28.18: Signal Transduction - Taste (Gustation)

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Learning Goals (ChatGPT o3-mini)

• Understand Taste GPCR Classification and Structure:

  • Describe the two main families of taste GPCRs (Type I and Type II), including the heterodimeric TAS1Rs for sweet and umami tastes and the monomeric receptors for bitter and kokumi sensations.
  • Identify key structural domains (e.g., Venus flytrap domain, cysteine-rich domain, transmembrane domain) and explain how these contribute to receptor function and ligand specificity.

• Explain Taste Signal Transduction Pathways:

  • Outline the intracellular signaling cascades activated by taste GPCRs, emphasizing the roles of heterotrimeric G proteins (e.g., Gα‑gustducin, Gβγ), PLCβ2, IP3-mediated Ca²⁺ release, and subsequent events leading to ATP release and neurotransmission.
  • Compare the mechanisms by which sweet, umami, bitter, and kokumi receptors transduce signals from tastants to the gustatory cortex.

• Analyze Receptor–Ligand Interactions:

  • Discuss how orthosteric agonists bind to specific receptor domains (e.g., VFT in TAS1Rs) to induce conformational changes necessary for receptor activation.
  • Evaluate the impact of allosteric modulators—including positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs)—on receptor conformation and signal transduction, using examples such as lactisole, NHDC, and various synthetic peptides.

• Explore Molecular Modeling and Mutagenesis Approaches:

  • Explain how heterologous expression, site-directed mutagenesis, and computational docking (e.g., using AlphaFold and AutoDock Vina) contribute to identifying ligand binding sites and deciphering the structural basis of receptor activation and modulation.
  • Interpret data from molecular models (e.g., interactive iCn3D structures) to determine how key residues in receptor domains influence ligand binding and receptor function.

• Examine Receptor Trafficking and Cell Surface Expression:

  • Describe the importance of proper receptor folding, dimerization, and trafficking in ensuring effective cell surface expression of taste GPCRs, with an emphasis on the roles of specific extracellular and transmembrane regions.

• Understand Bitter Taste Receptor Diversity and Specificity:

  • Compare the ligand-binding properties of broadly tuned versus narrowly tuned bitter taste receptors, and discuss how receptor sequence variability influences agonist specificity and affinity.
  • Evaluate the pharmacological strategies used to identify bitter receptor inhibitors and the implications for reducing off-target bitterness in foods and drugs.

• Investigate the Role of the Calcium-Sensing Receptor (CaSR) in Kokumi Sensation:

  • Explain how CaSR, a class C GPCR, mediates kokumi taste through interactions with various ligands (e.g., calcium ions, amino acids, glutathione) and discuss its downstream signaling pathways.
  • Analyze how allosteric modulation of CaSR by endogenous and synthetic compounds contributes to flavor enhancement and the potential extraoral roles of kokumi peptides.

• Integrate Physiological and Pharmacological Perspectives:

  • Relate the molecular mechanisms of taste GPCR activation to physiological taste perception and ingestion decisions.
  • Discuss the current challenges in taste receptor research, including difficulties in crystallizing taste GPCRs, and explore how emerging structural biology techniques (e.g., cryo-EM and serial femtosecond crystallography) may overcome these obstacles.

By achieving these goals, students will gain a comprehensive understanding of the structure, function, and pharmacology of taste GPCRs, enabling them to appreciate how these receptors contribute to taste perception and how they can be targeted to modulate taste responses in food science and medicine.

This chapter section is taken in large part from Ahmad and Dalziel. Front. Pharmacol., 30 November 2020 | https://doi.org/10.3389/fphar.2020.587664. Creative Commons Attribution License (CC BY)

G Protein-Coupled Receptors in Taste Physiology and Pharmacology

Heterotrimeric G protein-coupled receptors (GPCRs) comprise the largest receptor family in mammals and regulate most physiological functions. Besides mediating the sensory modalities of olfaction and vision, GPCRs also transduce signals for three basic taste qualities: sweet, umami (savory taste), and bitter, as well as the flavor sensation kokumi. Taste GPCRs reside in specialized taste receptor cells (TRCs) within taste buds. Type I taste GPCRs (TAS1R) form heterodimeric complexes that function as sweet (TAS1R2/TAS1R3) or umami (TAS1R1/TAS1R3) taste receptors, whereas Type II are monomeric bitter taste receptors or kokumi/calcium-sensing receptors. Sweet, umami, and kokumi receptors share structural similarities in containing multiple agonist binding sites with pronounced selectivity. In contrast, most bitter receptors contain a single binding site broadly tuned to a diverse array of bitter ligands in a non-selective manner. Tastant binding to the receptor activates downstream secondary messenger pathways, leading to depolarization and increased intracellular calcium in TRCs, innervating the brain's gustatory cortex. Despite recent advances in understanding the relationship between agonist binding and the conformational changes required for receptor activation, several major challenges and questions remain in taste GPCR biology, discussed in the present review. In recent years, intensive integrative approaches combining heterologous expression, mutagenesis, and homology modeling have provided insight regarding agonist binding site locations and orthosteric and allosteric modulation molecular mechanisms. In addition, studies based on transgenic mice, utilizing either global or conditional knock-out strategies, have provided insights into taste receptor signal transduction mechanisms and their roles in physiology. However, the need for more functional studies in a physiological context is apparent. A crystal structure of taste receptors would enhance it for a more complete picture of their pharmacological mechanisms.

Introduction

G protein-coupled receptors (GPCRs) are eukaryotes' largest and most diverse group of membrane receptors. A wide variety of ligands activate them in the form of light energy, lipids, sugars, peptides, and proteins, which convey information from the outside environment into the cell to mediate their corresponding functional responses. The conformational changes of GPCRs upon ligand binding initiate a series of biochemical reactions within the cell. These intracellular reactions regulate sensory functions of smell, taste, and vision, as well as a wide variety of physiological processes such as secretion, neurotransmission, metabolism, cellular differentiation, inflammation, and immune responses. Taste is one of the most important sensations in human life, enabling us to perceive different tastes from the diverse range of food available in nature, and is a major determinant of our ingestion decisions.

The anatomical units of taste detection are taste receptor cells (TRCs) assembled into taste buds distributed across different tongue and palate epithelium papillae. Taste processing is first achieved at the level of TRCs that specific tastants activate. They transmit information via sensory afferent fibers to the gustatory cortex in the brain for taste perception, as shown in Figure \(\PageIndex{1}\).  Three different morphologic subtypes of TRCs in taste buds sense the different tastes we perceive. Type I glial-like cells detect salty taste, while type II cells expressing GPCRs detect sweet, umami, and bitter tastes. Type III cells sense sour stimuli.

Figure \(\PageIndex{1}\): A schematic diagram shows taste signal transmission between the tongue and brain. Taste buds in different tongue and palate papillae contain taste receptor cells (TRC), which contain taste G protein-coupled receptors (GPCRs). The left side shows how afferent nerves transmit a signal to the gustatory cortex in the brain via cranial/glossopharyngeal nerves. The right side shows taste buds with taste TRCs and a simplified signal transduction pathway of taste receptors. Taste GPCRs are activated by a tastant that recruits a specific G protein that further induces intracellular calcium release (created with BioRender.com).

Type 1 taste GPCRs transduce sweet and umami stimuli, while Type 2 taste GPCRs sense bitter taste.  See Figure \(\PageIndex{2}\) and Table \(\PageIndex{1}\) for details.  The more recently described kokumi sensation is mediated by another GPCR, the calcium-sensing receptor (CaSR). Taste GPCRs are activated by specific taste ligands present in foods and recruit G proteins to activate downstream signaling effectors, as shown in Figure \(\PageIndex{3}\) and Table\(\PageIndex{1}\).

 

Figure \(\PageIndex{2}\): (created with BioRender.com).

Table \(\PageIndex{1}\): Taste GPCRs classification and their downstream signaling regulators.

 

Figure \(\PageIndex{3}\):  Schematic representation of signal transduction pathway of sweet, umami, bitter, and kokumi-calcium sensing receptors (CaSR) in taste receptor cells on the tongue.

Ligand-induced stimulation of the sweet (TAS1R2/TAS1R3), umami (TAS1R1/TAS1R3), bitter receptors (TAS2Rs), and kokumi sensation expressed in type II taste cells within taste buds activate a trimeric G protein composed of α-gustducin (Gα-gust) in sweet, umami, bitter, and Gα-q/11 in kokumi-receptor and a complex consisting of Gβγ proteins. The released Gβγ-complex activates phospholipase C isoform β2 (PLCβ2) which then induces the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); the second messenger IP3, in turn, activates the IP3 receptor (IP3R), an intracellular ion channel that allows Ca²⁺ release from the intracellular endoplasmic reticulum (ER store). An increase in intracellular Ca²⁺ then activates the complex of transient receptor potential cation channel subfamily M member 4 and 5 (TRPM4/5), which are plasma membrane-localized sodium-selective channels, leading to depolarization and subsequent activation of voltage-gated sodium channels (VGSC). The combined action of increased Ca²⁺ and membrane depolarization activates the complex of calcium homeostasis modulator 1 and 3(CALHM1/3) and pannexin1 channels, thus releasing the neurotransmitter ATP. Increased ATP, in turn, activates P2X ionotropic purinergic receptors 2 and 3 (P2X2/P2X3) on the afferent cranial nerve, generating an action potential that subsequently signals to the gustatory cortex for sensory perception. Besides well-known taste GPCR pathways, connecting proteins semaphorin 7A (Sem 7A) and 3A (Sem 3A) are depicted in close contact with sweet and bitter receptors as they provide instructive signals that fine-tune to sweet or bitter ganglion neurons, respectively. VFT, Venus flytrap domain; CRD, cystine-rich domain; ECD, extracellular domain. (created with BioRender.com).

