CHAPTER TEN

Constitutive Activity of Bitter Taste Receptors (T2Rs) Sai P. Pydi, Rajinder P. Bhullar, Prashen Chelikani1 Department of Oral Biology, University of Manitoba, Winnipeg, Manitoba, Canada 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 1.1 Taste sensory proteins 1.2 Bitter taste receptors 1.3 Expression and localization of T2Rs 2. Activation Mechanism of T2Rs 2.1 Role of highly conserved TM residues in T2R activation 2.2 Role of T2R-specific residues in agonist-induced activation 3. Constitutive Activity in GPCRs 3.1 Strategies used to identify CAMs in T2Rs 3.2 T2R CAMs in the TM domain 3.3 T2R CAMs in ICL3 4. Role of CAMs in Discovery of Bitter Taste Blockers 5. Conclusion Conflict of Interest Acknowledgments References

304 304 305 306 307 310 311 311 313 315 316 318 319 320 320 320

Abstract G protein-coupled receptors (GPCRs) play a vital role in transmitting an extracellular stimuli or signal into an intracellular response in various cells. In some scenarios, GPCRs or their mutants can also signal in the absence of an agonist or an external stimulus, referred to as basal or constitutive activity, and those mutants are termed constitutively active mutants (CAMs). Bitter taste is one of the five basic tastes and is mediated by bitter taste receptors (T2Rs), which belong to the GPCR superfamily. The 25 T2Rs present in humans do not belong to any of the major GPCR classes, and their classification is ambiguous. The characterization of T2Rs in many extraoral tissues including the airways and upper respiratory tract, where they were shown to cause bronchodilation and influence host susceptibility to infection, underscores the therapeutic relevance of these receptors. Recent structure–function and pharmacological studies on T2Rs led to the identification of CAMs. In this review, we summarize the major findings on constitutive activity of T2Rs and their diverse roles. We discuss the usefulness of the T2R CAMs in

Advances in Pharmacology, Volume 70 ISSN 1054-3589 http://dx.doi.org/10.1016/B978-0-12-417197-8.00010-9

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2014 Elsevier Inc. All rights reserved.

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terms of the discovery of bitter taste blockers, elucidating the mechanisms of T2R activation and dissecting the physiological pathways.

ABBREVIATIONS 3DHC 3b-hydroxydihydrocostinolide 3HP 3b-hydroxypelenolide Ca2+ calcium CAMs constitutively active mutants ER endoplasmic reticulum GPCRs G protein-coupled receptors ICLs intracellular loops IP3 inositol triphosphate PLC phospholipase C TAS1R/T1Rs sweet/umami taste receptors TAS2R/T2R bitter taste receptors TM transmembrane

1. INTRODUCTION 1.1. Taste sensory proteins The sense of taste has a crucial role in evaluating the nutritional value of food prior to ingestion. Recognition of nutritionally important food components is facilitated by specialized proteins expressed on the surface of taste receptor cells present in taste buds on the tongue. Mammals can sense many compounds but distinguish between only five basic taste qualities, sour, salt, umami, sweet, and bitter. The existence of different taste sensations implies that each taste quality has a specific protein to mediate these tastes. The salt and sour tastes are sensed by ion channels, whereas sweet, umami, and bitter tastes are sensed by G protein-coupled receptors (GPCRs) (Chandrashekar et al., 2000). Salt taste (Na+) is predominantly transduced by a sodiumselective channel, an amiloride-sensitive epithelial sodium channel (ENaC) (Canessa et al., 1994; Matsunami, Montmayeur, & Buck, 2000). Sour taste is mediated by two different groups of acid-sensitive integral membrane proteins (Chaudhari & Roper, 2010; Lindemann, 2001; Roper, 2007, 2013). Sweet taste and umami tastes are sensed by heterodimers of T1Rs that belong to the class C GPCR superfamily (Isberg et al., 2014). Sweet taste is sensed by a heterodimer of T1R2 and

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T1R3 (Hoon et al., 1999; Nelson et al., 2001), whereas umami taste is sensed by a T1R1 and T1R3 heterodimer. In humans, umami taste is evoked by monosodium glutamate and aspartate, and it was only in the 1990s that this taste was accepted as one of the five basic tastes (Ikeda, 2002). Amino acid sequence reveals that both sweet and umami receptors have a long N-terminal sequence referred to as the venus flytrap domain, a characteristic feature of the class C GPCRs (Isberg et al., 2014). The venus flytrap domain forms the orthosteric site for ligand binding in T1Rs (Tomchik, Berg, Kim, Chaudhari, & Roper, 2007; Yarmolinsky, Zuker, & Ryba, 2009; Fig. 10.1).

