in a decrease in cellular invasion, migration in HNSCC (Shimizu et al., 2008), or tumor growth of the human breast cancer cells (Souaze et al., 2006a). The two experimental designs demonstrate a contribution of NTS in these oncologic processes, through its interaction with NTRS1. At present, more knowledge is needed on the distribution and the coupling of NTSR2 in tumoral cells, as well as on the pharmaceutical tools specifically inhibiting NTSR2, to determine if NTSR2 is implicated in any oncogenic process.

#### **TARGETED APPROACHES FOR NTS/NTSR1**

A recent approach under investigation is to use NTSR1-specific overexpression as a means to target the delivery of therapeutic or visualizing molecules in tumoral cells. This approach is made possible since NTSR1 endocytosis permits the introduction of molecules of interest inside the targeted cell and several strategies have been developed. In the first case, a non-viral gene transfer particle bearing six NTS molecules covalently bound to poly-lysine, enabled the transfer of a reporter and therapeutic gene to those tumor cells expressing NTSR1 when injected in the tumor or into the blood circulation of xenografted nude mice (Arango-Rodriguez et al., 2006). Interestingly, both injection sites showed a high proportion of transfected cells. In a second case, a therapeutic effect was also detected on experimental neuroblastoma tumors transfected with a thymidine kinase gene associated with ganciclovir. A severe reduction of tumor growth was also observed (Rubio-Zapata et al., 2009). In the same vein, a protein interfering with the cell synthesis machinery by inducing irreversible ADP-ribosylation of elongation factor 2, exotoxin A, was fused with NTS. This recombinant protein was able to specifically recognize NTSR1 positive cells and to exert a real *in vitro* cytotoxicity (Wick and Groner, 1997). In another strategy, oligo-branched peptides were used because of their proteolysis resistant nature. Tetra-branched NTS, armed with 5-FdU, increased *in vitro* and *in vivo* tumoral cell cytotoxicity as compared to thefree drug (Falciani et al., 2010a,b). The same idea was applied to carry the *cis*,*cis*,*trans*diamminedichloridodisuccinatoplatinum(IV)–neurotensin bioconjugate specifically to tumor cells. The cytotoxicity of this molecule was tested on cell proliferation and found efficient when compared to the non-targeted platinum (Gaviglio et al., 2012).

Neurotensin-based radiopharmaceuticals have also been developed for tumor localization and therapy. Several strategies using different NTSR1 ligands were constructed whose main objectives were to limit the degradation of the molecule while maintaining the ligand specificity and a good affinity for NTSRs. Thus, the analog NT-XIX, with the three enzymatic cleavage sites stabilized showed a high specificity for NTSR1. A low accumulation of activity in the kidneys and a proper tumor-to-tissue ratio radioactivity clearance was observed *in vivo*, as well as a decrease in xenograft tumor growth (Garcia-Garayoa et al., 2009). More recently, new NTS analogs, DOTA-NT-20.3, and DOTA-NT-20.4 showed promising characteristics for imaging of NTS receptor-positive tumors and therapy (Alshoukr et al., 2009, 2011).

#### **NTSR1/NTS COMPLEX EXPRESSION IN HUMAN TUMORS**

"fendo-03-00184" — 2013/1/12 — 17:45 — page 5 — #5

Several experiments have investigated the expression of NTSR1 in human tumors, but most of these studies remain too small or local. In normal colonic tissue, NTSR1 was not detected by immunohistochemistry, whereas in human colonic adenomas expression, NTSR1 was associated with cytoplasmic beta-catenin localization. This was one of the first suggestions that NTSR1 expression was an early event in colon carcinogenesis (Souaze et al., 2006b). NTSR1 mRNA expression studied by *in situ* hybridization showed a higher level of expression in adenocarcinoma as compared to adenomas. In addition, a higher intensity of NTSR1 expression was observed in filtrated adenocarcinomas into and beyond the muscularis propria as compared with tumors that were localized to the mucosa or submucosa, suggesting a contribution of NTS/NTSR1 complex in tumor expansion (Gui et al., 2008).

In pancreas, NTS binding sites were first studied by autoradiography, and found specifically in pancreatic cancer but not in normal pancreas and chronic pancreatitis. Nevertheless, northern blots showed NTSR1 mRNA in normal pancreatic cells, but an increase of transcript level in chronic pancreatitis and pancreatic cancer. Within the tumors, NTSR1 expression was higher in advanced stages as compared to early stages (Wang et al., 2000). NTSR1 and NTS protein expression was confirmed by immunohistochemistry in normal and tumoral tissue. In this latter case 80% expressed both NSTR1 and NTS (Wang et al., 2011).

More recently, a correlation between NTSR1 or NTS expression with outcome of the disease was evoked. In non-small lung cancer cells, NTS and NTSR1 expression were not detectable, whereas 60% of patients with stage 1 adenocarcinoma expressed either NTS or NTSR1 and 40% expressed both markers. Patients treated only by surgery did not receive adjuvant therapy and, in this cohort, NTRS1 expression was correlated with a worse prognosis of the disease (Alifano et al., 2010b). In contrast, NTS was found to be expressed in normal epithelial cells of breast tissue, whereas NTSR1 was absent. In breast invasive carcinomas high expression of NTSR1 was correlated with pejorative clinical parameters and disease outcome (Dupouy et al., 2009). In head and neck squamous cell carcinomas, NTS and NTSR1 mRNA high levels were significantly correlated with higher rates of distant metastasis as well as with the survival rate (Shimizu et al., 2008). In normal pleura, NTS and NTSR1 were found in 30 and 77%, respectively. In malignant pleural mesothelioma, expression increased to 71.1 and 90.4%, respectively. Interestingly in benign tissue, NTSR1 was located at the cell membrane whereas in tumoral cells, NTSR1 expression was granular and mainly restricted to the cytoplasm. The lack of presence at the cell surface suggested a state of permanent activation. In these cases, NTS high expression was associated with poor disease outcome (Alifano et al., 2010a).

# **CONCLUSION**

Cancer is more often regarded as a generalized disease and its treatment is mainly based on chemotherapy.  In these cases only a prolongation of survival associated with a better quality of life can be expected. Therefore, it is necessary to improve our strategies. Recent treatment developments include the customization and selection of drugs specifically directed against biological and genetic abnormalities expressed by the tumor. However, as with conventional chemotherapy, these treatments are still insufficiently targeted and are often accompanied by serious side effects.

Two potential approaches (amongst others) to improve this situation include the highlighting of abnormalities specific to cancer cells, or the delivery of specific drugs against these pathological processes. This latter avenue would allow for effective treatment without side effects or benefit from a metabolic pathway in which it would be possible to connect conventional medicines and transfer them inside the cancer cells.

The neurotensin and neurotensin 1 receptor system have provided insights for certain oncological situations. As seen above, NTS/NTSR1 is overactive in many tumors (**Figure 1**). The development of active antagonists, and those well tolerated by the patient, in human clinical trials will be a necessary step. NTS/NTSR1 may also be considered to become a carrier of chemotherapy drugs for tumor cells. In this regard, platinum salts, such as cisplatin, whose digestive and renal toxicity is well documented, are a prime target for the development of such tests. In all cases, the effectiveness of treatment can only be improved if the selection of patients is likely to benefit from such treatment. The last 45 years of work on the NTS/NTSR1 system have allowed for a better understanding of normal and cancer cells. This system can be further exploited for therapeutic purposes and for improving the management of cancer.

"fendo-03-00184" — 2013/1/12 — 17:45 — page 6 — #6

# **REFERENCES**

GTP-binding component of adenylate cyclase. *Mol. Pharmacol.* 29, 489–496.

B. M. (1998). DNA methylation contributes to expression of the human neurotensin/neuromedin N gene. *Am. J. Physiol.* 274, G535– G543.

"fendo-03-00184" — 2013/1/12 — 17:45 — page 7 — #7

trans-diamminedichloridodisuccinatoplatinum(IV)-peptide bioconjugates. *Metallomics* 4, 260–266.

Gully, D., et al. (1997). Inhibition of neurotensin-induced pancreatic carcinoma growth by a nonpeptide neurotensin receptor antagonist, SR48692. *Cancer* 79, 1787–1793.

et al. (2012). PI3K p110alpha/Akt signaling negatively regulates secretion of the intestinal peptide neurotensin through interference of granule transport. *Mol. Endocrinol.* 26, 1380–1393.

and pathologic processes. *Curr. Opin. Endocrinol. Diabetes Obes.* 18, 75–82.

"fendo-03-00184" — 2013/1/12 — 17:45 — page 8 — #8

and endocrine hormone. *Prog. Histochem. Cytochem.* 16, 1–172.

*Neuropathol. Appl. Neurobiol.* 32, 575–584.

pancreatic cancer, MIA PaCa-2. *Surg. Oncol.* 2, 267–272.

"fendo-03-00184" — 2013/1/12 — 17:45 — page 9 — #9

T., et al. (2012). Circulating microR-NAs in serum of human K-ras oncogene transgenic rats with pancreatic ductal adenocarcinomas. *Pancreas* 41, 1013–1018.

**Conflict of Interest Statement:** 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.

*Received: 23 October 2012; accepted: 26 December 2012; published online: 17 January 2013.*

*Citation: Wu Z, Martinez-Fong D, Trédaniel J and Forgez P (2013) Neurotensin and its high affinity receptor 1 as a potential pharmacological target in cancer therapy. Front. Endocrin. 3:184. doi: 10.3389/fendo.2012.00184*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2013 Wu, Martinez-Fong, Trédaniel and Forgez. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# *David Chatenet 1,2\*, Thi-Tuyet M. Nguyen1,2, Myriam Létourneau1,2 and Alain Fournier 1,2\**

1 Laboratoire d'études moléculaires et pharmacologiques des peptides, INRS – Institut Armand-Frappier, Université du Québec, Ville de Laval, QC, Canada 2 Laboratoire International Associé Samuel de Champlain (INSERM/INRS-Université de Rouen), France

#### *Edited by:*

Hubert Vaudry, University of Rouen, France

#### *Reviewed by:*

Stacia A. Sower, University of New Hampshire, USA Ishwar S. Parhar, Monash University, Malaysia

#### *\*Correspondence:*

David Chatenet and Alain Fournier, Laboratoire d'études moléculaires et pharmacologiques des peptides, INRS – Institut Armand-Frappier, Université du Québec, 531 Boulevard des Prairies, Ville de Laval, QC H7V 1B7, Canada. e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
The urotensinergic system plays central roles in the physiological regulation of major mammalian organ systems, including the cardiovascular system. As a matter of fact, this system has been linked to numerous pathophysiological states including atherosclerosis, heart failure, hypertension, diabetes as well as psychological, and neurological disorders. The delineation of the (patho)physiological roles of the urotensinergic system has been hampered by the absence of potent and selective antagonists for the urotensin II-receptor (UT). Thus, a more precise definition of the molecular functioning of the urotensinergic system, in normal conditions as well as in a pathological state is still critically needed. The recent discovery of nuclear UT within cardiomyocytes has highlighted the cellular complexity of this system and suggested that UT-associated biological responses are not only initiated at the cell surface but may result from the integration of extracellular and intracellular signaling pathways. Thus, such nuclear-localized receptors, regulating distinct signaling pathways, may represent new therapeutic targets. With the recent observation that urotensin II (UII) and urotensin II-related peptide (URP) exert different biological effects and the postulate that they could also have distinct pathophysiological roles in hypertension, it appears crucial to reassess the recognition process involving UII and URP with UT, and to push forward the development of new analogs of the UT system aimed at discriminating UII- and URP-mediated biological activities. The recent development of such compounds, i.e. urocontrin A and rUII(1–7), is certainly useful to decipher the specific roles of UII and URP in vitro and in vivo. Altogether, these studies, which provide important information regarding the pharmacology of the urotensinergic system and the conformational requirements for binding and activation, will ultimately lead to the development of potent and selective drugs.