In this review, we will first explore the basic architecture of the gustatory sensory system and its peripheral signal transmission. Then we will discuss taste GPCR signal transduction mechanisms for the different taste modalities, their molecular structure, and the conformational changes following orthosteric/allosteric binding of endogenous and food-derived ligands.

Taste Buds and Neural Transmission

In mammals, taste buds on the tongue comprise 50–100 elongated epithelial cells and a small number of proliferative basal cells. Ultrastructural studies and gene expression patterns with cell function reveal three distinct anatomical types of TRCs within each taste bud: Type I, Type II, and Type III.  See Figure \(\PageIndex{2}\): and Figure \(\PageIndex{2}\) for details.

Table \(\PageIndex{2}\) Summary of taste receptor cell characteristics.

Type II TRCs express either sweet, umami, or bitter taste receptors at their cell surface. These receptors share some commonality with their signal transduction mechanisms intrinsic to TRCs. Taste GPCRs (sweet, umami, and bitter) couple to heterotrimeric G proteins that include Gα-gustducin, Gβ3, and Gγ13 and initiate a series of signal transduction cascades involving activation of phospholipase C-β2 (PLCB2), production of inositol-1,4,5-triophosphate (IP3), and IP3-dependent Ca²⁺ release from the endoplasmic reticulum (ER) via the IP3 receptor (IP3R). The increased intracellular [Ca²⁺]_i then activates the transient receptor potential cation channel subfamily M member 4 and 5 (TRPM4/5) in the basolateral plasma membrane, leading to membrane depolarization that triggers Na⁺ action potential firing, and depolarization-induced release of ATP. In turn, ATP acts as the primary neurotransmitter, stimulating purinergic receptors 2 and 3 (P2X2 and P2X3) on afferent cranial nerves, whose activation triggers an action potential that subsequently activates the gustatory cortex in the brain. α-gustducin is a distinct G protein selectively expressed in ∼30% of type II TRCs and shares 80% identity with retinal protein α-transducin and is a key contributor to signal transduction for sweet and bitter taste receptors.

An important aspect of taste transduction is how ATP signaling is conducted. Recent studies have discovered that calcium homeostasis modulators 1 and 3 (CALHM1/3) are enriched in type II TRCs, where they interact and form a functional complex. Their genetic deletion abolishes responses to sweet, bitter, and umami tastes, supporting the requirement of the CALHM1/3 complex as an ATP release channel for the GPCR-mediated tastes.

New information has provided insight into how specific taste qualities are fine-tuned to recognize their partner ganglionic neurons in the brain. Lee et al discovered semaphorin proteins, 7A and 3A, as the physical links between sweet and bitter TRCs, respectively, and their partner ganglion neurons in the brain. It remains to be determined what physical links exist between umami TRCs and their corresponding neurons in the brain. Delineating the underlying molecular basis for this interaction would provide a further understanding of purinergic transmission in the taste system. In addition, whether these mechanisms are relevant for kokumi sensation has not been investigated, despite CaSR having distinct expression in TRCs and significant functional synergy with other prominent taste qualities (sweet, umami, and salty). Moreover, there is still debate regarding the recognition of kokumi as a sixth taste entity; consequently, the calcium-sensing receptor (CaSR) is not yet included in the nomenclature for any subtypes of taste GPCRs, although it would best fit with Type 1 taste receptors.

Type 1 Taste G Protein-Coupled Receptors (Sweet and Umami)

Type 1 taste receptors (TAS1Rs) belong to the class C GPCRs, which possess a large N-terminal extracellular domain (ECD) fused to the heptahelical seven transmembrane domain (TMD). The ECD is further divided into two ligand-binding domains (LBD1 and LBD2), having a bilobed structure called a Venus flytrap domain (VFT) due to its resemblance to this shape. Except for GABA_B receptors, a cysteine-rich domain (CRD) connects the VFT to the TMD.

In contrast to other receptors from this class C of GPCRs, such as the metabotropic glutamate receptor (mGluR) or γ-aminobutyric acid type B receptors (GABABRs) which function as homo- or heterodimers, respectively, the TAS1Rs function as obligatory heterodimers. The distinct expression pattern of TAS1R1 and TAS1R2 in different subsets of murine cells led to the idea that they could detect two different taste profiles. However, following the discovery of the TAS1R3 subtype, it was clear that when TAS1R1 heterodimerizes with TAS1R2, the receptor detects sweet taste substances. On the other hand, if heterodimerized with TAS1R3 (TAS1R1/TAS1R3), it is responsible for umami or amino acid taste detection. Please refer to Figure \(\PageIndex{4A}\) for the basic structure of sweet and umami receptors.

 

 

Figure \(\PageIndex{4}\): depicting basic structural features of sweet/umami/kokumi dimeric receptor (A) and monomeric bitter receptor (B) (created with BioRender.com).

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of Taste receptor type 1 member 2 (TAS1R2) AlphaFold model (uniprot Q8TE23)

Figure \(\PageIndex{5}\): Taste receptor type 1 member 2 (TAS1R2) AlphaFold model (uniprot Q8TE23). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...mkhYb2uKoR68b9

The gray is the predicted transmembrane helices. The cyan is the intracellular domain. The blue is the extracellular domain. The predicted model of the structure has high confidence except for the yellow/orange at the distal end of the extracellular domain. Key residues in the ligand binding domain are shown as sticks, CPK colors, and labeled.

Sweet Taste Signal Transduction Mechanisms

The TAS1R2/TAS1R3 receptor recognizes a wide variety of sweet substances, including natural sugars, artificial sweeteners, amino acids, and proteins, as summarized in Table \(\PageIndex{3}\).  This was demonstrated in studies using heterologous expression systems as well as knockout mice for TAS1R2 and/or TAS1R3 subtypes that showed a blunted response to sugars, sweeteners, and D-amino acids, confirming the TAS1R2/TAS1R3 heterodimer as the main sweet taste receptor in vivo.

TABLE 3 | Agonists of sweet taste receptors along with their EC50 values.

Agonists	Nature	Binding pocket	EC50 (mM)	References
Sucrose	Natural carbohydrate	VFT (TAS1R2 and TAS1R3)	62	(Li et al., 2002; Servant et al., 2010; Zhang et al., 2010; Zhang
				et al., 2003)
Aspartame	Peptide	VFT (TAS1R2)	0.75	(Li et al., 2002; Liu et al., 2011; Masuda et al., 2012)
Neotame	Peptide	VFT (TAS1R2)	5	(Li et al., 2002; Masuda et al., 2012)
Cyclamate	Sulfamate	TMD (TAS1R3)	3.1	(Xu et al., 2004; Jiang et al., 2005c)
Brazzein	Protein	CRD (TAS1R3)	0.08	(Li et al., 2002; Jiang et al., 2004; Ide, et al., 2009; Masuda et al.,
				2012)
Thaumatin	Protein	CRD (TAS1R3)	0.005	Masuda et al., 2012; Jiang et al., 2004
Monellin	Protein	VFT (TAS1R3), VFT (TAS1R2)	0.01	Koizumi et al., 2007; Jiang et al., 2004
Neoculin	Protein	VFT (TAS1R2)	0.001	(Jiang et al., 2004; Koizumi et al., 2007)
Saccharin	N sulfonyl amide	VFT (TAS1R2)	0.19	(Li et al., 2002; Masuda et al., 2012; DuBois, 2016)
Suosan, cyanosuasan	Arylurea	VFT (TAS1R2)	ND	(Tinti and Nofre, 1991; Du Bois, 2016)
SC-45647	Guanidinoacetic acid	VFT (TAS1R2)	0.3	(DuBois, 1995; Sanematsu et al., 2014)
Sucralose	Halogenated carbohydrate	VFT (TAS1R2 and TAS1R3)	0.06	(Li et al., 2002; Masuda et al., 2012)
Acesulfame K	Sulfamate ester	VFT (TAS1R2)	0.54	(Li et al., 2002; Masuda et al., 2012)
Perillartine	Oxime, ethoxyphenyl urea, alkoxyaryl urea,	TMD (TAS1R2)	15	(Li et al., 2002; Servant et al., 2010)
Dulcin	Ethoxyphenyl urea	TMD (TAS1R2)	0.01	(Servant et al., 2010)
S819	Alkoxyaryl urea	TMD (TAS1R2)	0.025	(Zhang et al., 2008)
D-tryptophan	Amino acid	VFT (TAS1R2)	2.09	(Li et al., 2002; Masuda et al., 2012)
Xylitol, sorbitol	Polyols	VFT (TAS1R2)	ND	(Mahalapbutr et al., 2019)
Maltotriose, acarbose	Oligosaccharide, pseudotetrasaccharide	ND	ND	(Pullicin et al., 2017; Pullicin et al., 2019)

Where VFT, Venus flytrap domain; TMD, transmembrane domain; ND, not determined.

Table \(\PageIndex{3}\): Agonists of sweet taste receptors and their EC50 values.

The sweet receptor couples to heterotrimeric Gα-gustducin, which includes Gβ3 and Gγ13, as mice lacking Gα-gustducin showed a reduced response to sweet substances, either natural or artificial. Moreover, a point mutation in the C-terminal region of gustducin (G352P) (critical for its receptor interaction) results in the loss of its ability to activate taste GPCRs while keeping other functions intact. Further, G352P acts as a dominant negative to block heterotrimeric G protein interaction with taste receptors. It disrupts the responses to sweet and bitter compounds in wild-type (WT) and null mice. In addition, the G352 mutant further reduces any residual sweet/bitter taste responses of the null mice by acting as a “βγ sink” to bind all unbound βγ-subunits and remove them from the viable pool of G protein heterotrimers available to the receptor. These observations confirm the essential requirement of Gα-gustducin in sweet and bitter taste transduction.