1.2. Bitter taste receptors Bitter taste is the most complex and the least understood of all the five basic tastes and provides protection against ingestion of toxic substances. Plants secrete various bitter-tasting secondary metabolites to protect themselves

Bitter

So u

r

Umami

t

ee Sw

Salt

Figure 10.1 Schematic representation of the five basic taste sensory proteins. Umami and sweet tastes are sensed by heterodimers T1R1–T1R3 and T1R2–T1R3, respectively. The ligand binds to the N-terminal domain of umami and sweet receptors. Bitter taste is sensed by T2Rs, and the ligand binds within the extracellular and transmembrane domains. Salt and sour are sensed by ion channels.

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from herbivores, and herbivores have developed a taste against these bitter compounds. Some of the bitter compounds are known to be toxic; however, not all bitter-tasting compounds are toxic, and some of these compounds are known to have medicinal properties (Drewnowski & Gomez-Carneros, 2000; Potter, 1997). Mammals sense a wide variety of compounds, which include peptides, esters, phenols, lactones, flavonoids, terpenes, sulfimides, and organic salts as bitter (Behrens et al., 2004; Brockhoff, Behrens, Massarotti, Appendino, & Meyerhof, 2007; Drewnowski, 2001; Meyerhof et al., 2010). Binding of these compounds to the respective bitter taste receptors (T2Rs) results in the initiation of the bitter taste signal transduction. The canonical bitter taste signal transduction in the oral tissues is as follows: The activated T2R in turn activates a heterotrimeric G protein (Gagust, b1/b3, and g13) present on the intracellular surface of the taste receptor cell (McLaughlin, McKinnon, Spickofsky, Danho, & Margolskee, 1994; Spickofsky et al., 1994). Upon dissociation from Ga, the Gbg subunits activate phospholipase C (PLC) b2, which cleaves phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol triphosphate (IP3) (Caicedo, Pereira, Margolskee, & Roper, 2003; Huang et al., 1999; Kusakabe et al., 2000; McLaughlin, McKinnon, & Margolskee, 1992; Ruiz-Avila et al., 1995; Spielman, Huque, Nagai, Whitney, & Brand, 1994; Yan et al., 2001). IP3 facilitates release of calcium (Ca2+) from endoplasmic reticulum (ER) by activating type 3 IP3 receptors, present on ER. Released Ca2+ activates the transient receptor potential channel M5 that causes influx of cation and membrane depolarization, leading to the neurotransmitter release (Clark, Liggett, & Munger, 2012; Ming, Ruiz-Avila, & Margolskee, 1998; Pydi, Upadhyaya, Singh, Pal Bhullar, & Chelikani, 2012; Spielman et al., 1996).

1.3. Expression and localization of T2Rs Expression of T2Rs in the oral cavity varies across the species and so does the ability of vertebrates to taste different compounds (Davis et al., 2010; Go, Satta, Takenaka, & Takahata, 2005; Wooding et al., 2006). Initial studies in different mouse strains showed differences in bitter taste aversion, which suggested the existence of a group of bitter taste sensing genes (Lush, Hornigold, King, & Stoye, 1995). Few years later, the proteins encoded by these genes were discovered and functionally characterized in both humans and rodents and were referred to as T2Rs (Adler et al., 2000; Chandrashekar et al., 2000). The bitter taste sensing genes are referred to

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as TAS2Rs. Humans have 25 TAS2Rs that are intronless and distributed on chromosomes 5, 7, and 12 (Andres-Barquin & Conte, 2004; Conte, Ebeling, Marcuz, Nef, & Andres-Barquin, 2002, 2003). In humans, majority of the 25 TAS2Rs are clustered on two chromosomes, 7 and 12. 9 and 15 TAS2Rs were found clustered on chromosomes 7 and 12 each, respectively, and with only TAS2R1 found to be present on chromosome 5. In rodents, bitter taste sensing system is highly developed in both mouse and rat; 31 TAS2R genes are present (Singh, Vrontakis, Parkinson, & Chelikani, 2011). In mouse, these genes are distributed on chromosomes 2, 6, and 15, and in rat, these genes are present on chromosomes 2, 3, and 4 (Andres-Barquin & Conte, 2004; Bachmanov & Beauchamp, 2007; Wu, Chen, & Rozengurt, 2005). In addition to the oral cavity, T2Rs are expressed in various extraoral tissues. In the past few years, numerous reports characterized the expression and function of T2Rs in nongustatory tissues, summarized in Table 10.1. However, the distribution (i.e., number of T2Rs) and expression levels of these receptors varied from tissue to tissue. In upper respiratory tract, T2Rs are expressed on full-form SCCs and regulate the airway reflex in response to toxic substances or irritants in mice (Finger et al., 2003; Tizzano et al., 2010). In the lower respiratory tract, T2Rs are expressed on cholinergic brush cells and regulate the respiratory rate in mice (Krasteva et al., 2011). In humans, respiratory epithelial cells express T2Rs that help in clearing pathogens or irritants by increasing the movement of cilia (Shah et al., 2009). In airway smooth muscle cells of human and mouse, T2Rs were found to be involved in bronchodilation and relaxation (An et al., 2012; Clark et al., 2012; Deshpande et al., 2010). T2Rs are also expressed in human and rodent heart cells and were hypothesized to function as nutrient sensors (Foster et al., 2013). T2R expression was upregulated in the leukocytes isolated from severe and therapy-resistant asthma patients and were reported to have anti-inflammatory roles in asthma (Orsmark-Pietras et al., 2013). Recently, T2Rs were found to be involved in spermatogenesis (Li, 2013). The wide distribution of T2Rs in the human body offers new avenues for research, directed at understanding their pharmacological and physiological relevance (Clark et al., 2012).