**Keywords: urotensin II, urotensin II-related peptide, allosteric modulation, biased agonist, nuclear receptors**

#### **THE UROTENSINERGIC SYSTEM**

During the last decade, the urotensinergic system has drawn the attention of the scientific community due to its marked involvement in various pathological states including cardiovascular diseases. Initially isolated from the caudal neurosecretory system of the teleostean fish *Gillichthys mirabilis*, urotensin II (UII), a somatostatin-like peptide, was first characterized as a spasmogenic agent (Pearson et al., 1980). During more than 15 years, this peptide and its unknown receptors were thought to be restricted to fishes until it was demonstrated that UII was able to induce the relaxation of the mouse anococcygeus muscle (Gibson et al., 1984) and provoke the contraction of rat aortic strips (Gibson, 1987). These results, suggesting the presence of an homologous peptide in higher vertebrates, led to the isolation and characterization of UII in the frog *Rana ridibunda* (Conlon et al., 1992). Following this discovery, UII isoforms were either characterized or isolated in various vertebrate species including humans (Vaudry et al., 2010). A few years later, a peptide paralog, termed urotensin II-related peptide (URP), was isolated in rat brain extracts and subsequently identified in other mammalian species (Vaudry et al., 2010). Sequence comparison of all UII and URP isoforms revealed a striking conservation of the C-terminal cyclic hexapeptide (Vaudry et al., 2010). Conversely, the N-terminal region is highly variable both in length, ranging from 11 residues in humans to 17 residues in mice, and sequence composition (**Figure 1**) (Vaudry et al., 2010). In the human genome, UII and URP genes are respectively found at position 1p36 and 3q29 (Sugo et al., 2003). Those two genes are primarily expressed in motoneurons located in discrete brainstem nuclei and in the ventral horn of the spinal cord (Vaudry et al., 2010). However, UII and URP mRNAs have also been detected, although at a much lower level, in various peripheral tissues including the pituitary, heart, spleen, lung, liver, thymus, pancreas, kidney, small intestine, adrenal, and prostate (**Figure 2**) (Vaudry et al., 2010).

Both peptides are endogenous ligands of a G protein-coupled receptor initially identified as the orphan GPR14 receptor (Ames et al., 1999; Liu et al., 1999; Mori et al., 1999; Nothacker et al., 1999). Structural studies of this urotensin II receptor (UT) showed that, in addition to the common features found in the 1A GPCR family, such as the existence of a disulfide bridge between extracellular loops 1 and 2, N-linked glycosylation sites in the N-terminus portion, and phosphorylation sites in intracellular

**FIGURE 1 | Amino acid sequences of UII and URP in mammalian species;** *<***Gln, pyroglumatic acid.** Modified from Vaudry et al. (2010).

loops (Douglas et al., 2000), this protein also possesses a palmitoylation site located in the C-terminal segment of the rodent isoform that is not present in the human isoform (**Figure 3**). Worth to mention, the rat UT, consisting of 386 amino acids, shows only 75% homology with the human protein while sequences of human and monkey receptors, comprising 389 residues, are almost identical (Elshourbagy et al., 2002). Like UII and URP, UT is widely expressed in the central nervous system as well as

in various peripheral organs including the cardiovascular system, kidneys, bladder, pancreas, and adrenal gland (**Figure 2**) (Vaudry et al., 2010).

The urotensinergic system plays a seminal role in the physiological regulation of major mammalian organ systems, including the cardiovascular system (Vaudry et al., 2010). As a matter of fact, UII exerts potent haemodynamic effects (Krum and Kemp, 2007), positive inotropic and chronotropic responses (Watson et al., 2003), and osmoregulatory actions (Song et al., 2006), induces collagen and fibronectin accumulation (Dai et al., 2007; Zhang et al., 2008), modulates the inflammatory response (Shiraishi et al., 2008), plays a role in the induction of cardiac and vascular hypertrophy (Papadopoulos et al., 2008), causes a strong angiogenic effect (Guidolin et al., 2010) and inhibits the glucose-induced insulin release (Silvestre et al., 2004). Thus, the urotensinergic system was linked to numerous pathophysiological states including atherosclerosis, heart failure, hypertension, pre-eclampsia, diabetes, renal and liver diseases, variceal bleeding, ulcers, as well as psychological, and neurological disorders (Ross et al., 2010).

The present review focuses on the latest findings about the urotensinergic system in terms of receptor localization and pharmacology as well as receptor activation with the conception of new urotensinergic ligands aimed at discriminating UII- and/or URP-mediated biological actions.

# **DISCOVERY OF AN INTRACRINE PHARMACOLOGY OF THE UROTENSINERGIC SYSTEM**

#### **PRESENCE OF NUCLEAR UT IN THE HEART AND IN THE CENTRAL NERVOUS SYSTEM**

In many ways, UII exhibits actions similar to other key neurohormonal factors, *i.e.* angiotensin II (Ang-II) and endothelin-1 (ET-1), in driving a variety of cardiac and vascular disease processes (Maguire and Davenport, 2002). These include vasoconstriction as well as mitogenic, trophic and pro-fibrotic effects (Vaudry et al., 2010). A clear interaction of the urotensinergic system with the renin-angiotensin-aldosterone and endothelin systems is acknowledged in terms of regulation of systolic and diastolic functions (Fontes-Sousa et al., 2009). However, key differences were observed between these systems. In particular, UII induces a rather weak or absent vasoconstriction in a variety of human vascular beds (Maguire et al., 2000; Hillier et al., 2001) while it can also acts as a vasodilator in some vascular beds, such as those in the pulmonary vasculature (Stirrat et al., 2001). The recent discovery of specific intracellular receptors associated with the physiological and pathophysiological actions of Ang-II and ET-1 highlighted a high level of complexity for these peptidergic systems in the regulation of cardiovascular homeostasis. Traditionally, GPCRs are located at the plasma membrane where they modulate the activity of membrane-associated second messengers. As such, GPCRs can exert their effects through the regulation of ion channels, second messenger production, and protein kinase cascades in order to control cellular activity, gene expression, plasticity, differentiation, morphogenesis, and migration. However, in the recent years, the presence of functional intracellular receptors has almost become "a classic GPCR paradigm" (Boivin et al., 2008). These intracellular GPCRs could be involved in the control of several cellular processes including regulation of gene transcription, ionic homeostasis, cellular proliferation, and remodeling (Boivin et al., 2008). Intracellular GPCRs may be constitutively active, or may be activated by ligands internalized from the extracellular space or synthesized within the cell (**Figure 4**). Besides, they can regulate signaling pathways distinct from those activated by the

same receptor at the cell surface (Re, 1999). As such, biological outcomes might result from the integration of extracellular and intracellular signaling events (Terrillon and Bouvier, 2004; Hanyaloglu and von Zastrow, 2008; Sorkin and von Zastrow, 2009). This new paradigm for cellular signaling provides more complexity to study the function and physiological roles of GPCRs.

In a recent report, specific UII binding sites were observed on heart and brain cell nuclei from rat and monkey tissues (Doan et al., 2012; Nguyen et al., 2012). Except those two tissues and the spinal cord, none of the tested tissues including kidneys, lung, and skeletal muscle, all expressing UT at the cell surface, presented a subcellular localization of UT (Doan et al., 2012; Nguyen et al., 2012). Supporting the presence of such nuclear expression also in humans, the presence of nuclear UT was also observed in two human cell lines, *i.e.* SH-SY5Y neuroblastoma and U87 astrocytoma cell lines (Nguyen et al., 2012).

# **NUCLEAR UT ACTIVATION CAN MODULATE TRANSCRIPTION INITIATION**

As previously reported for nuclear Ang-II (Eggena et al., 1993), β3-adrenergic (Boivin et al., 2006; Vaniotis et al., 2011), and ET-1 receptors (Boivin et al., 2003), nuclear UT receptors can initiate transcription (Doan et al., 2012; Nguyen et al., 2012). Although UII and URP stimulated the transcription in isolated brain cell nuclei (Nguyen et al., 2012), only UII was able to trigger a similar effect in rat cardiac nuclei (Doan et al., 2012). Two-dimensional gel electrophoresis clearly indicated the occurrence of different immunoreactive species in both brain and heart membrane and nuclear fractions (Doan et al., 2012; Nguyen et al., 2012). Nuclear and membrane proteins extracted from heart tissues expressed three major UT-immunoreactive spots with an apparent molecular weight of 60 kDa at a pI value of 6–7 (Doan et al., 2012). Interestingly, a different pattern was observed in brain tissue (Nguyen et al., 2012). Since the UT gene is intronless, the various immunoreactive species were principally ascribed to post-translational modifications (Doan et al., 2012; Nguyen et al., 2012). Whether or not these UT species are involved in distinct UII-associated biological activities will require further investigation. However, it is well-known that glycosylation can modulate the cellular compartmentalization and functionality of the receptor, thereby influencing its intracellular trafficking and biological activity (**Figure 4**) (Duverger et al., 1995; Rondanino et al., 2003; Gobeil et al., 2006).