In addition to the Gα-gustducin pathway, sweet taste transduction occurs via two additional signaling pathways involving different secondary messengers. The first one involves cAMP, and the second one involves IP3. Normally, sugars elevate the cAMP level, while sweeteners stimulate IP3 production. Sucrose or other sugars bind to either TAS1R2 or TAS1R3 and recruit Gαs protein, leading to increased cAMP levels, which initiate the influx of cations through ion channels. Alternatively, cAMP activates protein kinase A, leading to TRC cell depolarization and an influx of calcium ions and neurotransmitter release. Sweetener binding to the TAS1R2/TAS1R3 heterodimer recruits Gα-gustducin proteins that stimulate PLCβ2, which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and IP3. IP3R3-induced Ca²⁺ release from ER stores activates TRPM5, leading to an action potential and subsequent release of neurotransmitters.  See Figure \(\PageIndex{3}\) for details.

Interestingly, confirmed that TRPM4 also mediates taste signaling independent of TRPM5, and knocking out both channel proteins (TRPM4/5) abolishes the sweet, umami, and bitter taste response completely. This revealed another layer of complexity to sweet signal transmission. This in-depth mechanistic research has increased our understanding of sweet and bitter receptors. It also presents a challenge to dissect the taste signal transmission pathways for umami and kokumi.

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Recent Updates: September 16, 2024

Sweet Proteins

Figure \(\PageIndex{x}\) shows an interactive iCn3D model of Taste receptor type 1 member 3 (TAS1R3, also abbreviated T1R3) AlphaFold model (UniProt Q7RTX0 ) docked with sucrose.  The PDB file was obtained using SwissDock and AutoDock Vina within SwissDock for docking.

Sucrose is shown in spacefill bound about halfway down the T1R3 receptor.  

Now let's compare this docked structure to Figure \(\PageIndex{x}\) below, which shows an interactive iCn3D model of Taste receptor type 1 member 3 (TAS1R3, also abbreviated T1R3) AlphaFold model (uniprot Q7RTX0 ) docked with protein called brazzein (recommended name Defensin-like protein) from Pentadiplandra brazzeana. Abramson, J et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature (2024).

 

Brazzein (54 amino acids, molecular weight 6500) is bound in the same region as the spacefill model of sucrose in the first iCn3D model.  The protein is digested in the gut, so it theoretically has no negative metabolic consequences compared to sucrose and non-protein artificial sweeteners.  This protein is 2000x sweeter than sugar and is used in some commercial products.  The PDB file was obtained using AlphaFold Server (beta) to dock the two proteins after inputting their amino acid sequences (an extraordinary feat possible since 2023 but now easily available online for free!).  

Figure \(\PageIndex{6}\) below shows an interactive iCn3D model showing the interaction interface between TAS1R3 (T1R3) and brazzein.

 

Figure \(\PageIndex{6}\):  

For an interactive iCn3D model of this rendering:

• download this file to your computer's download folder (easiest in Chrome)
• open iCn3D
• In the top menu bar, choose File, Open File, iCn3D PNG Image, and navigate to your download folder to select and open the file.

Note the diverse types of noncovalent interactions between amino acids from the receptor and brazzein (in CPK colors).

Compare this with the interactions between docked sucrose and TAS1R3 (T1R3) shown in the figure below.  Docking of the TAS1R3 (T1R3) AlphaFold structure and sucrose was performed using AutoDock Vina through Swiss Dock.  Figure \(\PageIndex{7}\)

Figure \(\PageIndex{7}\):  For an interactive iCn3D model, download this file to your computer's download folder (easiest in Chrome) and follow the instructions above.

Note that the bound ligand (sucrose) interactions occur in the same region of T1R3 as for bound brazzein, but involve just hydrogen bonding.

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Structural, Molecular, and Conformational Changes of Sweet Receptor

Since the sweet taste receptor has not yet been crystallized, determining the sweetener binding site's structure and activation mechanism has been challenging. Based on homology with other class C GPCRs (mGluRs and GABA_BRs), multiple studies propose similar activation mechanisms for the sweet receptor. The many sweet agonists and their diverse binding sites across receptor domains (VFT, TMD, and CRD) (see Table\(\PageIndex{3}\)) may explain its complex yet broadly tuned nature. For example, a single residue in VFT (I60) of TAS1R3 of the TAS1R2/TAS1R3 heteromer is required for a saccharin preference in inbred mouse strains.

Several studies utilizing homology and computational modeling based on the crystal structure of mGluR and GABA_BRs have predicted structural and functional aspects of orthosteric and allosteric binding sites for the sweet receptor. They reported that both VFT regions undergo ligand-dependent conformational changes and intersubunit interactions between ECDs, further stabilizing heterodimer formation for subsequent downstream signaling. The binding of orthosteric agonists to VFT of TAS1R2 leads to major conformational changes that form a TMD6/TMD6 interface between TMDs of TAS1R2 and TAS1R3, which is consistent with the activation process observed biophysically on the mGluR2 homodimer. The initial role of the bound agonist is to pull the bottom part of VFT3 (VFT of TAS1R3) toward the bottom part of VFT2 (VFT of TAS1R2) to transmit this movement from VFT2 (where agonists bind) through the VFT3 and the CRD3 (VFT and CRD of TAS1R3) to the TMD3 (TMD of TAS1R3). This facilitates G protein coupling and downstream signaling. The CRDs are crucial in this streamlined relay of structural changes, where disulfide bonds provide rigidity to the CRD and amplify the mechanical constraints that help attain an active conformation. This is empirically supported by a study in which a single mutation (A537P) in the CRD of TAS1R3 abolished the response to all sweeteners, indicating that the CRD3 must couple ligand binding in VFT2 to the conformational changes required in TMD3 for receptor activation.

Trafficking and cell surface expression are also crucial factors for sweet taste transduction. Molecular modeling with mutagenesis scanning revealed specific regions consisting of hydrophobic residues in ECD (site II; at the tip of CRD) and TMD regions (site IV; includes TMD6 and the cytoplasmic base of TMD5) of the TAS1R2 subunit to be important for dimerization with TAS1R3. Moreover, the CRD region and ECL2 domain of the transmembrane region seem important for surface co-expression of the TAS1R2/TAS1R3 dimer. In particular, the cytosolic C-terminus portion of the CRD region of TAS1R2 needs to be properly folded for coexpression and trafficking. This reflects the difficulty in expressing these receptors consistently in mammalian cell lines.

Positive Allosteric Modulation of Sweet Receptor

Class C GPCRs pose an ideal target for allosteric modulation, either positive (PAM) or negative (NAM). PAMs show little or no agonist activity but significantly enhance agonist activity. Sweet taste is a major global target of the food industry, and non-caloric sweeteners are highly sought after to exploit a huge commercial market. Novel PAMs (see Table \(\PageIndex{4}\)) for the sweet heteromer were reported that were not sweet on their own but significantly enhanced the sweetness of sucralose or sucrose. Agonist binding to the VFT region of TAS1R2 facilitates a closed conformation, constituting an active state of the sweet receptor, while its open conformation represents an inactive state. Molecular modeling and mutagenesis studies revealed that these PAMs follow a similar binding mode to that reported for umami PAMs (IMP and GMP). They bind near the opening of the binding pocket of the VFT region adjacent to their agonists, through Van der Waals and hydrogen bonding interactions, and utilize several critical residues for their activity. Although these residues are not in direct contact with any receptor-bound sweetener, mutation of some of them (K65, Y103, L279, D307, and R383) diminishes the response to sweeteners, suggesting that these residues normally stabilize the closed conformation. The initial closing of the VFT region by agonist binding and further stabilization of the closed conformation by subsequent binding of SE modulators occurs in two steps. First, by interacting with the ECD region of TAS1R2, and second, by strengthening the hydrophobic interactions between the two lobes of ECD and lowering the free energy needed for their closure.

TABLE 4 | Sweet taste receptors’ positive allosteric regulators with concentration (used in cell-based assays in studies) and negative allosteric modulators with their IC50 values.

Positive allosteric modulators (PAMs)	Nature	Binding pocket	Conc. (mM)	References
SE1, SE2, SE3	Undisclosed	VFT (TAS1R2)	0.05	(Servant et al., 2010; Zhang et al., 2010)
Neohesperidin dihydrochalcone (NHDC)	Flavonoid	TMD (TAS1R3)	0.25	(Jiang et al., 2005c; Winnig et al., 2007)
Unnatural tripeptides (several)	Biaryl derivative tripeptides	ND	2 – 20	Yamada et al., 2019
Sodium, cholesterol	Cation, lipid	TMD (TAS1R2)	ND	Perez-Aguilar et al., 2019
NAMs	—	—	IC50 (mM)	—
Lactisole	Carboxylic acid salt	TMD (TAS1R3)	0.041	(Jiang et al., 2005c)
(2-(2,4-dichlorophenoxy)propionic acid)	Carboxylic acid salt	TMD (TAS1R3)	0.006	(Nakagita et al., 2019)
Gymnemic acid	Triterpenoid glycoside	TMD (TAS1R3)	6.9	(Sanematsu et al., 2014)
Clofibric acid	Herbicide	TMD (TAS1R3)	1.4	(Maillet et al., 2009; Kochem and Breslin, 2017)
Amiloride	Diuretic	TMD (TAS1R2)	0.87	(Imada et al., 2010; Zhao et al., 2018)
Umami compounds: MSG, Glu-Glu, Glu-Asp	Peptides	VFT (TAS1R2)	ND	(Shim et al., 2015)

Where VFT, Venus flytrap domain; TMD, transmembrane domain; ND, not determined.