2. ACTIVATION MECHANISM OF T2Rs The most interesting question in bitter taste research is how relatively few T2Rs are capable of detecting hundreds of structurally diverse

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Table 10.1 Distribution of bitter taste receptors in various systems/tissues/organs, cell types they are expressed in, and their function (Clark et al., 2012; Foster, Roura, & Thomas, 2014) Tissue/organ/ system Cell type Species Function References

Brain Brain stem, cerebellum

C6 glial cells, primary neuronal cells

Frontal cortex Nasal cavity

Rat

Singh, Vrontakis, et al. (2011)

Rat

Dehkordi et al. (2012)

Human

Garcia-Esparcia et al. (2013)

Mouse, Solitary chemosensory rat cells

Vomeronasal organ

Trigeminal nerve response and respiratory depression

Mouse

Tizzano et al. (2010) Tizzano, Cristofoletti, Sbarbati, and Finger (2011) Voigt et al. (2012)

Tongue

Taste receptor Human, Sensing bitter taste Adler et al. (2000), Chandrashekar cells rat, et al. (2000), mouse Roper (2013)

Trachea

Mouse Cholinergic chemosensory cells

Thymus

Mouse Human

Krasteva et al. Bitter ligandinduced regulation (2011) of breathing Voigt et al. (2012)

Immune system

Leukocytes

Anti-inflammatory Orsmark-Pietras role in asthma et al. (2013)

Airways

Airway Human epithelial cells

Clearance of inhaled pathogens using cilia

Shah, Ben-Shahar, Moninger, Kline, and Welsh (2009)

Human Upper respiratory epithelial cells

Antibacterial effects

Lee et al. (2012)

Human, Bronchodilation, Airway decreased airway smooth muscle mouse obstruction in a cells mouse model of asthma

Deshpand et al. (2010), Zhang et al. (2013)

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Table 10.1 Distribution of bitter taste receptors in various systems/tissues/organs, cell types they are expressed in, and their function (Clark et al., 2012; Foster, Roura, & Thomas, 2014)—cont'd Tissue/organ/ system Cell type Species Function References

Aorta

Rat Vascular smooth muscle cells

Lund et al. (2013)

Heart

Myocytes (rat) Human, rat, mouse

Foster et al. (2013)

Bone marrow Bone marrow Human stromal cells

Lund et al. (2013)

Stomach

Colombo, Trevisi, Gandolfi, and Bosi (2012), Wu et al. (2005)

Small intestine (duodenum, jejunum)

Rat, mouse, pig STC-1 cells

Large intestine (colon, cecum) Testis

Multiple cell types

Rat, mouse

Regulation of gut peptide secretion

Jeon, Zhu, Larson, and Osborne (2008), Wu et al. (2005)

Human, Anion secretion rat

Dotson et al. (2008), Kaji, Karaki, Fukami, Terasaki, and Kuwahara (2009)

Mouse

Li (2013), Voigt et al. (2012)

Spermatogenesis

compounds (Meyerhof et al., 2010; Pronin, Tang, Connor, & Keung, 2004). Some T2Rs are activated by a wide range of compounds, whereas some are activated by a single bitter compound (Behrens et al., 2009; Born, Levit, Niv, Meyerhof, & Behrens, 2013; Brockhoff et al., 2007; Sakurai, Misaka, Ueno, et al., 2010). T2R31, T2R43, and T2R46 have around 85% sequence homology, but they bind to different agonists (Brockhoff, Behrens, Niv, & Meyerhof, 2010), giving credence to the hypothesis that each T2R might have a unique ligand-binding pocket. It was shown previously in class A GPCRs that there are three levels of amino

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acid conservation that can be considered in understanding the activation mechanism of a given subfamily (Chelikani et al., 2007; Smith, 2010, 2012). To understand the molecular mechanism of activation of T2Rs, we described the molecular determinants in terms of conserved residues, by targeting both highly conserved residues in transmembrane (TM) helices and receptor-specific residues to understand the agonist-specific activation of individual T2Rs.