A growing body of evidence supports the presence of GPCRs at the surface of the nuclear membrane, their orientation within this membrane, however, remains controversial. If they maintain the topology adopted in the endoplasmic reticulum during protein synthesis, the ligand binding site would be located in the lumen of the nuclear envelop (perinuclear space) with the C-terminal of the protein being localized either within or outside the nucleus. In fact, the topology of the nuclear membrane lumen is very similar to the extracellular space, which makes it a favorable environment for a binding site (Jong et al., 2005; Bootman et al., 2009). Since signaling starts with the recruitment of specific proteins to the C-terminal portion of the receptor, signals would be sent toward the cytosol or into the nucleus in accordance with the adopted GPCR orientation within the nuclear shell (**Figure 5**). Hence, the orientation of those nuclear GPCRs would determine the direction in which the signal is transmitted. As recently reported, nuclear UT receptors are able to regulate gene transcription (Doan et al., 2012; Nguyen et al., 2012). Furthermore, it is well-known that calcium ions play an important role in the control of gene expression (Bootman et al., 2009). In isolated nuclei, nuclear calcium levels can regulate gene transcription by interacting with the cyclic AMP response element-binding protein (CREB) and the downstream regulatory element antagonist modulator (DREAM), which are constitutively present in the nucleus. Changes in nucleoplasmic calcium can be achieved by triggering inositol(1,4,5)-triphosphate receptors (IP3Rs) located on the inner nuclear membrane (Bootman et al., 2009). Because it is generally accepted that UT activation is associated with the recruitment of Gαq/11 proteins to its C-terminal tail resulting in an IP3 increase (Proulx et al., 2008), it is highly probable that this portion of the receptor is located into the nucleoplasm (**Figure 5**). Interestingly, IP3Rs are concentrated in the nuclear membrane of heart ventricular cells and their activation was shown to initiate a pro-hypertrophic pathway (Arantes et al., 2012). These findings are well-correlated with the UII-induced cardiomyocyte hypertrophy and the presence of nuclear UT receptors in cardiac tissues (Gruson et al., 2010; Doan et al., 2012). Nevertheless, these intracellular UT receptors may have the capacity to regulate signaling pathways that differ from those of their plasma membrane counterparts, as recently demonstrated for the metabotropic glutamate receptor 5 (Jong et al., 2009), and the renin–angiotensin system (De Mello, 2008). As such, this intracrine pharmacology of the urotensinergic system represents a complementary system that could potentially involve the regulation of physiological functions.

#### **INTRACELLULAR TRAFFICKING OF UT**

This new intracrine pharmacology clearly highlights the complexity of this peptidergic system where UII and URP can trigger not only common but also different biological activities (Prosser et al., 2008; Jarry et al., 2010; Doan et al., 2012). Previous studies have detected the presence of GPCRs, such as Ang-II receptors, at the nucleus in an agonist-independent manner (Lee et al., 2004). Confocal microscopy of heart and brain tissue sections as well as various non-transfected cell lines clearly revealed a constitutive nuclear localization for UT (Doan et al., 2012; Nguyen et al., 2012). However, it is also possible that following their agonist-stimulated internalization, GPCRs relocate at the nuclear membrane (Lee et al., 2004). In such a case, the translocation is initiated by the presence of a nuclear localization signal (NLS), a short stretch of basic amino acid residues often localized within the intracellular loops that is recognized by importins α and/or β (**Figure 4**). For example, a NLS was observed in the seventh transmembrane domain and the carboxy-terminal segment of the Ang-II receptor subtype1 (Lys-Lys-Phe-Lys-Arg) and the third intracellular loop of the apelin receptors (Arg-Lys-Arg-Arg-Arg) (Lee et al., 2004). Interestingly, a similar sequence, *i.e.* Lys-Arg-Ala-Arg-Arg, is also observed in the third intracellular loop of human and monkey UT isoforms (**Figure 3**) while a Lys-Gln-Thr-Arg-Arg segment is observed in rat and mouse UT. However, it is important to note that many NLS signals are still unknown and that the presence of an obvious NLS motif may mask the existence of still uncharacterized NLS sequences. Specific post-translational modifications such as palmitoylation were reported to be involved in the addressing of the receptor either to the membrane or the nuclei. For instance, it was demonstrated that de-palmitoylation of GRK6A promoted its translocation from the plasma membrane to both the cytoplasm and nucleus (Jiang et al., 2007). Such a putative palmitoylation site is also found within the seventh transmembrane domain (Cys339) of rat and mouse UT isoforms but is absent in the primate (human and monkey) receptor (Marchese et al., 1995; Tal et al., 1995; Ames et al., 1999). Under chemically mediated hypoxic conditions, an increase of total UT expression, was observed suggesting that hypoxia might induce *de novo* synthesis of the peptide receptor. However, a significant decrease in nuclear UT expression was reported that could be interpreted as an increase in translocation of the protein to the membrane or a decrease of internalization with concomitant nuclear translocation (Nguyen et al., 2012). Altogether, it could be noted that the subcellular UT localization could be either attributed to translocation from the cell surface and/or *de novo* synthesis (**Figure 4**).

#### **UII AND URP AS INTRACRINE LIGANDS**

UII, and by extension URP, were originally thought to act as autocrine and paracrine modulators rather than as hormones (Yoshimoto et al., 2004). The term "intracrine" ligand relates to intracellular molecules binding to and activating intracellular receptors (**Figure 4**). Such ligands can be synthesized and targeted to the Golgi apparatus for secretion or act intracellularly either before secretion or following reuptake. The intracrine gene product might also arise from an alternative transcription initiation site, differences in mRNA maturation or translation leading to a gene product lacking secretory signals and consequently active only in the intracellular space (**Figure 4**) (Kiefer et al., 1994; Lee-Kirsch et al., 1999; Xu et al., 2009). To this extent, it is interesting

to note that two isoforms of the human UII precursor, differing mostly by their peptide signal, were discovered (Coulouarn et al., 1998; Ames et al., 1999).

A recent study demonstrated that FITC-conjugated hUII and URP were both internalized in non-expressing UT cell lines through receptor-independent mediated endocytosis (Doan et al., 2012) (**Figure 4**). This receptor-independent endocytic mechanism brought a new perception of the pseudo-irreversible binding characteristics often described for the urotensinergic system. Indeed, the lack of rapid UT desensitization through classic mechanisms (acid wash or trypsin treatments) was thought to reflect a strong, pseudo-irreversible binding of the ligands (Douglas and Ohlstein, 2000). However, this pseudo-irreversible character could also be due to the ability of both endogenous peptides to reach the internal compartment of the cell. Moreover, it is yet possible that following the internalization of ligand-receptor complexes, ligands are subsequently released from internalized endosomes within the cell. As such, internalized peptide-receptor complexes can be dissociated under the acidic environment found in endosomes, giving rise to receptor recycling at the plasma membrane (**Figure 4**) (Giebing et al., 2005). At this point, the fate of the peptide is unknown but based on the results published by Doan et al. (2012), it is conceivable that UII, and to a lesser extend URP, could leak from the vesicle and ultimately activate intracellular receptors.

#### **PROSPECTIVE ROLES OF NUCLEAR UT**

The precise role of this new intracrine urotensinergic system has yet to be elucidated both in physiological and pathological conditions. However, as for other GPCRs including Ang-II and ET-1 receptors, these intracellular receptors are important regulators of physiological and pathological functions and could therefore represent new targets for therapeutic interventions (Boivin et al., 2008; Tadevosyan et al., 2012).

Elevated UII plasma levels were observed in numerous disease conditions, including hypertension, atherosclerosis, heart failure, pulmonary hypertension, diabetes, renal failure, and metabolic syndrome (Ross et al., 2010). As demonstrated, the cellular uptake of UII but not URP is increased at lower pH (Doan et al., 2012). Pathological conditions such as cancer, ischemic stroke, inflammation, and atherosclerotic plaques often induce an increase in metabolic activity and hypoxia associated with an elevated extracellular acidity (Andreev et al., 2010). In these conditions, UII would enter more easily than URP inside the cell triggering transcription of UII-associated genes by activating the nuclear receptor. Thus, the elevated concentration of UII observed during the etiology of various diseases could sustain specific cellular responses while an intracellular feedback loop could maintain a particular cellular state (Petersen et al., 2006). Interestingly, known intracrines do not present any structural or chemical similarities but are generally growth regulators that can directly or indirectly modulate angiogenic or anti-angiogenic actions. Therefore, the angiogenic actions of the urotensinergic system, reported both *in vivo* and *in vitro* (Spinazzi et al., 2006), could thus involve the activation of nuclear UT.

The urotensinergic system is also highly expressed in the central nervous system, but its physiological function is still poorly understood. UT was observed in cortical astrocytes (Castel et al., 2006), a ubiquitous type of glial cell that greatly outnumbers neurons and occupies 25% to 50% of brain volume (Bignami et al., 1991). It is noteworthy that glioblastoma multiform (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression and that UII, URP, and UT mRNAs were systematically found to be expressed in different glioma and glioblastoma tumors (Diallo et al., 2007). These results support a role for the urotensinergic system, and in particular nuclear UT, in human brain tumorogenesis possibly via angiogenesis regulation. Finally, in the CNS, UII is able to induce norepinephrine, dopamine, and serotonin release in noradrenergic neurons (Ono et al., 2008). Intracerebroventricular UII administration modulates cardiac homeostasis via β-adrenoreceptor activation (Hood et al., 2005). These observations bring up the idea that the presence of nuclear UT receptors could also be associated to excitatory neurotransmission. In accordance with this hypothesis, various intracrines were reported to act as neurotransmitters within the CNS (Re, 2004).

Whether specific UII or URP biological actions on the CNS and the cardiovascular system are mediated totally or in part by the nuclear UT will need further studies as well as the development of specific nuclear UT probes. Although still poorly understood, the diverse functions exerted by agonists and hormones acting on intracellular GPCRs suggest that intracrine signaling might activate cellular responses distinct from those at the cell surface for a given receptor. In the last decade, biological actions of intracrines in heart and vasculature, including those of the renin–angiotensin-system in cardiac pathology, dynorphin B in cardiac development, as well as endothelin, highlighted the importance of intracrine physiology in pathological processes such as left ventricular hypertrophy, diabetic cardiomyopathy, and arrythmogenesis. So, the presence of functional UT receptors at the cell membrane and at the nucleus will probably be a new aspect to take into account during the development of therapeutic compounds for the treatment of pathologies associated with the urotensinergic system.

# **NEW INSIGHTS INTO UT ACTIVATION**

The precise definition of the (patho) physiological roles of the urotensinergic system *in vivo* was hampered by the absence of potent and selective UT antagonists. Indeed, the lack of efficacy observed with Palosuran (ACT-058362) (Clozel et al., 2004, 2006), the only UT antagonist that reached a phase II clinical trial in patients with diabetic nephropathy, was clouded by its low antagonist potency (Behm et al., 2008). Therefore, drug discovery programs continued to focus on the identification of potent and selective UT antagonists suitable for assessment in both preclinical species and man (Maryanoff and Kinney, 2010). As reported earlier this year, Sanofi launched a phase I clinical trial regarding a long acting UT antagonist, derived from a 5,6-bisaryl-2-pyridine-carboxamide scaffold (European patent application *EP2439193*), for the treatment of diabetic nephropathy. Similarly, GlaxoSmithKline started a phase I clinical trial for the use of an UT antagonist, *i.e.* SB1440115 (United States Patent application *12,373,901*), for the treatment of asthma. Finally, over the past few years, Boehringer-Ingelheim (European patent application *EP2155748*) as well as Janssen Pharmaceutical (United States Patent application *8,193,191*) filled several patents regarding UT antagonists but no phase I clinical trial was yet reported. With the recent discovery that UII and URP could exert common as well as different biological activities (Prosser et al., 2008; Hirose et al., 2009; Jarry et al., 2010), development of selective UT antagonists has become a more complex task.