Table \(\PageIndex{4}\): Sweet taste receptor’s positive allosteric regulators with concentration (used in cell-based assays in studies) and negative allosteric modulators with their IC50 values.

Several unnatural tripeptides with a novel core biaryl structure were found as potential sweet enhancers using a high-throughput chemical screening approach and heterologous expression of the TAS1R2/TAS1R3 heteromer. This study divided the potential molecule into three parts: "head and linker,” which are essential for its sweet enhancer activity, while the “tail” determines the activity level. This approach provided some useful inputs toward the synthesis of potent PAMs. Firstly, an amine incorporated at the α-position of the carbonyl group in the tail structure interacts with the TAS1R2 subunit, increasing allosteric activity. Secondly, additional hydrophobic substitutions in the tail structure increased allosteric activity in the molecule. Lastly, the distance between the head and linker and the insertion of an amide bond is crucial for its synthesis. Although their binding characteristics and allosteric mechanisms are unknown, these observations provide a starting point for identifying and synthesizing new sweet PAMs in the future.

Unlike an agonist, which binds to the extracellular domain, small-molecule PAMs can also bind to the transmembrane domain in class C GPCRs. For example, the flavonoid sweetener, neohesperidin dihydrochalcone (NHDC) binds to TMD regions to enhance the agonist-induced sweet response. It interacts with a receptor binding pocket in the TMD of TAS1R3 and requires seventeen critical residues in TMDs and extracellular loop 2 for its allosteric activity. These residues also contribute to cyclamate and lactisole binding sites. Among seventeen residues, eight alter receptor activation by NHDC (Q637^3.29, S640^3.32, H641^3.33, Y699^4.60, W775^6.48, F778^6.51, L782^6.55, and C801^7.39) and influence lactisole-mediated inhibition. Similarly, nine of the seventeen residues (Q637^3.29, H641^3.33, H721^ex2, S726^5.39, F730^5.43, W775^6.48, F778^6.51, L782^6.55, and C801^7.39) mediate activation by cyclamate. In contrast, six (Q637^3.29, H641^3.33, W775^6.48, F778^6.51, L782^6.55, and C801^7.39) influence receptor inhibition by lactisole as well as receptor activation by cyclamate [superscript refers to the nomenclature suggested for class C GPCRs, where the first number denotes TMD region. The second number denotes the residue position from the most conserved residue.

Notably, three critical residues in TMD6 (W775^6.48, F778^6.51, L782^6.55) and one in TMD7 (C801^7.39) of TAS1R3 were found crucial for allosteric binding, as their mutation to alanine altered the receptor's sensitivity to NHDC and cyclamate, as well as to the inhibitor lactisole. Therefore, TMD6 and TMD7α helices of TAS1R3 are integral to allosteric modulation of the sweet receptor, implicating them in TAS1R2 and TAS1R3 subunit interactions and indicating an important role for this structural region in the conformational changes involved in receptor activation. Furthermore, these residues are conserved across mammalian species.

Negative Allosteric Modulation of Sweet Receptor

Like PAMs, negative allosteric modulators (NAM) such as lactisole and gymnemic acid bind to the TMD region of TAS1R3 and inhibit sweet substance-induced responses. Lactisole, an aralkyl carboxylic acid, inhibits sweet and the umami receptor response in humans and presents a rare opportunity to study the structural cross-talk between these two taste qualities. Using heterologous expression and mutagenesis, Jiang et al. reported that lactisole's sweet inhibition might be mediated by its binding to TMD3, TMD5, and TMD6 of TAS1R3 and induce a conformation change that restricts the movement required to stabilize the active state. Residues A733^5.46 in TMD5, L798^7.36 in TMD7, and R790^ex3 in extracellular loop 3 were crucially important for sensitivity to lactisole in humans. These observations were confirmed in a recent study where 2-(2,4-dichlorophenoxy)propionic acid (2,4-DP) was found to be a more potent antagonist and utilize the same residues as well as four additional ones (H641^3.37, H734^5.43, F778^6.53, and Q794^7.32) in binding to TAS1R3. Moreover, the (S)- isomer of both compounds was found to be more strongly bound to the TMD of TAS1R3 and be a more effective inhibitor [lactisole; (S)-lactisole IC50, 20 µM, while (R)- lactisole exerted no inhibition at this concentration.; 2,4-DP: (S)-isomer was 10-fold more effective than (R)-2,4-DP. The (S)- lactisole isomer interacts with the TMD via its carboxyl group and stabilizes in only one orientation in the binding pocket, which does not allow for very strong binding. In contrast, (S)-2,4-DP binds through two moieties simultaneously, a carboxyl group and an aromatic ring with two Cl⁻ groups, and stabilizes in several different orientations through hydrophobic interactions that allow stronger binding, resulting in stronger negative allosteric modulation.

These observations provide information about the relevance of structural modification in NAM compounds that could affect their interaction with the receptor. Although TMDs of TAS1R3 are the most likely regions responsible for allosteric modulation, TMDs and VFT regions of TAS1R2 cannot be ruled out completely. For example, the diuretic amiloride binds to TAS1R2 (TMD3, TMD5, TMD7) and inhibits the sweet response in a species-dependent manner. Further, the umami compound [monosodium glutamate (MSG)] and peptides (Glu-Asp, Glu-Glu) bind to the VFT region of TAS1R2 and inhibit the sweet-induced response. These observations suggest that both subunits are important for the allosteric activity of TAS1R2/TAS1R3, and further structural studies are required to design novel sweet allosteric modulators.

Umami Taste Signal Transduction Mechanisms

In contrast to four well-known basic human tastes (sweet, bitter, salty, and sour), umami or ‘savory taste’ is relatively recent and was introduced in the early 2000s by Kikuna Ikeda as a new seasoning element in food. The main stimulus for the umami taste is the amino acid L-glutamate, present in the diet mainly as MSG. Glutamate was first extracted from konbu/kombu (dried kelp of Fucus vesiculosus) and described as having a “unique taste” and “very different from other tastes”. The terminology “umami” comes from the Japanese word “umai” meaning “delicious.” Moreover, the taste of umami is also produced by food such as mushrooms and soy sauce that contain amino acids (L-aspartate), peptides, and synthetic ingredients similar to glutamate and some organic acids, as shown in Table \(\PageIndex{5}\).

TABLE 5 | Umami receptor agonists with their EC50 values and other pharmacological properties.

Agonist	Nature	EC50 (mM)	Binding pocket	References
L-amino acids (glutamate, aspartate, alanine, serine, asparagine, arginine, histidine, threonine, glutamine) L-theanine	Amino acids Amino acid (plant origin)	3 (glutamate), ND for others ND	VFT (TAS1R1) VFT (TAS1R1)	(Li et al., 2002; Nelson et al., 2002; Zhang et al., 2008; Toda et al., 2013) (Narukawa et al., 2014)
VFT, venus flytrap domain; ND, not determined.

Table \(\PageIndex{5}\): Umami receptor agonists with their EC50 values and other pharmacological properties.

The umami receptor (TAS1R1/TAS1R3) is a heteromeric member of the class C GPCRs, whereas most other receptors of this class exist as homodimers. TAS1R1/TAS1R3 is the predominant umami taste receptor, and the TAS1R1 subtype is critical for sensing umami taste, as its deletion abolishes the response to umami taste stimuli. However, TAS1R1/TAS1R3 is not the only receptor capable of detecting umami ligands. Studies using heterologous expression, afferent nerve recordings, and behavioral experiments have confirmed that metabotropic glutamate receptors 1 and 4 (taste-mGluR1 and taste-mGluR4) also sense umami stimuli. Notably, TAS1R3 knock-out mice show a strongly diminished response to glutamate and sweet stimuli, and taste cells isolated from these mice respond to IMP and glutamate, which is abolished in the presence of mGluR antagonists (Pal Chaudhry et al., 2016). TAS1R1/TAS1R3 is not only activated by glutamate, but this activation is strongly enhanced in the presence of 5′-ribonucleotides (inosine 5′ monophosphate; IMP), a response that is a hallmark of umami taste.

The main transduction components following the activation of TAS1R1/TAS1R3 are similar to those for sweet taste, i.e., α-gustducin (and γ13/β1 or β3), PLCβ2, IP3R, and TRPM4/5. Cyclic nucleotides may also contribute to the transduction of umami taste in TRCs. When taste tissue is stimulated with umami, its cyclic AMP level is decreased. However, the consequence of decreased cAMP in TRCs has not yet been fully elucidated. Both α-transducin and α-gustducin are involved in umami taste signal transduction, as mice lacking the gene for one of these proteins showed a reduced response to this taste. In the taste palate, fungiform papillae, α-gustducin, and α-transducin activate PDE that reduces cAMP levels. Ligand binding to the TAS1R1/TAS1R3 heterodimer releases Gβγ subunits to stimulate PLCβ2, which hydrolyzes PIP2 to DAG and IP3. IP3 then activates IP3R3, releasing calcium ions from intracellular compartments, as shown in Figure \(\PageIndex{3}\). Calcium ions activate TRPM5 and TRPM4 channels, leading to an influx of sodium ions, subsequent cell membrane depolarization, and finally release of ATP, which activates ionotropic purinergic receptors located in sensory fibers. This pathway was confirmed when mice devoid of TRPM5, TRPM4, PLCβ2, and IP3R3 showed a reduced response to umami taste perception following glutamate stimuli.