2.1. Role of highly conserved TM residues in T2R activation Amino acid sequence analysis of 188 T2Rs across the species identified 13 highly conserved residues in TM1, TM2, TM3, TM5, and TM7 (Pydi, Bhullar, & Chelikani, 2012; Singh, Pydi, Upadhyaya, & Chelikani, 2011). Strikingly, there was no similarity between the highly conserved motifs present in class A GPCRs and T2Rs. For example, class A GPCRs have highly conserved LxxD motif in TM2, D/ERY motif in TM3, CNxP motif in TM6, and NPxxY motif in TM7 (Arakawa et al., 2011). However, these motifs are completely absent in T2Rs (Favre et al., 2005; Lu, Coetsee, White, & Millar, 2007; Mirzadegan, Benko, Filipek, & Palczewski, 2003; Zhang, Mizrachi, Fanelli, & Segaloff, 2005). Interestingly, T2Rs have two unique and conserved motifs, which are LxxxR in TM2 and LxxSL in TM5 (Singh, Pydi, et al., 2011). The only two TM residues that are conserved in both T2Rs and class A GPCRs are N1.50 in TM1 and L2.50 in TM2. The number in superscript is the Ballesteros and Weinstein number (Ballesteros & Weinstein, 1995). Highly conserved residues in TM1 of T2Rs are N1.50 and I1.53. Alanine replacement of Asn1.50 in T2R1 resulted in complete loss of function (Singh, Pydi, et al., 2011). Molecular modeling analysis showed that Asn1.50 is involved in a key hydrogen bonding (H-bond) network that connected TM1 with TM2 and TM7. A similar intra- and interhelical H-bond interaction of Asn1.50 was observed in class A GPCRs (Deupi & Kobilka, 2007; Kobilka & Deupi, 2007; Park, Scheerer, Hofmann, Choe, & Ernst, 2008; Rasmussen et al., 2007; Scheerer et al., 2008). Other conserved residue in TM1 of T2Rs was I1.53 and mutation of this residue to alanine in T2R1 leads to receptor hyperactivity (Singh, Pydi, et al., 2011). The most conserved motif (97% conserved) in T2Rs is LxxSL present at the intracellular end of TM5. Mutational and molecular modeling studies of residues in this motif showed that this motif plays a crucial structural role in stabilizing the cytoplasmic end of TM5 and a functional role by interacting

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with residues in the third intracellular loop (ICL3) (Pydi, Singh, Upadhyaya, Bhullar, & Chelikani, 2014; Singh, Pydi, et al., 2011).

2.2. Role of T2R-specific residues in agonist-induced activation T2Rs bind to structurally diverse compounds, and to accommodate these compounds, the binding pocket of T2Rs should be flexible and selective. To understand this ligand specificity of T2Rs, a recent study focused on a subfamily of eight closely related T2Rs, T2R31, and T2R44 to T2R50 (Brockhoff et al., 2010). Among these eight T2Rs, T2R31, T2R44, and T2R46 have
85% sequence identity, mainly in the TM helices with some variation toward the extracellular side of the receptors. Chimeric receptors of T2R31 and T2R46 were generated to identify the receptor regions involved in ligand-specific activation. Two residues present at the extracellular side of TM7 were swapped, and the chimeric receptors assayed for agonist activity. Double-mutated T2R46 was activated by the T2R31 agonist, aristolochic acid, and the chimeric T2R31 was activated by T2R46 agonists, absinthin and strychnine (Brockhoff et al., 2010). This study confirmed the agonist specificity of two closely related T2Rs and the role of these key residues in receptor activation. Interestingly, some T2Rs show high specificity in ligand binding and differentiate between anomers of the same compound. Gentiobiose (6-Ob-D-glucopyranosyl-D-glucose) and isomaltose (6-O-a-D-glucopyranosylD-glucose) are anomers; the former one tastes bitter, whereas the latter tastes sweet. T2R16 showed robust intracellular calcium mobilization when treated with gentiobiose; however, it was not activated by isomaltose (Sakurai, Misaka, Ishiguro, et al., 2010; Sakurai, Misaka, Ueno, et al., 2010).