# **UT AS SHAPESHIFTING PROTEINS**

GPCRs represent the largest and most diverse family of cell surface receptors. These plasma membrane proteins bind their endogenous ligands in order to activate an intracellular signaling cascade that will result in a biological action. Conventional views of ligand-receptor activation considered all components of the signaling cascade to be linearly related, *i.e.* to emanate from the initial activation of the receptor. However, multiple studies pointed out the ability of some ligands to selectively trigger specific signaling pathways, therefore having collateral and not linear efficacy (Roettger et al., 1997; Kohout et al., 2004). As such, GPCRs cannot be considered as pharmacological on/off switches anymore. Their intrinsic nature rather suggests that dynamic changes in the receptor conformation, resulting from ligand binding, are a mean of information transfer (Kenakin and Miller, 2010). Hence, the propensity of GPCRs to assume multiple conformations make them allosteric proteins that are able to select specific subsets of secondary messengers depending on the ligand-induced adopted conformation. As such, various ligands were reported to possess differential functional profiles for a given receptor, as it was initially described for the CCR7 receptor (Kohout et al., 2004).

The URP sequence, strictly conserved throughout species, supports the concept that specific receptor interactions were maintained despite variation in the receptor amino acid sequence (Elshourbagy et al., 2002). Based on the specific expression of

URP mRNA in several cerebral structures (rostroventrolateral medulla) and tissues (heart, seminal vesicle), it was suggested that URP rather than UII would be the biologically active peptide in the UT-associated regulation of autonomic, cardiovascular and reproductive functions (Dubessy et al., 2008). Moreover, distinct pathophysiological roles for UII and URP in hypertension have been suggested (Hirose et al., 2009). Indeed, mRNA expression of both UII and URP was up-regulated in the atrium of spontaneously hypertensive rats (SHR) when compared with age-matched Wistar Kyoto (WKY) rats. However, the specific upregulation of URP but not UII mRNA in aorta and kidney of SHR rats supported the idea that these peptides might act individually in various biological systems (Hirose et al., 2009). Accordingly, it was demonstrated that UII and URP were able to exert not only common but also divergent physiological actions clearly suggesting the propensity of these two endogenous ligands to select a specific UT conformation (Prosser et al., 2008; Jarry et al., 2010; Doan et al., 2012). The concept of biased agonism has recently emerged from various studies, putting forward the notion that specific ligand-induced conformational changes can lead to particular signaling (Patel et al., 2010). In isolated ischaemic heart experiments, UII and URP were both able to reduce myocardial damages through creatine kinase reduction but only UII was able to reduce atrial natriuretic peptide (ANP) production (Prosser et al., 2008). Supporting the idea that UII and URP interact with UT in a distinct manner, it was recently demonstrated that URP, an equipotent UII paralog, was able to accelerate the dissociation rate of membrane bound 125I-hUII while hUII had no noticeable effect on URP dissociation kinetics (Chatenet et al., 2012a). Altogether, these results suggest that each ligand is able to select a specific UT conformation that triggers definite biological activities for each of these two peptides.

The peptide N-terminal region was initially pointed out as potentially involved in the observed biological activity differences between UII and URP (Prosser et al., 2008). However, it is only recently that this region was clearly associated with a putative differential binding mode of hUII and URP (Chatenet et al., 2012a). Using exocyclic Ala-derivatives of hUII, acting as very potent ligands of the UT receptor (Brkovic et al., 2003), dissociation kinetics experiments revealed a putative interaction between UT and the glutamic residue at position 1 of hUII. Indeed, it was observed that the replacement of this residue by an alanine moiety, *i.e.* [Ala1]hUII, provoked an increase of the dissociation rate of hUII but not URP (Chatenet et al., 2012a). In agreement with this view, an important electrostatic interaction between Glu1 of hUII and its receptor was previously reported in docking studies (Lescot et al., 2008). No other substitution was able to induce such pharmacological changes. Because this compound was reported to exert almost equipotent contractile activity compared to hUII and URP, the lost of a putative specific interaction with UT was thought to have generated an analog behaving as an URP derivative therefore acting at the URP-associated orthosteric binding site. Supporting this hypothesis, hUII(4–11), considered as the minimal hUII fragment to exert full biological activity, was also able to alter hUII but not URP dissociation rate (Chatenet et al., 2012a). This N-terminal segment could thus be crucial for signaling pathway selection upon activation and its deletion could lead to signaling mechanism misinterpretation, all UII truncated analogs potentially acting as URP derivatives.

Overall, these results support the presence of specific pockets/interactions within UT, aimed at selecting distinct UT conformations that can differentiate UII and URP biological activities (**Figure 6**). Briefly, it is hypothesized that upon the initial UII-UT interaction, involving the N-terminal region of UII, UT undergoes conformational changes aimed at welcoming the C-terminal domain of UII, characterized by an intracyclic β-turn (Carotenuto et al., 2004). To the opposite, URP, lacking this N-terminal portion and characterized by the presence of an intracyclic γ-turn (Chatenet et al., 2004), would bind UT in a slightly different manner; ultimately triggering a slightly different subset of signaling pathways. These observations have clearly highlighted the crucial need to reassess the development of pan UT antagonists, *i.e.* blocking UII- and URP-mediated receptor activation, and to develop new analogs of the urotensinergic system aimed at discriminating UII- or URP-mediated biological activities. Such compounds would allow a better understanding of the pathophysiological roles of the urotensinergic system and also expand our knowledge on allosteric modulation of class A GPCRs.

#### *Allosteric modulation of the urotensinergic system*

As demonstrated, the urotensinergic system is far more complex than previously thought with the presence of nuclear receptors and UII/URP specific as well as common actions. For the past two decades, identification of peptidic and non-peptidic agonists and antagonists of the urotensinergic system has gathered much interest for the treatment of various cardiovascular pathologies. An extensive review regarding the various peptidic and non-peptidic ligands of the urotensinergic system, all acting as competitive compounds, is beyond the scope of this review but more details can be found elsewhere (Maryanoff and Kinney, 2010).

Additional biological complexity, but also novel opportunities for drug discovery, has arisen from the fact that many GPCRs possess allosteric binding sites (Christopoulos and Kenakin, 2002). Similar to the initial concept of agonism, *i.e.* linear efficacy, antagonism has been historically viewed as a simple "turning off " of the receptor. As such, this non-accommodating mechanism does not allow any agonist to impart information to the receptor, the orthosteric binding site being occupied by the competitive antagonist. However, an allosteric modulator binds to its own site, different from the orthosteric site, forming a complex characterized by the concomitant presence of the endogenous agonist and the allosteric modulator. Such modulators can alter the biological properties of the endogenous orthosteric ligand either via changing its affinity, its efficacy, or both (Leach et al., 2007; May et al., 2007). This type of antagonism, termed permissive, can modify the reactivity of the receptor toward the agonist probably through conformation selection and stabilization of one or

part of the receptor states. Targeting receptor allosteric sites can offer the possibility of greater selectivity due to a lower sequence conservation within allosteric pockets across subtypes of a given GPCR, as well as the potential to fine-tune physiological signaling in a more spatial and temporally-selective manner (Kenakin, 2011).

The urotensinergic system, encompassing two endogenous peptides, provides potential for allosteric compounds to differentially modulate individual peptide responses, a behavior termed "probe dependence" (Kenakin, 2008). During the course of structure-activity relationship studies on URP derivatives, two compounds, *i.e.* [Bip4]URP and [Pep4]URP, termed urocontrin and urocontrin A (UCA) respectively, showed a specific behavior that has set them apart from known UT antagonists (**Figure 7**) (Chatenet et al., 2012a,b). Indeed, these compounds were able to selectively and significantly reduce hUII-induced contraction without altering URP-mediated vasoconstriction (Chatenet et al., 2012a,b). For instance, the efficacy of hUII-induced rat aortic ring vasoconstriction was significantly reduced (∼31%) by a pretreatment with a nanomolar concentration of UCA (**Figure 8**). Interestingly, this ability to selectively and significantly reduce UII-induced contraction was not specie-dependent since a similar effect was observed on cynomolgus monkey aortic rings. To the best of our knowledge, only two other UT ligands exerted insurmountable activity (Herold et al., 2003; Behm et al., 2010). However, none of them could differentially alter hUII and URP biological activity. The insurmountable nature of urocontrin and UCA antagonism was attributed to an allosteric modulation of UT. Indeed, an excess of UCA accelerated the 125I-hUII dissociation rate, thus suggesting that the binding of the antagonist changes the receptor conformation in such a way that the radioligand is released from the receptor. Accordingly, no difference in 125I-URP dissociation kinetics was observed in similar conditions (**Figure 8**). The apparent absence of effect on the URP pharmacological profile by UCA was attributed to its ability to select a receptor conformation through functional allosteric modulation that impairs hUII-associated actions but not URP-mediated

biological activities. As stated above, for a given receptor, an allosteric modulation that depends on the type of orthosteric ligand used is referred to as "probe dependence" (Kenakin, 2005; Keov et al., 2011). This probe dependence phenomenon supports the idea that the two endogenous ligands, despite depicting a high structure homology and recognizing a similar binding pocket, represent chemically distinct entities interacting in different structural environments within the orthosteric pocket. Because hUII and URP differ only by the length and composition of their N-terminal domain (Vaudry et al., 2010), it was postulated that this region could be involved in their putative different binding modes. Corroborating this hypothesis, the hUII counterpart of UCA, *i.e.* [Pep7]hUII,

**FIGURE 8 | Schematic representation of a proposed allosteric modulation of the urotensinergic system by urocontrin A and rUII(1–7).** By acting at an allosteric binding site, UCA is able to modify the receptor topography preventing the proper interaction of UT with the linear UII N-terminal region ultimately leading to an inefficient activation characterized

by a reduced efficacy. On the opposite, such a receptor conformational change has no effect on URP-mediated action. Conversely, binding of the rUII(1–7) N-terminal segment, initiates a topographical change that antagonizes the effect of URP, but not UII. Modified from Chatenet et al. (2012a).

acted as a weak but full agonist of the UT receptor (Chatenet et al., 2012a). This N-terminal domain is thus able to modulate the topology of the receptor in such a manner that the C-terminal domain of UII is able to trigger receptor activation. However, could this N-terminal segment be biologically active? As an agonist, the N-terminal domain of rat UII [rUII(1–7)], *i.e.* Pyr-His-Gly-Thr-Ala-Pro-Glu-amide (**Figure 7**), was unable to induce the contraction of rat aortic rings (Chatenet et al., 2012a). Amazingly, pre-treatment of rat aortic rings with rUII(1–7) induced an apparent increase in rUII contractile efficacy while reducing the potency and the efficacy of URP-mediated vasoconstriction (Chatenet et al., 2012a). These results clearly suggested that rUII(1–7) acted as a probe dependant allosteric modulator on rUII- and URP-mediated vasoconstriction (**Figure 8**) (Chatenet et al., 2012a). Since all UII isoforms possess different N-terminal domains, it is hypothesized that these regions could act as specie-selective specific URP modulators but there is currently no clue regarding an endogenous production of those N-terminal UII domains *in vivo*.