Structural, Molecular, and Conformational Changes of Umami Receptor

In the last decade, several in-depth modeling and mutagenesis approaches have improved structural and molecular understanding of the umami receptor. The VFT regions of both subunits of TAS1R1/TAS1R3 comprise orthosteric and allosteric ligand binding sites for umami stimuli.

Mutagenesis and molecular modeling studies reveal that the cognate agonist glutamate binds in the VFT region of the TAS1R1 subunit of TAS1R1/TAS1R3 and stabilizes the closed active receptor conformation. Moreover, four residues in the TAS1R1 VFT region (S172, D192, Y220, and E301) showed no detectable response to glutamate when they were mutated to alanine, suggesting that they are critical for glutamate binding. The glutamate binding and stabilization of the closed conformation of TAS1R1 activate the downstream signaling pathway, while TAS1R3 remains in an open (inactive) conformation. Therefore, closure of the VFT is the key event that sensitizes umami taste receptor signal transduction. Apart from glutamate, other L amino acids were also found to elicit functional responses by binding to the corresponding VFT region of TAS1R1. Six residues that contributed to the acidic amino acid agonist (L-glutamate and L-alanine) responses have been identified (S148, R151, A170, E174, A302, and D435).

Allosteric Modulation of Umami Receptor

Because of significant advancements in understanding and food industry application of umami taste, its allosteric modulators are sought after. Several allosteric umami ligands have been discovered with varying potency, only a few of which have been characterized at the molecular level. The best-characterized umami PAMs, the 5′-ribonucleotides: inosine 5′-monophosphate (IMP) and guanosine 5′-monophosphate (GMP), interact with the VFT region of the TAS1R1 subunit to enhance the glutamate-induced response that is the hallmark of umami taste, as shown in Table \(\PageIndex{6}\). IMP and GMP binding sites in the VFT are adjacent to that for glutamate binding. The mutation of four residues (H71, R277, S306, and H308) abolished the IMP/GMP-induced glutamate response, suggesting their involvement in the allosteric binding of these nucleotides. Structurally, IMP and GMP stabilize the closed form of the TAS1R1 VFT region through electrostatic interactions and coordinate the positively charged residues that act as pincers. The ability of IMP and GMP to interact with the VFT region (as opposed to the TMD region) represents a unique mechanism of positive allosteric regulation within class C GPCRs.

TABLE 6 | Umami receptor allosteric modulators with concentrations used in cell-based assays and other pharmacological properties.

Allosteric modulators	Nature	Conc. (mM)	Binding pocket	References
IMP/GMP	Nucleotide	1	VFT (TAS1R1)	(Li et al., 2002; Nelson et al., 2002; Zhang et al.,
				2008)
Cyclamate	Sodium	8	TMD (TAS1R3)	(Xu et al., 2004)
	cyclohexylsulfamate
Methional (3-methylsulfanylpropanal)	—	0.12	TMD (TAS1R3)	(Toda et al., 2018)
Lactisole (2-4-methoxyphenoxy propionic acid)	Carboxylic acid salt	5	TMD (TAS1R3)	(Xu et al., 2004)
Clofibric acid (4- chlorophenoxy)-2-methylpropanoic	Herbicide acid	4	TMD (TAS1R3)	(Maillet et al., 2009; Kochem and Breslin, 2017)
acid

Where VFT, Venus flytrap domain; TMD, transmembrane domain.

Table \(\PageIndex{6}\): Umami receptor allosteric modulators with concentrations used in cell-based assays and other pharmacological properties.

In contrast to IMP and GMP, which bind to the TAS1R1 extracellular domain, the well-known flavor compound methional and its analogs bind to the TMD region and allosterically regulate the umami receptor in a species-dependent manner. Importantly, methional utilizes several distinct residues in different TAS1R1 transmembrane domains (TMD2-7) to act as a PAM in the human umami receptor, yet it behaves as a NAM in the mouse counterpart. This unusual phenomenon provided an opportunity to study the mechanisms of both positive and negative modulation in TAS1R1 simultaneously.

Construction of chimeric receptors between human (h) and mouse (m) and their functional analysis demonstrated that the TMD of TAS1R1 is the key domain for switching methional's PAM/NAM activities. Point mutation substitutions between these species identified four residues (h/m; F768/L769, N769/H770, S799/T800, and S802/G803) that are collectively required to switch PAM/NAM activities. A similar mode of allosteric regulation and PAM/NAM mode switching has been reported for mGluR5, suggesting this is an unusual and distinct phenomenon of the class C GPCRs. Further, alanine scanning mutagenesis in TAS1R1 of the corresponding residues vital for the activity of other taste inhibitors (sweetener inhibitors; NHDC and cyclamate; sweet and umami taste inhibitors; lactisole) revealed three residues required for PAM (W697^4.50, F728^5.40, and F732^5.44) and a single residue (F642^3.40) for NAM. These results suggest that methional's PAM and NAM activities are conferred by residues distinct from those required for the PAM/NAM switch. Knowing that methional is an important part of food seasoning globally, these observations could help maximize its use in enhancing flavors, amino acids, and nucleotides.

Despite PAMs being a central focus for umami allosteric modulation, there has also been considerable research on negative allosteric modulation, where lactisole emerged as a prominent NAM of the umami receptor, TAS1R2/TAS1R3. Because umami and sweet receptors share the TAS1R3 subunit, findings from studies on sweet receptor lactisole binding are relevant. A comprehensive study on the sweet receptor identified critical residues within the TMD regions (S640^3.32, H641^3.33 in TMD3 and F778^6.51, L782^6.55 in TMD6) of TAS1R3 required for the lactisole binding pocket and showed a large effect on sensitivity to lactisole. Because lactisole shares structural similarities with two other classes of compounds: fibrates and phenoxy-herbicides, researchers studied them to search for novel sweet/umami inhibitors. The lipid-lowering drug, clofibric acid, inhibits the TAS1R3 umami receptor-mediated in vitro and in vivo response, as shown in Table \(\PageIndex{6}\).  Like lactisole, clofibrate inhibits the umami taste from glutamate by binding with a similar affinity to TAS1R1/TAS1R3. However, its specificity against the umami receptor must still be validated alongside other umami taste receptors (mGluR1, mGluR4, or NMDA).

TYPE 2 TASTE G PROTEIN-COUPLED RECEPTORS (BITTER RECEPTORS)

Type 2 taste GPCRs are represented by bitter taste receptors with a distinct subset of bitter-sensing cells in type II TRCs. Notably, 25 bitter taste receptors (TAS2Rs) are reported to be expressed in humans. Much work has been done to explore the diversity among TAS2Rs and their agonists in taste biology. Some TAS2Rs (TAS2R3, TAS2R5, TAS2R13, TAS2R50) are narrowly tuned to structurally similar bitter compounds, whereas others are broadly tuned (TAS2R10, TAS2R14, TAS2R46), responding to several bitter compounds. Initially, it was believed that each bitter-sensitive type II TRC expressed every TAS2R isoform, but other studies suggest that TAS2Rs can be expressed differentially, allowing for possible discrimination among bitter compounds. Please refer to Figure \(\PageIndex{4B}\) for the basic structure of the bitter receptor.

Bitter Taste Signal Transduction Mechanisms

The bitter taste is the most complex of all the five basic tastes and protects against the ingestion of toxic substances by eliciting an innate aversive response across species. The TAS2Rs that mediate bitter taste perception are among ∼50 TAS2Rs identified in mammals, and 25 are known to be expressed in humans. The TAS2R family is the most diverse and binds to a wide range of agonists compared to other taste GPCRs.

TAS2Rs are distinctive among class A GPCRs in that many bind agonists with low apparent affinity in the micromolar range, rather than the nanomolar range. The activation of TAS2Rs by harmless, minute amounts of bitter compounds such as those contained in most vegetables would limit the availability of food resources appearing safe for consumption and therefore could negatively affect survival. Hence, the concentration ranges at which bitter taste receptors are activated are well-balanced to allow species to maintain a healthy diet yet avoid ingesting spoiled food containing strongly bitter ligands.

Hundreds of bitter compounds have been reported to evoke bitterness and activate human bitter receptors in different cell-based assays. These bitter agonists include plant-derived and synthetic compounds such as peptides, alkaloids, and many other substances. Many compounds activate some TAS2Rs, whereas others show strict specificity for a single bitter compound. Interestingly, TAS2R31, TAS2R43, and TAS2R46 have around 85% sequence homology, but they bind to different agonists, reinforcing the idea that each TAS2R might have a unique ligand-binding pocket.

The canonical TAS2R signal transduction cascade signaling molecules shared among bittersweet and umami receptors include the heterotrimeric G protein subunits (Gα-gustducin, Gβ3, and Gγ13), a phospholipase C (PLCβ2), an inositol trisphosphate receptor (InsP3R), and the TRPM5 ion channel. Upon receptor activation by bitter ligands, the G protein α-gustducin dissociates from its βγ subunits. The latter activates PLCβ2, releasing Ca²⁺ from IP3-sensitive Ca²⁺ stores, resulting in Na⁺ influx through TRPM5 channels. This Na⁺ influx depolarizes the cells and causes the release of neurotransmitter ATP through gap junction hemichannels or CALHM1 ion channels, as shown in Figure \(\PageIndex{3}\).