3. CONSTITUTIVE ACTIVITY IN GPCRs Constitutive or spontaneous activity is the production of a second messenger or a downstream signal by a receptor in the absence of an agonist. In GPCRs, the first evidence about constitutive activity was presented for the d opioid and b2-adrenergic receptors in the 1980s (Cerione et al., 1984; Costa & Herz, 1989; Koski, Streaty, & Klee, 1982). More than 60 GPCRs exhibit constitutive activity (Seifert & Wenzel-Seifert, 2002). Based on available literature, around 50% of the known constitutively active wildtype GPCRs couple with Gi/Go proteins and 25% couple with Gs and Gq proteins, respectively. Constitutive activity of a receptor also depends on the cell type in which they are expressed. For example, histamine

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receptor (H2), serotonin receptor (5-HT1), and cannabinoid receptors (CB1 and CB2) showed high basal activity when expressed in CHO cells compared to other cell types (Bouaboula et al., 1997; Smit et al., 1996; Varga et al., 1998). Mutations in GPCRs also lead to constitutive activity, and the mutants are known as constitutively active mutants (CAMs). In class A GPCRs, there are a number of regions, such as the D/ERY motif present at the cytoplasmic end of TM3, that are hot spots for CAMs (Scheer et al., 2000; Scheer, Fanelli, Costa, De Benedetti, & Cotecchia, 1996, 1997). Several receptor subtypes show constitutive activity, but it is very difficult to recognize this activity in physiological conditions due to low expression levels of the receptors. CAMs can be classified into four broad types based on their response to agonist at intrinsic or saturation concentration compared to a wild-type receptor treated with the same agonist concentration (Fig. 10.2). (1) Normoresponsive CAMs, their agonist response is similar to wild type. (2) Hyperresponsive CAMs, their agonist response is more than wild type. (3) Hyporesponsive CAMs, their agonist response is less than the wild type. (4) Nonresponsive CAMs, these CAMs do not show an agonistdependent calcium release (Seifert & Wenzel-Seifert, 2002).

nre sp CA onsiv M e No

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po Hy

rre s CA pons ive M

pe Hy

ore s CA pons M ive

No rm

Wil d

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e

Calcium mobilized (DRFU)

Basal Agonist

Figure 10.2 General classification of constitutively active mutants based on their response to agonist. White columns represent the basal activity and black columns represent the agonist-dependent stimulation (intrinsic value).

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3.1. Strategies used to identify CAMs in T2Rs We studied more than 100 mutants in different regions of T2R1 and T2R4 and characterized their constitutive activity. T2R CAM constitutive activity was assessed by measuring the basal (agonist-independent) second messenger levels in the whole cell expressing the mutant receptor. In cell-based experiments, it is easier to amplify the receptor and measure the cellular responses. 3.1.1 Measuring basal activity of the receptor 3.1.1.1 Calcium mobilization assay

For Gs- and Gq-coupled GPCRs, constitutive activity is measured in terms of increase in basal intracellular cAMP and Ca2+ levels. While taste receptors are known to couple with their cognate Ga protein, gustducin, however, it was reported that the T2Rs can signal through pathways independent of gustducin, as gustducin knockout mouse was still able to sense bitter stimuli (Caicedo et al., 2003). As described in Section 1.2, the increase in IP3 through the activation of PLC b2 by the Gbg subunits is thought to be the main mode of signal transduction by T2Rs. In T2R studies using heterologous systems, a chimeric Ga protein Ga16/gust44 designed and characterized by Dr. Udea is used routinely to produce the second messenger (IP3) signal (Ueda, Ugawa, Yamamura, Imaizumi, & Shimada, 2003). T2R4 and/or mutants are cotransfected with the chimeric Ga16/gust44 protein in HEK293T cells (Pydi, Bhullar, et al., 2012; Pydi, Chakraborty, Bhullar, & Chelikani, 2013; Pydi et al., 2014). The basal activity of the receptor was represented in terms of calcium mobilized in the absence of any ligand. Intracellular calcium levels were measured by incubating cells with calcium-binding dye Fluo-4 NW and the fluorescence readout was measured using FlexStation-3 fluorescence plate reader (Molecular Devices, CA). Cells transfected with empty vector and Ga16/gust44 were used as a control, and the calcium levels of wild type and mutants were compared statistically by normalizing to wild-type receptor cell surface expression as determined by ELISA. Mutants with significantly high basal calcium might be possible CAMs, but it is not true in all cases. The constitutive activity of the receptor was further characterized in detail; see Section 3.1.1.2. 3.1.1.2 Effect of receptor density on calcium mobilization

In our studies, several T2R mutants showed high basal activity in the heterologous expression system (Pydi, Bhullar, et al., 2012; Pydi et al., 2014). In order to confirm true CAMs, we characterized the constitutive activity of these mutants in detail by measuring the change in basal calcium levels with

314

Calcium mobilized (DRFU)

Sai P. Pydi et al.