# **CONCLUSIONS**

These latest findings about the urotensinergic system will probably generate a considerable interest within the scientific

# **REFERENCES**

intact cells and vascular tissues. *Br. J. Pharmacol.* 155, 374–386.

community. First, the discovery of UT on the nuclear membrane and the presence of intracellular ligands open up new avenues in UT signaling physiology. In general, nuclear-localized receptors may regulate distinct signaling pathways, suggesting that biological responses mediated by GPCRs are not only initiated at the cell surface but might result from the integration of extracellular and intracellular signaling pathways. These receptors are therefore well-positioned to play major roles in the physiological and pathophysiological responses associated with their endogenous ligands. Finally, the discovery of allosteric modulators of the urotensinergic systems such as urocontrin, UCA, and rUII(1–7), will surely enable a better understanding of the urotensinergic system by allowing to discriminate *in vitro* and *in vivo* specific biological actions mediated by UII and/or URP. Therefore, these unique derivatives will be useful as chemical templates for the rational design of novel UT receptor ligands, as well as pharmacological tools for *in vitro* and particularly *in vivo* studies aimed at clarifying the role(s) played by the UII/URP/UT receptor system in physiology and pathology.

# **ACKNOWLEDGMENTS**

This work was supported by the Canadian Institutes for Health Research (awarded to Alain Fournier).

allosterism and complexing. *Pharmacol. Rev.* 54, 323–374.

transforming growth factor-beta1 secretion in urotensin II-induced collagen synthesis in neonatal cardiac fibroblasts. *Regul. Pept.* 140, 88–93.

Molecular and pharmacological characterization of genes encoding urotensin-II peptides and their cognate G-protein-coupled receptors from the mouse and monkey. *Br. J. Pharmacol.* 136, 9–22.

T., et al. (2009). Increased expression of urotensin II, urotensin II-related peptide and urotensin II receptor mRNAs in the cardiovascular organs of hypertensive rats: comparison with endothelin-1. *Peptides* 30, 1124–1129.

Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3. *EMBO J.* 13, 4126–4136.

M., Nishioka, R. S., et al. (1980). Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes. *Proc. Natl. Acad. Sci. U.S.A.* 77, 5021–5024.

pancreatic extracts and inhibits insulin release in the perfused rat pancreas. *Eur. J. Endocrinol.* 151, 803–809.

**Conflict of Interest Statement:** 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.

*Received: 26 September 2012; paper pending published: 30 October 2012; accepted: 10 December 2012; published online: 02 January 2013.*

*Citation: Chatenet D, Nguyen T-TM, Létourneau M and Fournier A (2013) Update on the urotensinergic system: new trends in receptor localization, activation, and drug design. Front. Endocrin. 3:174. doi: 10.3389/fendo.2012.00174*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2013 Chatenet, Nguyen, Létourneau and Fournier. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any thirdparty graphics etc.*

# Taltirelin is a superagonist at the human thyrotropin-releasing hormone receptor

# *Nanthakumar Thirunarayanan, Bruce M. Raaka and Marvin C. Gershengorn\**

Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

#### *Edited by:*

Hubert Vaudry, University of Rouen, France

#### *Reviewed by:*

Hubert Vaudry, University of Rouen, France

Ludovic Galas, Institut National de la Santé et de la Recherche Biomédicale, France

#### *\*Correspondence:*

Marvin C. Gershengorn, Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 50 South Drive, Room 4134, Bethesda, MD 20892, USA. e-mail<EMAIL_ADDRESS>
Taltirelin (TAL) is a thyrotropin-releasing hormone (TRH) analog that is approved for use in humans in Japan. In this study, we characterizedTAL binding to and signaling by the human TRH receptor (TRH-R) in a model cell system. We found that TAL exhibited lower binding affinities than TRH and lower signaling potency via the inositol-1,4,5-trisphosphate/calcium pathway than TRH. However, TAL exhibited higher intrinsic efficacy than TRH in stimulating inositol-1,4,5-trisphosphate second messenger generation. This is the first study that elucidates the pharmacology of TAL at TRH-R and shows that TAL is a superagonist at TRH-R. We suggest the superagonism exhibited by TAL may in part explain its higher activity in mediating central nervous system effects in humans compared to TRH.

**Keywords: taltirelin, thyrotropin-releasing hormone, human TRH receptor**

"fendo-03-00120" — 2012/10/6 — 19:44 — page 1 — #1

# **INTRODUCTION**

Based on evidence that thyrotropin-releasing hormone (TRH) modulates a number of central nervous system (CNS) activities including arousal, antidepressant activity, anxiolytic effects, increase in locomotor activity, antagonism of pentobarbitalinduced sedation, thermoregulation, and cardiovascular and gastrointestinal autonomic functions (Horita et al., 1986; Horita, 1998; Khomane et al., 2011), many analogs of TRH were synthesized and studied. Taltirelin (TAL) hydrate [(1-methyl-(S)-4,5 dihydroorotyl)-histidyl-prolinamide, TA-0910] is an analog that showed improved CNS activity (Suzuki et al., 1990; Yamamura et al., 1991, 1997) and lower thyrotropin (TSH)-releasing activity (Yamamura et al., 1997) compared to TRH in rodents. Based on these characteristics, TAL was studied as a treatment for neurodegenerative disorders and is the only TRH analog that has been approved for use in humans; it is used in Japan to treat patients with adult spinal muscular atrophy (Ceredist®).

In some mammals, including rodents, there are two subtypes of G protein-coupled (or seven-transmembrane-spanning) receptors for TRH. These are TRH receptor 1 (TRH-R1), which is the primary (or only) receptor in TSH-secreting cells, and TRH-R2, which is expressed throughout the CNS along with TRH-R1 but typically in different areas of the brain (O'Dowd et al., 2000; Sun et al., 2003). In humans, by contrast, only a single type of receptor for TRH, TRH-R, is expressed that is more similar to TRH-R1 than TRH-R2 (Duthie et al., 1993; Matre et al., 1993). It has been reported that TAL binds with lower affinity than TRH to receptors in rat or mouse pituitary and in brain tissue preparations (Kinoshita et al., 1997; Asai et al., 1999, 2005). However, the pharmacology of TAL at TRH-R has not been characterized.

In this study, we characterized TAL binding and signaling by TRH-R in a model cell system and show that TAL is a superagonist at TRH-R.

# **MATERIALS AND METHODS MATERIALS**

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were purchased from Biosource (Rockville, MD, USA). TRH (pyroGlu-His-ProNH2) and MeTRH (pGlu-His(1(τ ) methyl)-ProNH2) were purchased from Sigma (St. Louis, MO, USA). [3H]MeTRH was purchased from PerkinElmer (Waltham, MA, USA). TAL (N-[[(4S)-Hexahydro-1-methyl-2,6-dioxo-4 pyrimidinyl]carbonyl]-L-histidyl-L-prolinamide) was obtained from Tocris (San Diego, CA, USA).

# **CELL CULTURE AND GENERATION OF CELLS STABLY EXPRESSING TRH-R**

HEK-EM 293 (human embryonic kidney) cells stably expressing TRH-R were generated as follows. The human TRH-R cDNA in pcDNA3.1(+) was obtained from the Missouri S&T cDNA Resource Center (Rolla, MO, USA) and was subcloned into the pcDNA3.1(+)/hygromycin vector. HEK-EM 293 cells were transfected with the cDNA of TRH-R using FuGENE 6 transfection reagent (Roche Diagnostics GmbH, Mannheim, Germany) and the cell clones stably expressing TRH-R were selected using hygromycin (250 μg/ml). HEK-EM 293 cells stably expressing TRH-R were grown in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, 10 μg/ml streptomycin, and 200 μg/ml hygromycin B (Invitrogen, Carlsbad, CA, USA) at 37◦C in a humidified 5% CO2 incubator.

#### **COMPETITION BINDING**

Competition binding assays were performed in monolayers of intact HEK cells expressing TRH-Rs. The cells (220,000 cells/well in 24-well plates) were preincubated with various concentrations of unlabeled TAL, TRH, or MeTRH for 15 min before addition of radioligand and then incubated at 37◦C for 1 h with 4 nM [3H]MeTRH as described elsewhere (Engel et al., 2006). Non-specific binding was determined in incubations with excess non-radiolabeled MeTRH. IC50 is the concentration of unlabeled ligand that reduces specific binding of [3H]MeTRH by 50%. The receptor number per cell was calculated from competition binding curves of various doses of unlabeled MeTRH and 4 nM [3H]MeTRH (**Figure 1**) and found to be 16,000/cell.

# **MEASUREMENT OF INTRACELLULAR CALCIUM MOBILIZATION**

Cells stably expressing TRH-R were seeded in black-walled, clearbottomed 96-well plates (Corning, NY, USA) at a density of 60,000 cells/well in DMEM with 10% fetal bovine serum and incubated for 24 h at 37◦C in 5% CO2. The following day, the culture media was replaced with 100 μl of Hank's balanced salt solution with 20 mM HEPES, pH 7.5 and the cells were loaded with 100 μl of calcium 4 fluorescent dye (Molecular Devices, Sunnyvale, CA, USA) for 1 h at room temperature before addition of compounds. Transient changes in intracellular [Ca++] induced by TAL, TRH, or MeTRH were measured using the FLIPRTETRA system (Molecular Devices, Sunnyvale, CA, USA). Changes in fluorescence were detected at the emission wavelength ranges from 515 to 575 nm. The agonistic responses of ligands were assessed

immediately upon their addition in a concentration range from 0.1 nM to 30 μM. Responses were measured as peak fluorescent intensity minus basal fluorescent intensity at each compound concentration and are presented as % of the maximum response.

# **MEASUREMENT OF IP1 PRODUCTION**

Cells were seeded at 220,000/well in white, solid bottom, tissue culture-treated 24-well plates and cultured at 37◦C with 5% CO2 overnight. Serial dilutions of TAL, TRH, or MeTRH, in Hank's balanced salt solution with 20 mM HEPES and 50 mM LiCl, pH 7.4, were added at 200 μl/well on the second day. After 60 min incubation at 37◦C in 5% CO2, inositol monophosphate (IP1) content was measured using the IP-One ELISA kit (Cisbio International, France) according to the manufacturer's protocol. The results were calculated as IP1 nanomoles/well and are presented as % of the maximum response.

# **DATA ANALYSIS**

The dose–response data were analyzed by non-linear regression of curve fit with one-site competition using GraphPad Prism software version 4 (GraphPad, Inc., San Diego, CA, USA) and the significance was determined by *t*-test or ANOVA.