Structural, Molecular, and Conformational Changes of Bitter Receptors

Classification of TAS2Rs has always been ambiguous because they were initially considered a distinct family or grouped with the frizzled receptors. Still, most recent analyses support their classification with Class A GPCRs. The ability of bitter taste receptors to interact with numerous structurally diverse substances compared to other GPCRs is remarkable. It includes many drugs/antibiotics, polyphenols, bacterial metabolites, salts, and metal ions. Therefore, exploring the criteria for identifying highly heterogeneous bitter compounds with pronounced selectivity has become a significant research area. Some of these studies rely solely on in silico homology/computational modeling, and others on in vitro genetic modification and functional assay systems.

As a group of over ∼50 receptor subtypes, TAS2Rs recognize structurally diverse agonists, where some are broadly tuned (TAS2R46, TAS2R14, TAS2R10, and TAS2R43) recognize diverse agonists, while others (TAS2R1, TAS2R4, TAS2R7) show strong selectivity and narrow tuning. The agonist binding cavity in most bitter GPCRs is located deep within their transmembrane domain (TMD), except TAS2R7, which resides on the extracellular surface. TAS2Rs are also distinct in containing highly conserved TMD regions, with thirteen key residues and two motifs (LXXXR in TMD2 and LXXSL in TMD5) that are absent in class A GPCRs, and may reflect their different activation mechanisms. LXXSL plays a structural role by stabilizing the helical conformation of TMD5 at the cytoplasmic end and a functional role by interacting with residues in intracellular loop 3 (ICL3), which is important for proper receptor folding and function. Moreover, mutation of the conserved residues in LXXSL and LXXXR motifs results in protein misfolding and poor surface expression.

The initial study highlighting bitter taste receptors' structure–activity relationship was performed with receptors belonging to a subfamily of closely related TAS2Rs. By physically swapping the extracellular loop 1 (ECL1) between TAS2R43 and TAS2R31, chimeric TAS2R31/TAS2R43 (ECL) gained responsiveness to the compound n-isopropyl-2methyl-5-nitrobenzenesulfonamide (IMNB), whereas the reverse chimera TAS2R31 (ECL)/TAS2R43 lost responsiveness to IMNB. Although this report supports an important contribution of residues located within the transmembrane region of the investigated receptors, the extracellular loops appear important for agonist selectivity. This empirical finding contrasts with earlier computational studies, which predicted the agonist binding site to lie within the helical bundle of TAS2Rs without particular contacts between extracellular loops and docked agonists.

Bitter Receptor Ligand Binding Pocket

The emergence of TAS2Rs as the most broadly tuned taste receptors might give the impression that their specific interaction with numerous agonists is due to several binding pockets that accommodate subgroups of bitter compounds. However, structure–function analysis of TAS2Rs (except for TAS2R7) has demonstrated the presence of only a single agonist binding pocket comprising the upper parts of TMD2, TMD3, TMD5, TMD6, and TMD7. Their broad tuning and recognition of such a broad spectrum of agonists might most likely be attributed to an additional extracellular binding site called a “vestibular site,” in addition to the orthosteric site, as reported for TAS2R46. This two-site architecture offers more ligand recognition points than a single one and thus might help in selecting the appropriate agonists. Moreover, the presence of the vestibular site may also help to discriminate among the wide spectrum of bitter ligands.

Although broadly tuned receptors (TAS2R46, TAS2R31, and TAS2R43) have high homology in amino acid sequence, their agonist profiles only slightly overlap, which suggests the involvement of key residues at different positions in agonist specificity. Consequently, when strychnine interacting positions in TAS2R46 (residues differ at this position in TAS2R31, TAS2R43) were exchanged between these two receptors, not only was the strychnine responsiveness transferred to the recipient receptor (TAS2R31, TAS2R43), but also sensitivity to additional TAS2R46 agonists (absinthin and denatonium). Sensitivity to activation by aristolochic acid was lost in the mutant receptors. This experimental evidence supports the presence of a common agonist binding pocket and agrees with other studies on TAS2R16, TAS2R14, and TAS2R7 receptors.

Recent studies used homology modeling and mutagenesis to elucidate the nature of the ligand-binding pocket in TAS2R7, TAS2R14, and TAS2R16 receptors. They reported that the binding pocket is flexible and wide open to accommodate molecules of diverse sizes and shapes, thus permitting chemical modifications among agonists. Although the molecular basis for the promiscuity of bitter receptors is attributed to their apparent flexible, spacious binding site, future work is required to elucidate the contact points between TAS2Rs binding site residues and their agonists in terms of additional binding locations.

Bitter Receptors Ligand Binding Domain and Amino Acid Residues

Most of the TAS2R studies are based on molecular modeling, mutagenesis, and heterologous expression systems, which suggest that the ligand binding pocket is formed by several key residues in most TMDs (TMD1, TMD2, TMD3, TMD5, TMD6, and TMD7), except for TMD4.

Studies show similarities and differences regarding residues and positions involved in agonist-receptor interactions. However, most of them agree that besides position N^3.36 in TMD3 (superscript as per Ballestros-Weinstein nomenclature for class A GPCRs) and other residues (L^3.32, L^3.33, and E^3.37) in its proximity, play a role in agonist activation of several broadly tuned TAS2Rs (TAS2R1, TAS2R16, TAS2R30, TAS2R38, TAS2R46). In contrast, for the narrowly tuned TAS2R7, one position in TMD3 (H94^3.37) and another in TMD7 (E264^7.32) were crucial for metal ion binding. Mutagenesis and molecular modeling revealed that these two residues contribute to the metal ion binding pocket in TAS2R7. Moreover, metal ions bind distinctively to residues lining the binding pocket, and interestingly, the presence of calcium in the assay solution appears to affect the TAS2R7 response to metal ions. It is not clear how calcium affects metal ion binding to TAS2R7, but it might work cooperatively with certain ions and not others. Future studies focusing on structural interactions between the receptor and metal ions will provide further insights into how they activate the receptor.

Two studies of TMD2 suggest that position N2.61 is critical for binding in TAS2R1 and TAS2R46. Likewise, in TMD7, position 2657.39 is implicated in binding to TAS2R46 (E265) and TAS2R1 (I263). In TMD5, position H5.43 is implicated in binding in TAS2R16 and E5.46 in TAS2R1, while in TMD7, position E^7.32 is crucial for metal ion binding. These residues represent putative contact points for agonist interaction and form a pattern of being spaced one helical turn from each other.

Recent mutagenesis studies performed in broadly tuned TAS2R14 with agonists (aristolochic acid, picrotoxinin, thujone) found several residues in TMDs to be involved in agonist binding. However, in contrast to TAS2R10 and TAS2R46, mutation of TAS2R14 did not result in a complete loss of function for all agonists but a varied reduction in responsiveness or selectivity toward agonists. Among several mutants, only the mutation of W89A resulted in a complete loss of responsiveness against picrotoxinin, while others showed more subtle agonist-selective changes. This indicates that TAS2R14 is not streamlined for the most sensitive detection of selected agonists, but rather tailored to detect numerous diverse agonists, with comparatively lower apparent affinity.

The binding characteristics of bacterial acyl homoserine lactones (AHLs) on TAS2Rs (TAS2R4, TAS2R14, and TAS2R20) suggest the presence of a single orthosteric site situated close to the extracellular surface and reinforce the significant role of the extracellular loop structure (ECL2) in TAS2R ligand binding and activation. The crucial AHL binding residues in TAS2R4 and TAS2R14 are predominantly located in ECL2, while in TAS2R20, they are present in TMD3 and TMD7 helices. The ECL2 residues, N165 in TAS2R4, and R160 and K163 in TAS2R14 were crucial for lactone binding. In contrast, TAS2R20 residues W88 (TMD3) and Q265 (TMD7) are essential for agonist binding. In addition, the hydrophobic amino acids in the three TAS2Rs are important in directing the orientation of the hydrophobic acyl chains of lactones that facilitate receptor activation.

The transmembrane domain in GPCRs is composed mainly of hydrophobic amino acids accommodated in the plasma membrane. Therefore, hydrophobic properties of the receptor binding pocket are important for any membrane-accessible agonist. Hydrophobic residues in TMD3 and TMD7 of TAS2R16 are important in forming a wide ligand-binding pocket that accommodates larger ligands like the β-glycosides. By using salicin analogs as TAS2R16 novel agonists (differing structurally from salicin in β-glucoside core constituents), several critical residues were identified that are required for signaling. Interestingly, these were identical to the residues critical for salicin signaling, except for W261, which was not required for activation by the analog 4-NP-β-mannoside. Importantly, all these residues are in the receptor's TMD helices or intracellular face, consistent with classical GPCR signal transduction. These results suggest that larger ligands bind to the wide binding pocket of TAS2R16 on the extracellular side, and then their signal is transduced via conserved residues on the intracellular side. This can account for the broad spectrum of ligand recognition conferred by TAS2R16.

Unlike broadly tuned receptors, narrowly tuned ones like TAS2R7 show two different types of critical residue in ligand binding. The first type includes D86, W170, and S181, which are agonist-independent. Their mutation significantly reduces the ability of TAS2R7 to bind an agonist. A second group, consisting of D65 and W89, is selective for quinine and enhances binding to a specific category of ligand.

Despite the variation in the amino acid type and location important for agonist binding among receptors of the bitter family, for the most part, ligand binding pockets are present on the extracellular surface of TMDs or ECL2. The function of the residues at these binding pockets is dictated by multiple factors, including the type of ligand, the movements in TMDs, and the associated movement of ECL2 to accommodate the ligand. Structure–function studies have identified a conserved KLK/R motif in the intracellular carboxyl-terminal domain of 19 TAS2Rs that is critical for cell surface expression, trafficking, and receptor activation.