WT – T2R CAM – T2R

3 ´ mg 2 ´ mg

1 ´ mg

Cell surface expression %

Figure 10.3 Pharmacological characterization of constitutive activity in T2Rs. A linear relationship was observed between increase in receptor expression and basal activity (i.e., calcium mobilized). With an increase in the amount of transfected DNA (mg), increased receptor expression was observed. Figure represents the typical slope of a wild-type T2R and a constitutively active mutant of T2R.

an increase in receptor density. We transfected HEK293T cells with 1, 2 , and 3  concentrations of T2R4 or mutant DNA, with the rationale that an increase in DNA concentration in transfection will increase receptor expression. As expected, an increase in receptor cell surface expression was observed (Fig. 10.3). If the mutant is a CAM, with an increase in receptor expression, the basal calcium levels will also increase. A linear relationship was observed between the receptor expression level and the constitutive activity (Fig. 10.3). Slopes of the T2R4 and mutants were compared, and some mutants showed 2- to 10-fold increase in constitutive activity when compared to wild-type T2R4 (Pydi, Bhullar, et al., 2012; Pydi et al., 2014). 3.1.1.3 IP3 assay

The calcium imaging dye used in the aforementioned experiments, Fluo-4 NW (no wash) is a fluorometric dye with a high Z0 factor and eliminates the need for the washing steps, which is usually required after loading the cells with the dye. Its main drawback is that it is not a ratiometric dye; hence, quantifying the absolute levels of basal calcium levels using this dye is not possible. Therefore, to quantify the basal levels of the second messengers generated, we measured the absolute levels of basal IP3 generated using a commercially available IP3 fluorescence polarization (FP) assay kit (HitHunter IP3 FP assay; DiscoveRx, Fremont, CA). First, an IP3 standard

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graph was constructed using known concentrations of IP3, and this graph was used to measure the basal IP3 levels in the wild-type GPCRs and mutants (Chakraborty et al., 2013, 2012; Pydi et al., 2014). 3.1.2 Molecular modeling In recent times, an increased number of molecular modeling studies have focused on understanding the constitutive activity of GPCRs. The activation of GPCRs is a dynamic process, and it involves multiple conformational changes to attain active confirmation. In conjunction with the experimental data, molecular modeling data provide useful information on the start and end-point trajectory of simulations. In the absence of T2R crystal structures and any biophysical studies, we modeled ground-state T2Rs using class A GPCR crystal structures as templates. Ground-state T2R4 model was built using rhodopsin crystal structure (PDB ID: 1U19) and the CAM– T2R4 models were built using the crystal structure of constitutively active rhodopsin mutant bound to C-terminal peptide (PDB ID: 2X72) (Standfuss et al., 2011). These models were energy-minimized using steepest descent and conjugate gradient algorithms. Molecular dynamic simulations of 10 ns were performed with time steps of 2 fs, collecting trajectory data at every 500 ps. These models were then analyzed to study the helical movements, rotational changes of the TM regions, and changes in the orientation and roles of various residues in inter- or intrahelical interactions (Pydi, Bhullar, et al., 2012; Pydi et al., 2014).

3.2. T2R CAMs in the TM domain Sequence alignment of 188 T2Rs showed five conserved residues in TM1, TM2, and TM7 (Singh, Pydi, et al., 2011). Ser7.47 is present in the TM7 and is conserved in 71% of T2Rs, 18% of T2Rs have proline, whereas 11% of T2Rs do not have any of the aforementioned two residues. Using T2R4 as the base receptor, we made three types of mutants to understand the role of this residue in receptor activation (Pydi, Bhullar, et al., 2012). First, we mutated serine to alanine, with the expectation that this mutant will have minimal effect on folding. Second, we mutated it to conserved threonine, and finally, the third substitution was a serine to proline, as proline is present at position 7.47 in 18% of the T2Rs. We carried out pharmacological characterization of these T2R mutants (Pydi, Bhullar, et al., 2012). All the three mutants were properly expressed and targeted to the cell surface. To characterize the basal and intrinsic activity of wild type and three mutants, we stimulated HEK293T cells transiently