# **RESULTS**

"fendo-03-00120" — 2012/10/6 — 19:44 — page 2 — #2

The pharmacology of TAL binding and signaling was studied in HEK-EM 293 cells, which do not endogenously express TRH-Rs, engineered to express 16,000 TRH-Rs/cell (**Table 1**). In competition binding assays using 4 nM [3H]MeTRH and various doses of unlabeled MeTRH, the concentration of MeTRH that halfmaximally inhibited [3H]MeTRH binding (IC50) was 3.0 nM (**Figure 1**). The half-maximally effective concentration (EC50) of MeTRH for stimulating an increase in cytosolic Ca2+ concentration (Ca2+ release) was 7.2 nM, which is not different from the IC50 (*p* > 0.1). Thus, MeTRH is an agonist at TRH-R with high potency. We compared the effects of TAL and TRH in competing for [3H]MeTRH binding and on stimulating Ca2+ release (**Figure 2**). The IC50 values were 910 and 36 nM for TAL and TRH, respectively, and the EC50 values were 36 and 5.0 nM for TAL and TRH, respectively. Thus, TRH was a high potency agonist and TAL was a moderate potency agonist at TRH-R. We noted, moreover, that the IC50/EC50 ratio was 25 for TAL (*p* < 0.02), 7.2 for TRH (*p* < 0.05), and approximately 0.4 for MeTRH. When comparing two agonists, the agonist with the higher IC50/EC50 ratio has a greater intrinsic efficacy (Engel et al., 2006). These findings suggested that TAL may be a more efficacious agonist than TRH and that TRH is more efficacious than MeTRH. We previously showed that TRH exhibited a higher intrinsic efficacy than MeTRH at

#### **Table 1 | Pharmacological parameters for TRH-R.**

mouse TRH-Rs (Engel et al., 2006). As TRH is the natural, full agonist, TAL is termed a superagonist, and MeTRH is a partial agonist.

sigmoidal dose–response method.

As Ca2+ release is a rapid and transient response to TRH-R activation, it is easier to compare relative intrinsic efficacies by quantifying activation of the inositol-1,4,5-trisphosphate pathway. Inositol-1,4,5-trisphosphate production is the step prior to Ca2+

release in signal transduction by TRH-Rs (Gershengorn, 1986) and can be quantified by measuring accumulation of its metabolic product IP1 over time by inhibiting IP1 degradation (**Figure 3A**). The EC50 values for IP1 production were found to be 150 nM for TAL and 3.9 nM for TRH. More importantly, TAL was clearly more efficacious than TRH in that TAL stimulated an increase in IP1 production that was 180% of that stimulated by TRH (*p* < 0.001).

Another way of demonstrating relative intrinsic efficacies of agonists is to show that the maximal response of a more efficacious agonist is inhibited by a less efficacious agonist (Engel et al., 2006). **Figure 3B** illustrates the IP1 responses to maximally effective doses of MeTRH, TRH, and TAL. As is evident, TAL (3.5 fold over MeTRH) is more efficacious than TRH (2.1-fold over MeTRH); MeTRH is the least efficacious. As predicted, the least efficacious agonist MeTRH inhibited the response to TRH (full agonist) and to TAL (superagonist).

#### **DISCUSSION**

In this study, we characterized the binding and signaling properties of TAL at TRH-R that, to our knowledge, have not been previously reported. The binding properties of TAL at rodent TRH receptors have been studied previously (Asai et al., 1999; Brown, 1999). In agreement with the findings with rodent receptors, we found that TAL binds to TRH-R with lower affinity than TRH. Our most interesting observation, however, is that TAL exhibits higher intrinsic efficacy than TRH; that is, TAL can stimulate the same level of signaling as TRH but at lower levels of receptor occupancy and could induce higher levels of signaling than TRH at full occupancy (**Figure 3**). Since TRH is the natural, full agonist for TRH-R, TAL is termed a superagonist. We previously reported that other TRH analogs displayed higher intrinsic efficacies than TRH at rodent TRH receptors (Engel et al., 2006). In the same study, we showed that MeTRH, the only TRH analog with higher affinity and potency than TRH, was a partial agonist that displayed lower intrinsic efficacy than TRH and that when high levels of TRH and MeTRH were added simultaneously the level of signaling was lowered to that of MeTRH. This is the predicted effect of adding a

"fendo-03-00120" — 2012/10/6 — 19:44 — page 3 — #3

**(B)** MeTRH inhibits IP1 formation stimulated by TAL and TRH. IP1 production was determined in the presence of TRH (0.1 μM), TAL (3 μM), MeTRH (3 μM), TRH + MeTRH, or TAL + MeTRH. The data are expressed as mean ± SEM performed in duplicate of two experiments.

partial agonist along with a full agonist. We used a similar experimental design herein to confirm that TAL is a superagonist at TRH-R; MeTRH antagonized IP1 production stimulated by both TRH and TAL (**Figure 3B**).

Previous reports in rodents showed that TAL displayed more activity in stimulating CNS effects than TRH (Suzuki et al., 1990; Brown, 1999). TAL may have similar CNS effects in humans (Gary et al., 2003; Khomane et al., 2011). The differences in the activities of TAL and TRH in the CNS have been attributed to the higher stability in blood and increased penetration of the blood–brain barrier of TAL compared to TRH. Although this is likely true, our new findings of the signaling efficacy of TAL at TRH-R suggest that the higher intrinsic efficacy of TAL may be contributing to its CNS activity in humans also.

# **REFERENCES**

(TRH) hypothesis of homeostatic regulation: implications for TRHbased therapeutics. *J. Pharmacol. Exp. Ther.* 305, 410–416.

In summary, we have described characterization of the pharmacology of TAL at TRH-R. Most importantly, we showed that TAL is a superagonist when signaling at TRH-R via the Gq/11 protein-phospholipase C-phosphatidylinositol-4,5-bisphosphateinositol-1,4,5-trisphosphate-calcium pathway. We suggest the superagonism exhibited by TAL may, in addition to its relative metabolic stability and ability to cross the blood–brain barrier, explain its higher activity in mediating CNS effects in humans compared to TRH.

#### **ACKNOWLEDGMENTS**

This work was supported by the Intramural Research Program of the National Institutes of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health (1 Z01 DK011006).

human thyrotropin-releasing hormone receptor. *Biochem. Biophys. Res. Commun.* 195, 179–185.

"fendo-03-00120" — 2012/10/6 — 19:44 — page 4 — #4

*Yakurigaku Zasshi* 110(Suppl. 1), P33–P38.

**Conflict of Interest Statement:** 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.

*Received: 10 July 2012; paper pending published: 20 August 2012; accepted: 20 September 2012; published online: 09 October 2012.*

*Citation: Thirunarayanan N, Raaka BM and Gershengorn MC (2012) Taltirelin is a superagonist at the human thyrotropin-releasing hormone receptor. Front. Endocrin. 3:120. doi: 10.3389/ fendo.2012.00120*

*This article was submitted to Frontiers in Neuroendocrine Science, a specialty of Frontiers in Endocrinology.*

*Copyright © 2012 Thirunarayanan, Raaka and Gershengorn. This is an openaccess article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# **Patricia M. Hinkle1\*, Austin U. Gehret 2 and BrianW. Jones 3**

1 Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY, USA

2 Department of Science and Mathematics, National Technical Institute for the Deaf, Rochester Institute of Technology, Rochester, NY, USA

3 Department of Pharmacology, University of Washington, Seattle, WA, USA

#### **Edited by:**

Liliane Schoofs, Catholic University of Leuven, Belgium

#### **Reviewed by:**

Perry Barrett, University of Aberdeen, UK Leo T. Lee, The University of Hong Kong, Hong Kong

#### **\*Correspondence:**

Patricia M. Hinkle, Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue Rochester, Rochester, NY 14642, USA. e-mail: patricia\_hinkle@

urmc.rochester.edu

The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5 negative recycling endosomes.The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation ofTRH receptors begins whenTRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for theTRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.

**Keywords: dephosphorylation, desensitization, internalization, phosphorylation, Rab, thyrotropin-releasing hormone, trafficking,TRH receptor**

# **INTRODUCTION**

Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, sits atop the hypothalamic/pituitary/thyroid axis, stimulating release of thyrotropin from the anterior pituitary gland. TRH also stimulates prolactin secretion (**Figure 1**). Initially characterized in clonal rat pituitary cell lines, TRH binding sites were later identified in brain membranes (Hinkle and Tashjian, 1973; Burt and Snyder, 1975). The link between TRH receptors and G proteins was made at a time when heterotrimeric G proteins were thought to transduce signals only from receptors coupled to adenylyl cyclase (Hinkle and Kinsella, 1984; Hinkle and Phillips, 1984). It quickly became clear that TRH receptors belong to the G protein-coupled receptor (GPCR) superfamily. A single TRH receptor gene has been found in humans and higher mammals and two genes encoding homologous receptors, TRHR1 and TRHR2, in rodents (Sun et al., 2003). TRHR1 predominates in the anterior pituitary gland while both TRHR1 and TRHR2 are found in rodent CNS (O'Dowd et al., 2000). Thyroid hormones exert powerful feedback inhibition over the TRH response system by inhibiting TRH synthesis and processing in TRH neurons in the paraventricular region of the hypothalamus and decreasing TRH receptors and responses in the pituitary gland (Gershengorn, 1978; Perrone and Hinkle, 1978; Hinkle and Goh, 1982; Segerson et al., 1987; Fekete and Lechan, 2007; Costa and Hollenberg, 2012). Prolonged hypothyroidism leads to a 40-fold increase in TRH receptor mRNA levels in pituitary glands (Costa et al., 2012). In addition, pyroglutamyl peptidase, a highly specific TRH-degrading ectoenzyme, is dramatically increased in hyperthyroid animals (Schomburg and Bauer, 1997; Marsili et al., 2011).

Genetic deletion of either the TRH peptide precursor or TRHR1 results in central hypothyroidism and mild growth retardation in mice, consistent with the established role of TRH in thyroid physiology (Yamada et al., 1997; Rabeler et al., 2004; Zeng et al., 2007). Interestingly, loss of the TRH receptor appears to result in a more severe phenotype than loss of the TRH peptide, possibly because the TRH receptor displays physiologically significant constitutive activity. Mice lacking TRHR1 also exhibit low prolactin and hyperglycemia, as well as increased anxiety and depression. Some of these behavioral effects may be secondary to hypothyroidism. Mice lacking TRHR2 display a subtle phenotype

consistent with depression and decreased anxiety (Sun et al., 2009). In humans,absence of the TRH receptor causes low free T3 and free T4 with thyroid stimulating hormone (TSH) levels inappropriately low for the degree of hypothyroidism (Bonomi et al., 2009). Lack of a TRH receptor does not result in infertility or failure to lactate.