Agonist, Antagonist Binding and Modulation of Bitter Receptors

In simple pharmacological terms, an antagonist is a ligand that inhibits the biological response induced by an agonist and does not induce any response of its own. In contrast, a ligand that reduces the constitutive/basal activity of a GPCR is considered an inverse agonist. An antagonist acts as a competitive inhibitor to block receptor activity. Many agonists have been identified for bitter receptors, but few antagonists have been found, as described in Table \(\PageIndex{7}\). Finding an antagonist/inhibitor for bitter taste would help understand the TAS2R signal transduction mechanism, but it also has potential use in foods to overcome unwanted bitterness in consumer products. Such bitter blockers have been proposed to increase the palatability of bitter-tasting food and beverages, increase compliance in taking bitter-tasting drugs, especially children’s formulations, and reduce or prevent off-target drug effects in extra-oral tissues.

TABLE 7 | Bitter taste receptor inhibitors with their IC50 values and other pharmacological properties.

Antagonist	Mode of action	Bitter receptors	Tested agonists	IC50 (µM)	References
GIV3727or 4-(2,2,3-trimethylcyclopentyl) butanoic acid	Competitive orthosteric inhibitor	31	acesulfameK	6.4	(Slack et al., 2010)
		43	Aristolochic acid	11.33
		4	Colchicine	108
		40	Cohumulone	6.24
Gamma-aminobutyric acid (GABA)	Orthosteric inhibitor	4	Quinine	3.2	(Pydi et al., 2014b)
3β-hydroxydihydrocostunolide (3HDC)	ND	46	Absinthin	14.1	(Slack et al., 2010; Brockhoff et al., 2011)
			Andrographolide	4.9
			Denatonium	6.8
			Picrotoxinin	4.7
			Strychnine	15.3
3-hydroxypelenolide(3HP)	ND		Absinthin	57.8	(Brockhoff et al., 2011)
			Andrographolide	44.5
			Denatonium	51.4
			Picrotoxinin	22.9
			Strychnine	84.9
Probenecid	Allosteric inhibitor	16	Salicin	292	(Greene et al., 2011)
Sakuranetin	ND	31	Saccharin	5.5	(Fletcher et al., 2011)
6-Methoxysakuranetin	ND	31	Saccharin	10.2	(Fletcher et al., 2011)
Jaceosidin	ND	31	Saccharin	11.7	(Fletcher et al., 2011)
6,3′-dimethoxyflavanone	ND	39	Epicatechin gallate (ECG)	4075	(Roland et al., 2014)
			Denatonium	240
6-Methoxyflavanone	ND	39	Epicatechin gallate (ECG)	479	(Roland et al., 2014
N,N-bis(carboxymethyl)-l-lysine(BCML)	ND	4	Quinine	0.059	(Pydi et al., 2014b)
(±) abscisic acid (ABA)	ND	4	Quinine	34.4	(Pydi et al., 2015)
ND, not determined.

Table \(\PageIndex{7}\): Bitter taste receptor inhibitors with IC50 values and other pharmacological properties.

To date, ∼12 bitter inhibitors have been reported to interact with only 10 TAS2Rs subtypes, as described in Table \(\PageIndex{5}\).  They do so by binding to transmembrane domains similar to agonists. GIV3727 (4-(2,2,3-trimethylcyclopentyl) butanoic acid) was the first TAS2R antagonist discovered and well-characterized structurally that acts as an orthosteric competitive antagonist for TAS2R31. It competes with the acesulfame K agonist both in vitro and in vivo. GIV3727 is moderately selective because it inhibits multiple bitter receptors, including TAS2R4, TAS2R40, and TAS2R43. Homology modeling revealed that the -COOH group in GIV3727 is important for ligand-receptor interactions, as its replacement with an ester or the corresponding alcohol abolished its antagonist activity. Moreover, a mutagenesis study in TAS2R31 and TAS2R43 revealed residues K265^7.39 and R268^7.39 in TMD7 to be crucial for its antagonistic activity. Similarly, another non-selective inhibitor, probenecid (p-(dipropylsulfamoyl) benzoic acid) was found to act as NAM of TAS2R16 activity and inhibits TAS2R38 and TAS2R43 as well. Two point mutations, P44T and N96T, in TMD3 of hTAS2R16 were found to suppress probenecid's ability to inhibit salicin activity significantly. Hydrophobicity seems important for their pharmacological activity, as observed for both probenecid and GIV3727. The sesquiterpene lactone, 3β-hydroxydihydrocostunolide (3HDC), is an interesting bitter blocker as it acts as a competitive antagonist of TAS2R46, TAS2R30, TAS2R40, yet activates TAS2R4, TAS2R10, TAS2R14, and TAS2R31 as an agonist.

Similarly, various flavanones were also noted as antagonists for TAS2R31 and TAS2R39 with varying efficacy. Taken together, most of the currently known antagonists are non-selective, and there is an urgent need for studies that focus on selective antagonists of major broadly tuned TAS2Rs (such as TAS2R10, TAS2R14, TAS2R16, and TAS2R46). To target bitterness in terms of food industry needs, potential peptide inhibitors from different protein sources such as hen protein hydrolysates (inhibits TAS2R4, TAS2R7, TAS2R14) and beef proteins (inhibits TAS2R4) are reported to be effective. Several umami glutamyl peptides isolated from soybeans have been found to act as non-competitive allosteric inhibitors of TAS2R16 against the salicin-induced response.

Constitutive Activity of Bitter Receptors

A phenomenon in GPCR activity is constitutive activity, essentially an active state occurring without an agonist, which has been demonstrated in more than 60 GPCRs. It is the production of a second messenger or downstream signaling by a receptor in a ligand-independent manner. The constitutive activity provides another possibility for taste inhibitor discovery using inverse agonists. Inverse agonists can inhibit both agonist-dependent and agonist-independent activity, while antagonists can inhibit only agonist-dependent activity. Interestingly, some mutations in GPCRs can lead to constitutive activity, and receptors with this characteristic (including constitutively active mutants or CAM) are important tools to investigate new bitter inhibitors. Although constitutive activity has not been observed naturally in TAS2Rs, when induced by mutation, these receptors provide a useful means to investigate the relationship between an active receptor conformation and inverse agonist pharmacology.

Molecular modeling and functional assays report five CAMs critical residues for TAS2Rs, one in TMD7 (S285^7.47) and four others in intracellular loop 3 (H214A, Q216A, V234A, and M237A). Of the five CAMs, only TAS2R4 with the H214A mutation shows a 10-fold increase in constitutive activity. This histidine residue is highly conserved in most TAS2Rs. Mutation of H214 (H214A) helped in finding two new inverse agonists (GABA and ABA) as summarized in Table \(\PageIndex{7}\). Similar pharmacological approaches can be used to generate mutants of all TAS2Rs to screen for their inverse agonist/bitter taste blockers. However, for better characterization and interpretation of TAS2Rs, future in vivo studies should be performed to understand the functional relevance of these CAMs. At the same time, it is worth noting that the potential presence of endogenous agonists makes it difficult to determine the true constitutive activity of GPCRs, including TAS2Rs.

Kokumi Sensation Signal Transduction

In addition to the five basic tastes, sensations beyond these add another dimension to taste perception. One such example is “kokumi,” which is distinct from the other five tastes in that it does not have a taste but instead induces a sensation of “mouthfulness,” depth, thickness, and aftertaste in the flavors. Although this flavor has been used historically and is well recognized in Japanese cuisine, it was first characterized by researchers who isolated a kokumi taste substance from water extracts of garlic and onion and identified γ-glutamylcysteinylglycine or glutathione (GSH) as the main active ingredient of kokumi flavor. GSH is abundant in food-grade yeast extract and has been used to make foods more flavorful.

Kokumi signal transduction was unknown until kokumi-calcium sensing receptors (CaSR) expression was reported in a subpopulation of taste cells in mice and rats, which suggested it could function as a taste receptor for calcium and amino acids. However, its apparent role in kokumi stimuli detection was not confirmed. Ohsu et al. for the first time reported that kokumi peptides (GSH, γ-Glu-Val-Gly, and various γ-glutamyl peptides (see Table \(\PageIndex{8}\)) signal through CaSR and can synergize with sweet, salty, and umami taste qualities to impart an augmented kokumi sensation, i.e., increased depth of flavor, which was further complemented by later studies. By using heterologous expression systems and human sensory analysis, these studies demonstrated that kokumi peptides impart kokumi sensation to sweet, salty, and umami taste via CaSR, as the kokumi component was specifically suppressed in the presence of the CaSR-specific NAM NPS-2143. Maruyama et al. identified a distinct population of taste cells expressing CaSR in mouse lingual tissue that did not express either sweet or umami receptors to validate this idea further. Notably, these cells are specifically responsive to kokumi substances and elicit a Ca²⁺ response to focally applied kokumi stimuli in mouse lingual slices. Moreover, this response was inhibited in the presence of NPS-2143. These findings support the idea that CaSR mediates kokumi sensation effects in TRCs

TABLE 8 | Kokumi sensation receptor agonists, allosteric modulators with concentrations used in cell-based assays.