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expressing these receptors with buffer alone (agonist-independent) and with single saturation concentration of agonist quinine (intrinsic), and the intracellular Ca2+ mobilization was measured. Interestingly, S285A mutant showed a twofold increase in intrinsic signal, when compared to wild-type T2R4 and S285T or S285P mutants. Furthermore, among the three mutants, only S285A showed threefold increase in basal activity. To characterize its CAM phenotype, we carried out a pharmacological assay described in detail in Section 3.1.1.2. Receptor expression levels were normalized to wild-type T2R4 cell surface expression and plotted against the basal Ca2+ levels. S285A showed fivefold increase in its slope value when compared to wild-type T2R4 (Pydi, Bhullar, et al., 2012). This confirmed the constitutive activity of S285A mutant and this was the first report of a CAM in any taste receptor (Pydi, Bhullar, et al., 2012). This was a hypersensitive CAM as it showed a twofold increase in intrinsic Ca2+ levels when compared to wild-type T2R4. In order to understand the molecular mechanism of activation of the S285A CAM, we built molecular models of the inactive state of wild-type T2R4 and constitutively active S285A using rhodopsin inactive state and constitutively active crystal structures. We also introduced conserved structural waters in our models. In the inactive state model, interhelical hydrogen bonding was observed between Ser7.47 and Arg2.54, whereas in the case of S285A model, these interactions were lost and Arg2.54 established contacts with Asn1.50. In addition, in the wild-type T2R4 model, backbone interactions were observed between Gly1.46 and Asn1.50, whereas these contacts were lost in the CAM model. This confirmed that Ser7.47 maintains the T2R4 in the inactive confirmations by forming intermolecular interactions connecting TM1–TM2–TM7. When this residue was mutated to alanine, the interactions were lost and the receptor attained active confirmation, whereas the conservative substitution with threonine (S285T) retained these interactions as observed in wild-type T2R4. Thus far, the S285A mutant is the only CAM reported that is present in the TM domain of T2Rs.

3.3. T2R CAMs in ICL3 Recent active and inactive crystal structures of GPCRs showed major structural rearrangements on the intracellular side, especially at ICL3. In addition, structure–function studies showed that ICL3 performs diverse functions in different GPCR classes. For example, in neuropeptide Y1 receptor, ICL3 locks the receptor in the ground (inactive) state in which mutations in this

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regions lead to constitutive activity (Chee et al., 2008), whereas in melanocortin-3 receptor, ICL3 is important for signaling and ligand binding (Wang & Tao, 2013). To understand the role of ICL3 in T2Rs, an alanine scan mutagenesis of this loop was recently performed (Pydi et al., 2014; Fig. 10.4). Statistically significant basal activity was observed for R213A, H214A, Q216A, N227A, V234A, and M237A mutants. However, only H214A, Q216A, V234A, and M237A exhibited a true CAM phenotype with constitutive activity ranging from 2- to 10-fold over wild-type T2R4 (Pydi et al., 2014). Among these four CAMs, H214A displayed 10-fold increase in constitutive activity, the highest reported for a T2R CAM thus far. Strikingly, His2145.53 is conserved in 96% of the T2Rs (i.e., 24 of the 25 T2Rs have a His at this position) and is present at the TM5–ICL3 interface. The four ICL3 CAMs were localized at the amino and carboxyl terminus of ICL3. Molecular modeling studies revealed an intricate network of sidechain and backbone interactions involving the CAMs with the conserved FLAG tag

Extracellular region

D D D K L

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T E R

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F F

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M

T

I V

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L

G

T

N

L

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Figure 10.4 Secondary structure representation of T2R4 amino acid sequence. Transmembrane regions are represented in cylinders and the helices present in intracellular surface are represented in small squares. T2Rs have a short N-terminal and a short C-terminal region. An octapeptide sequence, FLAG was attached at the N-terminal of the receptor to study cell surface receptor expression. Constitutively active mutants were represented in dark grey color. These residues are distributed in transmembrane and intracellular regions. The highly conserved transmembrane residues are represented in gray color.

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LxxSL motif on TM5 and movement of TM6 relative to TM5. In the inactive T2R4 model, Leu207 and Ser210 residues of LxxSL motif interact with ˚ Met237 and His214 of ICL3. While in the CAM model, there is a small 2 A outward movement of TM6 at the cytoplasmic end. This causes a major rearrangement of interactions involving the CAMs and residues of the LxxSL motif leading to the receptor adopting an active conformation (Pydi et al., 2014). However, the molecular mechanism underlying the observed 10-fold increase in constitutive activity for the H214A mutant remains to be determined.