This review focuses on aspects of TRH receptor signaling that have been elucidated in either pituitary cell models expressing endogenous receptors (pituitary tissue, primary cultures of pituitary cells, cell lines derived from pituitary tumors) or generic cell lines expressing transfected TRH receptors (HEK293, CHO, COS, Hela). In rat anterior pituitary tissue, TRH receptor mRNA is found not only in TSH-secreting cells but also in prolactin- and/or growth hormone-secreting cells (Konaka et al., 1997). Although the great majority of pituitary cells that bind rhodamine-labeled TRH and respond to TRH with an increase in intracellular calcium stain for prolactin or TSH-β, responses are occasionally observed in unexpected cell types (Ashworth et al., 1995; Villalobos et al., 1997; Yu et al., 1998). Signal transduction of endogenous TRH receptors has not been characterized in neurons, where no suitable model system has been identified. Even though there is only one TRH receptor in higher mammals, that receptor may be regulated quite differently in pituitary cells and neurons because of different patterns of TRH stimulation and different sets of G protein

subunits, effectors, receptors kinases, and phosphatases, arrestins, and downstream targets.

As outlined in **Figure 2**, TRH receptors are textbook calciummobilizing receptors: they are coupled to Gq and G11, which activate phospholipase Cβ (PLCβ), hydrolysis of phosphatidylinositol (4,5)bisphosphate, and release of the second messengers inositol (1,4,5)triphosphate (IP3) and diacylglycerol (DG) that in turn mobilize intracellular calcium and activate protein kinase C (Drummond et al., 1984; Drummond, 1985; Gershengorn, 1989). Two of the four forms of phospholipase Cβ (PLCβs 1 and 3) are found in anterior pituitary tissue, and calcium responses to TRH are intact in mice lacking PLCβ3 (Romoser et al., 2001). Thus PLCβ1, which is stimulated by Gαq/11-GTP but not Gβγ, generates second messengers in response to TRH. Maximal concentrations of intracellular free calcium are attained at concentrations of TRH at least an order of magnitude below the apparent *K*d of the receptor and the EC50 for IP3 production. These initial events are followed by complex changes that result in depolarization and a sustained influx of extracellular calcium through voltage-gated L-type calcium channels (Hinkle et al., 1996; Barros et al., 1997). When TRH receptors are expressed in embryonic fibroblasts from mice lacking Gαq and Gα11, TRH does not stimulate any increase in intracellular calcium.

The TRH receptor is a rhodopsin family GPCR with typical features including seven transmembrane domains, several extracellular glycosylation sites, an essential disulfide bond between the first and second extracellular loops, a fairly short third intracellular loop, and a cytoplasmic tail. The receptor has a canonical (D/E)RY at the cytoplasmic end of the third transmembrane segment, the usual NPxxY at the cytoplasmic end of the seventh transmembrane helix, and an intracellular eighth helix anchored by two palmitoylated Cys residues in the carboxyl tail (Du et al., 2005). Except for the most distal region, the cytoplasmic tail of the receptor is conserved among species. The C-terminal amino acids of numerous GPCRs form classical PDZ ligands, sequences that can interact with proteins bearing a PDZ domain; PDZ is an acronym for three proteins containing the domain, PSD98, Dlg1, and zo-1 (Romero et al., 2011). TRH receptors, however, do not contain sequences predicted to interact with PDZ domains. Using a combination of experimental data obtained with receptor mutants, multiple ligands, and molecular modeling, Gershengorn and his colleagues have developed a sequential model for receptor binding and activation in which the TRH tripeptide first interacts weakly with extracellular loops and then moves deeper into the receptor and contacts transmembrane domains to form a high affinity complex. The conformational changes that occur upon receptor activation involve movements in transmembrane helices 5 and 6 and the third intracellular loop that result in G protein coupling (Gershengorn and Osman, 1996).

#### **DESENSITIZATION OF THE TRH RECEPTOR**

Thyroid stimulating hormone displays a circadian pattern that is attributed to rhythmic release of hypothalamic TRH. As a consequence, desensitization and resensitization of the TRH signaling pathway are likely to play central roles in regulating TRH actions *in vivo*. TRH rapidly stimulates a large increase in IP3 mass, providing a proximal, easily quantified response. Measurements of IP3 production have established that the TRH receptor undergoes classical desensitization. Continuous application of TRH leads to a transient burst of IP3 that peaks within seconds and falls within a minute, though to levels that remain above baseline (Drummond et al., 1984; Gershengorn and Osman, 1996; Yu and Hinkle, 1997, 1998). When cells are exposed to TRH intermittently, the size of the

IP3 response diminishes with successive stimulations. The extent of desensitization depends on the method used to measure it and the cell type under study (Falck-Pedersen et al., 1994). Decoupling the receptor from G proteins contributes to the transient nature of the IP3 elevation, which is more sustained in cells expressing a TRH receptor that lacks most of the cytoplasmic tail and is thereby spared from usual desensitization processes (Yu and Hinkle, 1998; Jones and Hinkle, 2005), as shown in **Figure 3**. Changes further downstream in the signal pathway can also decrease TRH responses by mechanisms including protein kinase C-mediated inhibition of phospholipase Cβ and slow refilling of intracellular calcium stores (Yu and Hinkle, 1997). TRH receptors were the first GPCRs shown to undergo what is now termed homologous downregulation (Hinkle and Tashjian, 1975; Gershengorn, 1978). Incubation with TRH decreases the number of TRH binding sites without changing receptor affinity in pituitary GH3 cells. The molecular basis for downregulation is still not completely understood.

Like many rhodopsin family GPCRs (Bulenger et al., 2005; Milligan, 2009; Lohse, 2010), TRH receptors form oligomers when they are overexpressed in non-native cells (Kroeger et al., 2001; Hanyaloglu et al., 2002; Zhu et al., 2002). Bioluminescence resonance energy transfer (BRET), co-precipitation and biochemical analyses have all been used to document TRH receptor homodimers, and the two rodent TRH receptors (TRHR1 and TRHR2) form heterodimers (Hanyaloglu et al., 2002). TRH increases the apparent fraction of receptors in dimers quantified by all of these techniques, perhaps because TRH causes receptors to concentrate in clathrin-coated pits and endosomes. Truncated receptors that do not recruit arrestin or internalize can form dimers

incubated with TRH and IP3 mass was measured at intervals (graphs). To visualize arrestin, receptors were expressed with GFP-labeled arrestin3. To with them but it does not interact with truncated receptors, which do not internalize.

constitutively but not in response to TRH (Zhu et al., 2002). When receptors are coerced to form oligomers by addition of a chemical dimerizing agent, signaling is not altered but internalization is accelerated and recycling blunted (Song and Hinkle, 2005). It has been difficult to assess the physiological relevance of TRH receptor dimers because no mutations that prevent oligomerization have been identified and available methods are inadequate to characterize endogenous receptors in pituitary cells. Recent studies (Whorton et al., 2007) have provided compelling evidence that at least some rhodopsin family GPCRs can activate G proteins as monomers, yet there are clear examples of receptors that form heterodimers with unique pharmacology (Jordan and Devi, 1999). One possibility is that that only one member of a GPCR pair signals to a single G protein. The TRHR1/TRHR2 heterodimer interacts with arrestins and internalizes in a different pattern from either receptor alone (Pfleger et al., 2004), but the intriguing possibility that the TRH receptor forms heteromers with other GPCRs to impart novel pharmacology has not been explored.

# **OVERVIEW OF GPCR RECEPTOR DESENSITIZATION**

Most GPCRs become phosphorylated after they are activated, and the TRH receptor is no exception. GPCR phosphorylation is catalyzed by members of a family of Ser/Thr protein kinases, GPCR kinases, or GRKs. There are seven GRKs, four of them widely expressed (GRKs 1, 2, 5, and 6; Moore et al., 2007; Gurevich et al., 2012). What sets the GRKs apart from other kinases is their ability to discriminate between GPCRs in inactive versus activated conformations. They act preferentially on agonist-occupied and constitutively active receptors and do not have strict requirements for the amino acid sequence surrounding phosphorylation sites. There are important differences in how different GRKs are recruited to the plasma membrane, however, and examples of GPCRs that are phosphorylated at different sites by different GRKs. GRKs exert a number of kinase-independent effects on GPCR signaling and are known to phosphorylate substrates other than receptors (Moore et al., 2007; Premont and Gainetdinov, 2007; Evron et al., 2012; Gurevich et al., 2012). GPCRs can also be phosphorylated by kinases that become activated in response to signaling such as cAMP-dependent protein kinase and protein kinase C. Phosphorylation carried out by such downstream kinases can influence GPCR-G protein coupling, desensitization, and trafficking.

Once activated and phosphorylated, most GPCRs bind to arrestin, which interrupts the interaction between an activated receptor and its cognate G protein and terminates signaling via the G protein-mediated pathway. Arrestins 2 and 3 (also referred to as β-arrestin1 and β-arrestin2) are ubiquitously expressed. Like G proteins and GRKs, arrestins distinguish between inactive and activated receptor conformations. Arrestins bind preferentially to activated GPCRs; they also contain a positively charged pocket that interacts strongly with negatively charged phosphates (Gurevich and Gurevich, 2006; DeWire et al., 2007; Moore et al., 2007; Premont and Gainetdinov, 2007; DeFea, 2011; Shenoy and Lefkowitz, 2011). The arrestins therefore tend to bind with highest affinity to receptors that are both activated and phosphorylated. When arrestin engages receptor, a buried helical region in the arrestin carboxyl terminus undergoes a major translocation to expose binding

sites for AP2 and clathrin, targeting the arrestin-receptor complex to coated pit regions of the membrane destined to pinch off into endosomes in a dynamin-dependent process (Moore et al., 2007; Shenoy and Lefkowitz, 2011).

Arrestin is a scaffolding protein capable of anchoring a wide array of kinases, phosphatases, and other proteins to the arrestin-GPCR complex (Xiao et al., 2007; DeFea, 2011). A rapidly expanding body of literature shows that arrestin-GPCR complexes can initiate alternate signaling pathways, notably those leading to MAP kinase activation (Lefkowitz and Shenoy, 2005; Luttrell and Gesty-Palmer, 2010; Reiter et al., 2012). The Lefkowitz group has proposed that the phosphorylation pattern of a GPCR can act as a barcode in which different phosphosites recruit different arrestins to turn on different signal pathways (Liggett, 2011; Nobles et al., 2011). This model is appealing because it adds tremendous diversity to GPCR signaling, and it is supported by evidence that arrestin2 and arrestin3 bind preferentially to distinct phophosites on several receptors (Ren et al., 2005). There are also many examples of what are termed biased ligands that favor particular pathways (Rajagopal et al., 2010; Reiter et al., 2012). For example, biased ligands may preferentially activate G protein or arrestin cascades, activate one arrestin signaling function but not another (such as kinase activation and internalization), or act as an agonist in one pathway and an antagonist in another. Biased agonists and antagonists open new doors for the development of therapeutically useful GPCR ligands.