4. ROLE OF CAMs IN DISCOVERY OF BITTER TASTE BLOCKERS Antagonists and inverse agonists of T2Rs are referred to as bitter taste blockers or bitter blockers. T2Rs are activated by hundreds of bitter compounds, but only four bitter blockers are known thus far, which block a few T2Rs. High-throughput screening followed by competition dose–response assays identified a synthetic bitter antagonist GIV3727; this is the first antagonist discovered for a T2R. It is an orthosteric inhibitor for T2R31 with an IC50 value of 6 mM and completely inhibits the activity of T2R40 and T2R43 in the presence of their agonists humulone and aristolochic acid, respectively, whereas for T2R4 and T2R7, it reduced the activity by only 50% in the presence of colchicine and cromolyn (Slack et al., 2010). Probenecid was found to inhibit the agonist activity of T2R16, T2R38, and T2R43, and the IC50 values for T2R16 and T2R43 were found to be around 292 and 211 mM, respectively (Greene et al., 2011). 3bHydroxypelenolide (3HP) and 3b-hydroxydihydrocostinolide (3DHC) are sesquiterpene lactones and are known to activate various T2Rs but inhibit the activity of T2R46. In addition, 3HP inhibits T2R30, T2R31, and T2R43, whereas 3DHC inhibits agonist activity of T2R30 and T2R40 (Brockhoff et al., 2011; Greene et al., 2011). Costa and Herz demonstrated that even in the absence of a bound agonist, GPCRs could produce a downstream cellular signal. Furthermore, they reported on compounds that can reduce this activity, which they coined as negative antagonists, but later, these compounds were renamed as inverse agonists (Costa & Herz, 1989; Costa, Lang, Gless, & Herz, 1990). Negative efficacy or inverse agonism was not properly utilized in drug discovery, because of various technical difficulties. Significant advances in experimental methods and detection systems accelerated the discovery of CAMs and in

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identification of novel inverse agonists. CAMs have high basal activity when compared to wild-type GPCRs and inverse agonists inhibit this basal activity. However, the inhibition of basal activity ranges from 20% to 90%, which can depend on the compounds as well as the receptor the inverse agonist blocks (Black & Shankley, 1995). Inverse agonist are defined to have (1) efficacy, whereas the neutral antagonists have (0) efficacy (Bylund & Toews, 2014). Thus far, only eight CAMs were identified in T2Rs (Pydi, Bhullar, et al., 2012; Pydi et al., 2014). Of the eight T2R CAMs, only the H214A mutation in T2R4 showed up to 10-fold increase in constitutive activity, and interestingly, this histidine residue is highly conserved in T2Rs. The main challenge in finding appropriate ligands that can block or decrease T2R activity is lack of effective and robust assay technologies. An ideal assay should be economical and give information on whether a given ligand has the same effect on all the 25 T2Rs or shows T2R subtype-specific effects. The highly conserved nature of His2145.53 in all the 25 T2Rs enables a mutation to be made at position 5.53 in all the 25 T2Rs and to use these CAMs as pharmacological tools to screen for bitter taste blockers (Pydi SP, Bhullar RP, and Chelikani P. PCT application, CA2013/050313). Using the technique described in the preceding text, we can quickly assess the effect of a given ligand (whether it has agonist or antagonist or inverse agonist activity) on all the 25 T2Rs, by testing only the wild-type T2R and a CAM at position 5.53 (corresponds to His214 in T2R4) in each of the 25 T2Rs (Pydi SP, Bhullar RP, and Chelikani P. PCT application, CA2013/050313). This is more economical and less labor-intensive compared to the traditional competition-based dose–response assays for characterizing T2R ligands. Furthermore, smaller amounts of ligands are needed for the assays using CAMs.

5. CONCLUSION Although T2Rs were discovered in 2000, considerable advances in understanding the pharmacology of these receptors have been made only in the past 5 years. In the same time span, landmark studies showed that T2Rs are expressed in extragustatory systems with varying functions, including diseased and pathological conditions. Recent studies from our lab identified several conserved and nonconserved CAMs in TM and ICL regions of T2Rs. While discovery of new T2R CAMs is bound to grow in the next few years as more structure–function and pharmacological

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studies are pursued, some of these T2R CAMs can be used as pharmacological tools (as described in Section 4) to discover novel ligands (bitter taste blockers) that can reduce the activity of these receptors. The discovery of T2R blockers has both applied value (sought-after by the nutraceutical and flavor industry) and pharmacological importance as it would allow the dissection of signaling pathways that involve T2Rs.

CONFLICT OF INTEREST The authors have a patent PCT application CA2013/050313 submitted on bitter taste receptors described in this review article.

ACKNOWLEDGMENTS This work was supported by a discovery grant (RGPIN 356285) from the Natural Sciences and Engineering Research Council of Canada to P. C., a graduate studentship from MHRC/MICH to S. P. P., and an MMSF Allen Rouse Career Award to P. C.

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