G protein-coupled receptors have been broadly divided into two groups based on their interactions with arrestins (Oakley et al., 1999, 2000). Class A receptors bind arrestin 3 more strongly than arrestin 2 or visual arrestin, internalize without associated arrestin, and recycle rapidly. Examples include the well-characterized β2 adrenergic receptor, the µ-opioid receptor and dopamine D1 receptor. Class B receptors bind well to both arrestin 2 and arrestin 3, internalize with arrestin, and traffic to lysosomes where they undergo intracellular degradation or slow recycling. Examples include the V2 vasopressin receptor, type 1a angiotensin II receptor, and the TRH receptor.

#### **PHOSPHORYLATION OF TRH RECEPTORS**

Thyrotropin-releasing hormone receptors become phosphorylated rapidly once they are activated. Receptor phosphorylation has been demonstrated by 32P incorporation, an upward mobility shift on gel electrophoresis, and reactivity with phosphositespecific antibodies (Hanyaloglu et al., 2001; Zhu et al., 2002; Jones and Hinkle, 2005; Jones et al., 2007). All of these changes are reversed by phosphatase treatment. Phosphorylation sites in the TRH receptor have been partially mapped by characterizing 32P incorporation and antibody reactivity of wildtype receptors and receptors lacking various Ser and Thr residues in the cytoplasmic tail. Jones et al. generated antisera against multiply phosphorylated peptides from five different conserved regions of the TRH receptor tail and validated their specificity (Jones and Hinkle, 2008). None of the antibodies recognize TRH receptors from unstimulated cells, but four of them react with receptors from TRH-treated cells. Endogenous TRH receptors in pituitary cells are phosphorylated strongly at residues between amino acids 355 and 365 and less efficiently at two regions farther downstream. Current information about phosphosites in the TRH receptor C-terminus is presented in **Figure 4**. As discussed below, phosphorylation of Thr365 is particularly important for arrestin recruitment, internalization, and desensitization. There is no evidence for phosphorylation at Tyr residues. The distal regions of the TRH receptor are more heavily phosphorylated when the receptor is expressed in HEK293 or CHO cells compared to native receptors. The finding of different site usage in different settings suggests a need for caution when charactering phosphorylation of overexpressed epitope-tagged GPCRs in heterologous cells, an approach that is normally required for analysis by mass spectrometry.

Thyrotropin-releasing hormone receptor phosphorylation does not appear to be hierarchical because the rates of phosphorylation are the same for different phosphosites. TRH does not promote 32P incorporation into receptors truncated before the palmitoylation site and TRH does not alter mobility of truncated receptors, leading to the conclusion that either intracellular loops are not phosphorylated in response to TRH or phosphorylation in the intracellular loops somehow requires the receptor tail. Following TRH addition, nearly all receptors appear to be phosphorylated based on the mobility shift and near quantitative immunoprecipitation with phosphosite-specific antibodies (Jones et al., 2007).

In pituitary cells, endogenous receptors on the plasma membrane are not detectably phosphorylated in the basal state, but they become strongly phosphorylated within 10 s of TRH addition and endocytosis of phospho-receptors is readily apparent by 10 min (**Figure 5**). Interestingly, when rat pituitary tissue from

residues are shown in pink or red in regions known to undergo TRH-dependent phosphorylation. Phosphorylation at Thr365, in red, is essential for desensitization and internalization, and phosphorylation at other sites between amino acids 355 and 365 has been documented and shown to Phosphorylation in this region is not essential for desensitization and internalization, but it may be important for different, as yet unidentified signaling functions. Phosphorylation in the non-conserved distal region has not been examined.

an untreated animal is examined, phosphorylated TRH receptor is clearly visible. The intensity of the phospho-receptor signal increases greatly minutes after animals are injected with TRH and, as expected, the signal is seen cells that stain for TSH-β and prolactin (Jones et al., 2007).

Thyrotropin-releasing hormone receptor phosphorylation is exceedingly fast, with a half-time of 0.2 min (Jones et al., 2007; Gehret and Hinkle, 2010). This rapid phosphorylation is unusual among GPCRs and marks the TRH receptor as a superior substrate. Rapid phosphorylation is not simply a consequence of the sequence of the receptor tail. The transmembrane helices and intracellular loops of the TRH receptor are likewise important for rapid phosphorylation of the receptor tail even though they are not phosphorylated themselves. A chimeric TRH receptor with the β2-adrenergic receptor tail is phosphorylated at β2-adrenergic receptor GRK sites with kinetics typical of the TRH receptor, not the β2-adrenergic receptor (Gehret and Hinkle, 2010). The ratelimiting step for TRH receptor phosphorylation may be recruitment of GRKs to the activated receptor. In support of this idea, dimerization of a mutant TRH receptor that cannot undergo activation with a truncated TRH receptor that signals well but cannot undergo phosphorylation results in phosphorylation of the signaling-incompetent partner (Song et al., 2007). This is most easily explained if the activatable receptor recruits a kinase that acts on its partner in the receptor dimer. The predicted amphipathic eighth helix between the canonical NPxxY at the end of the seventh transmembrane domain and the palmitoylated Cys-X-Cys motif is not phosphorylated but the positively charged residues in this region are essential for phosphorylation at downstream sites and subsequent internalization (Gehret et al., 2010).

Considerable evidence points toward GRK2 as the kinase responsible for TRH receptor phosphorylation. Phosphorylation is inhibited by dominant negative, kinase-dead forms of GRK2 and by siRNAs targeting GRK2 (Jones and Hinkle, 2005; Jones et al., 2007). The effects of siRNA knockdown are seen both in pituitary cells and heterologous cells expressing transfected receptors. In addition, paroxetine, which has recently been recognized as an effective and relatively specific GRK2 inhibitor, delays phosphorylation of the TRH receptor (Thal et al., 2012). In keeping with the rapid rate of TRH receptor phosphorylation, GRK2 translocates to membrane fractions within 10 s of TRH addition (Jones and Hinkle, 2005). All of these experiments implicate GRK2 in TRH receptor phosphorylation, but because GRK2 knockdown and inhibition do not block receptor phosphorylation completely it seems likely that other GRKs are also able to phosphorylate the receptor.

Inhibitor data suggest that casein kinase II, which is predicted to phosphorylate Thr365 and two other downstream sites, may contribute to receptor phosphorylation (Hanyaloglu et al., 2001). Pharmacological activation of conventional protein kinase C isoforms leads to weak phosphorylation of some but not all sites in the TRH receptor tail, but protein kinase C inhibitors do not alter TRH-dependent phosphorylation (Jones et al., 2007). Furthermore, TRH receptor phosphorylation and internalization occur normally in cells lacking Gαq and Gα11, indicating that calcium- and diacylglycerol-activated kinases are not essential (Yu and Hinkle, 1999; Jones and Hinkle, 2005). Together these results suggest little role for downstream kinases in TRH receptor phosphorylation.

#### **ARRESTIN INTERACTIONS WITH THE TRH RECEPTOR**

Arrestin interactions with TRH receptors have been monitored by a variety of approaches including translocation of GFP-arrestin (Groarke et al., 1999, 2001; Oakley et al., 1999, 2000; Yu and Hinkle, 1999; Smith et al., 2001; Hanyaloglu et al., 2002), coprecipitation of arrestin and receptor (Jones et al., 2007), effect of arrestin on agonist affinity (Jones and Hinkle, 2005, 2008), and BRET (Kroeger et al., 2001; Hanyaloglu et al., 2002). GFP-arrestin is diffusely localized in the cytoplasm of cells expressing TRH receptors. When TRH is added, GFP-arrestin2 and GFP-arrestin3 move to the plasma membrane within a minute or two (see **Figure 3**). The ability of TRH receptors to recruit arrestins 2 and 3 places it in the Group B category of GPCRs. Although arrestin translocation is detectable with most GPCRs, arrestin movement seen with the TRH receptor is exceptionally robust (Oakley et al., 2000). GFP-arrestin translocation is not observed in cells expressing TRH receptors lacking palmitoylation sites or truncated before the major phosphorylation sites (Vrecl et al., 1998; Yu and Hinkle, 1998; Hanyaloglu et al., 2001; Smith et al., 2001; Jones and Hinkle, 2005, 2008).

As shown in experiments depicted in **Figure 6**, GFP-arrestin does translocate to receptors with Ala substitutions for the four Ser and Thr residues between 355 and 365, proving that downstream phosphosites are sufficient to bind arrestin (Jones and Hinkle, 2008). This is important because distal phosphosites are not sufficient for desensitization and internalization, as discussed below. Arrestin co-precipitates with activated TRH receptors even if Ser/Thr residues in the 355–365 region are Ala-substituted, again showing that arrestin interacts with at least two regions of the receptor. BRET studies document a close interaction between the type 1 TRH receptor and arrestins 2 and 3 that is lost if the receptor is truncated before the palmitoylation sites (Pfleger et al., 2004). **Figure 7** summarizes current understanding about the importance of different TRH receptor phosphorylation sites.

Because arrestins bind preferentially to activated GPCRs, theoretical considerations dictate that arrestin will increase the affinity of a GPCR for agonists, a prediction borne out by experimental evidence (Gurevich et al., 1997). Consistent with this, the apparent affinity of the potent agonist [3H]MeTRH, measured at room temperature or above in intact cells, is highly dependent on arrestin levels (Jones and Hinkle, 2005, 2008). In arrestin2/3 knockout cells, cotransfection with wildtype arrestin increases the apparent affinity for [3H]MeTRH 14-fold. Arrestin increases the agonist affinity of truncated and Ala-substituted receptors, supporting the concept of multiple arrestin binding sites and raising the possibility of phosphorylation-independent arrestin interactions.

#### **ROLE OF ARRESTIN IN TRH RECEPTOR SIGNALING, DESENSITIZATION AND INTERNALIZATION**

Thyrotropin-releasing hormone receptors expressed in arrestin2/3 knockout cells generate strong IP3 responses to TRH. TRHstimulated IP3 levels are much lower in cells expressing arrestin2, arrestin3, or both, proving that arrestin is important for desensitization and that either arrestin is sufficient for this function.

**FIGURE 6 | Arrestin recruitment and arrestin-dependent desensitization.** Cells expressing the receptors shown were incubated with GFP-arrestin and imaged before or after 3 min exposure to TRH. The right panels show TRH-stimulated IP3 production in arrestin-null cells expressing receptor with no arrestin (No Arr), wildtype arrestin 3 (wtArr), or R169E-Arr, which binds

activated GPCRs in a phosphorylation-independent fashion. TRH receptors are: Wt, wildtype; 4Ala, Ala substituted for 4 Ser/Thr residues from amino acids 355 through 365; and 4Ala-371Stop, 4Ala receptor truncated at amino acid 370. Even though arrestin binds robustly to receptors lacking phosphosites at amino acids 355–365, arrestin does not desensitize signaling.