Ca^2^+	Orthosteric agonist/cation	1^a	VFT
Mg^2^+	Orthosteric agonist/cation	10^a	VFT
^Gd2^+	Orthosteric agonist/cation	0.02^a	VFT
Al^2^+	Orthosteric agonist/cation	0.5^a	VFT
Sr^2^+	Orthosteric agonist/cation	0.5^a	VFT
Mn^2^+	Orthosteric agonist/cation	0.5^a	VFT
Ni^2^+	Orthosteric agonist/cation	0.5^a	VFT
Ba^2^+	Orthosteric agonist/cation	0.2^a	VFT

Ca^2^+	Orthosteric agonist/cation	1^a	VFT	Ca^2^+
Spermidine	Orthosteric agonist/polyamine	0.002^a	VFT	(Nemeth et al., 2018)
Neomycin	Orthosteric agonist/aminoglycoside antibiotic	0.06^a	VFT	(Katz et al., 1992)
Gentamicin	Orthosteric agonist/aminoglycoside antibiotic	0.15^a	VFT	Katz et al., 1992)
Kanamycin	Orthosteric agonist/aminoglycoside antibiotic	0.1	VFT	(Katz et al., 1992)
Amyloid β-peptides	Orthosteric agonist/Peptide	0.001–0.04	—	(Ye et al., 1997)
Poly-Lysine	Orthosteric agonist/peptide	0.03 µM^a	VFT	(Brown et al., 1991; Nemeth et al., 2018)
Poly L-arginine	Orthosteric agonist/peptide	0.004 µM^a	VFT	Brown et al., 1991; Nemeth et al., 2018)
Lysozyme	Agonist/protein	0.59^a	ND	(Yamamoto et al., 2020)
Thaumatin	Agonist/protein	0.07^a	ND	(Yamamoto et al., 2020)
Aromatic L-amino acids (Trp, Phe, His, Ala, Ser)	PAMs	10	VFT	(Conigrave et al., 2000; Mun et al., 2004; Geng et al., 2016)
Anions (SO4^2^-)	NAM	10	VFT	(Geng et al., 2016)
Cinacalcet	PAM/phenylalkylamine	0.051 µM^a	TMD	(Miedlich et al., 2002; Petrel et al., 2004; Nemeth et al., 2004)
Calindol	PAM/phenylalkylamine	0.31 µM^a	TMD	Miedlich et al., 2002; Petrel et al., 2004)
NPS R-568	PAM/phenylalkylamine	0.5 µM^a	TMD	(Miedlich et al., 2002; Petrel et al., 2004)
NPS R-467	PAM/phenylalkylamine	0.01	TMD	(Miedlich et al., 2002; Petrel et al., 2004)
γ-Glu-Val-Gly	PAM/Peptide	0.041 µM^a	—	(Ohsu et al., 2010)
γ-Glu-Cys-Gly (Glutathione)	PAM/Peptide	76.5 µM^a	VFT	(Ohsu et al., 2010; Wang et al., 2006
γ-Glu-Ala	PAM/Peptide	3.65 µM^a	ND	(Wang et al., 2006; Ohsu et al., 2010)
γ -Glu-Val	PAM/Peptide	1.34 µM^a	ND	(Wang et al., 2006; Ohsu et al., 2010)
γ -Glu-Cys	PAM/Peptide	0.45 µM^a	VFT	(Ohsu et al., 2010; Wang et al., 2006)
γ -Glu-α-aminobutyryl-Gly (Opthalmic acid)	PAM/Peptide	0.018 µM^a	ND	(Ohsu et al., 2010)
NPS2143	NAM	0.0003 (IC50)	TMD	(Gowen et al., 2000; Petrel et al., 2004)
Calhex 231	Mixed PAM/NAM	0.1–1 µM (PAM); 3–10 µM (NAM)	TMD	(Petrel et al., 2003; Petrel et al., 2004; Gregory et al., 2018)

Where VFT, Venus flytrap domain; TMD, transmembrane domain; ND, not determined. a shows EC50 value.

Table \(\PageIndex{8}\): Kokumi sensation receptor (kokumi-calcium sensing receptors (CaSR)) agonists, allosteric modulators with concentrations used in cell-based assays.

More recently, kokumi peptides have been found to have an extraoral physiological role in the gastrointestinal tract, where they stimulate the secretion of hormones (cholecystokinin and glucagon-like peptide 1 by activating CaSR. However, future studies with tissue-specific deletion of CaSR in taste buds would help delineate its role in taste physiology.

CaSR involvement in taste is a relatively recent discovery, but its central role in extracellular calcium homeostasis in mammals is well recognized. Diverse ligands activate CaSR, including cations (Ca²⁺ and Gd³⁺), peptides, polyamines, and amino acids, as listed in Table \(\PageIndex{8}\). Unlike other taste modalities (sweet, bitter, and umami), CaSR–ligand binding and recruitment of G protein result in the activation of an intricate, amplifying signaling network that initiates numerous intracellular functions. The functional diversity of CaSR results from its ability to activate multiple Gα proteins (Gq/11, Gi/o, G12/13, and Gs), which subsequently affect multiple signaling pathways related to the pathophysiology of parathyroid hormone secretion, cancer, and metastasis.

Kokumi substrates activate CaSR and transmit their signal through Gαq/11 proteins, which further activate PLCβ, releasing intracellular Ca²⁺ stored through activation of IP3 receptor channels in the ER. Whether the kokumi pathway strictly relies on Gαq/11 protein or can also use Gα-gustducin, like other taste modalities, for downstream signaling, is still unknown, as shown in Figure \(\PageIndex{3}\). The growing number of reports on kokumi flavor signal transduction sheds light on its potential use as a flavor enhancer.

Structural, Molecular, and Conformational Changes of Kokumi Receptor

The kokumi-calcium sensing receptors (CaSR) belong to the class C GPCR. Within this class, CaSR and metabotropic glutamate receptors (mGluRs) are known to function as disulfide-linked homodimers, as shown in Figure \(\PageIndex{4A}\). Structurally, the human CaSR is similar to sweet and umami taste receptors but differs in being a homodimer instead of a heterodimer. The ECD of CaSR not only senses nutrients (Ca²⁺, L-Phe, and polypeptides, as summarized in Table \(\PageIndex{8}\)) and allows ligands to modulate CaSR cooperatively, but is also required for its dimerization. The binding of Ca²⁺ and other ligands to the ECD changes the conformation of the seven transmembrane domains, causing alterations in the intracellular loops and the intracellular domain (ICD), further triggering downstream signaling pathways. The ICD is relatively diverse among species and helps to control CaSR signaling in multiple ways by modulating receptor expression, trafficking, and desensitization.

Homology modeling, mutagenesis, and heterologous expression revealed distinct and closely located binding sites for Ca²⁺ and aromatic L-amino acids, in VFT and the cleft of the VFT, respectively. Notably, four putative Ca2+ binding sites of varying affinity have been predicted in the VFT of the CaSR, and the interaction between site 1 and the other three sites plays a central role in positive cooperativity in sensing Ca²⁺. Besides Ca²⁺, aromatic L amino acids (L-Trp, L-Phe) also activate the CaSR by binding adjacent to the VFT region through three serine and one threonine residue (S169/S170/S171/T145). Interestingly, the double mutation T145/S170 selectively impaired L amino acid (Phe, Trp, His) sensing of CaSR, while Ca²⁺ sensing remained intact.

The recent crystal structure of the entire extracellular domain of CaSR identified four novel Ca²⁺ binding sites in each protomer of the homodimer, including one at the homodimer interface, which does not correspond to any of the sites reported previously by Huang et al. It is unclear why these additional calcium-binding sites were not found in earlier studies. This might be due to the different expression systems, crystallization conditions, and analysis methods. The conditions of the more recent studies may have stabilized an active conformational state in which these calcium sites become available. Among these four Ca²⁺-binding sites, site 4 seems most relevant to receptor activation as it directly participates in the active CaSR conformation. Moreover, a previously reported natural mutation G557E reduced the potency of Ca²⁺, possibly by affecting backbone conformation, thereby weakening the affinity of Ca²⁺ for this site. This confirms that a Ca²⁺ ion at site 4 stabilizes the active conformation of the receptor by facilitating homodimer interactions between the membrane-proximal LBD2 region and CRD of CaSR.

The most interesting aspect of Ca²⁺ and L-amino acid interplay was reported by Zhang et al. who studied L-Phe binding characteristics by monitoring intracellular [Ca²⁺]_i oscillations in living cells and performing molecular dynamic simulations. Their findings supported a previous observation that the L-Phe binding pocket is adjacent to the Ca²⁺ binding site 1. Importantly, by binding to this site, L-Phe influences all Ca²⁺ binding sites in the VFT region and enhances CaSR functional cooperativity through positive heterotropic cooperativity to Ca²⁺. Moreover, the dynamic communication of L-Phe at its predicted binding site in the hinge region with the Ca²⁺ binding sites not only influences the adjacent Ca²⁺ binding site 1 but also globally enhances cooperative activation of the receptor in response to alterations in extracellular Ca²⁺.

The crystal structures of the entire ECD region of CaSR in the resting and active conformations have provided additional information about the dynamics between calcium and L-amino acid binding. Most importantly, using L-Trp, the study provided direct evidence that L-amino acids are CaSR co-agonists, acting concertedly with Ca²⁺ to achieve full receptor activation. Several lines of evidence support this contention: 1) L-Trp binds at the interdomain cleft of the VFT, which is a canonical agonist-binding site for class C GPCRs and shares a common receptor-binding mode with the endogenous agonists (amino acids or their analogs) of mGluR and GABA_B receptors. 2) L-Trp interacts with both LBD1 and LBD2 in ECD to facilitate its closure, a crucial first step during CaSR activation. In contrast, no Ca²⁺ ion is found at the putative orthosteric agonist-binding site to induce domain closure. 3) Mutations of L-Trp-binding residues (S147A, S170A, Y218A, and E297K) severely reduced Ca²⁺ induced IP accumulation and intracellular Ca²⁺ mobilization , indicating that L-Trp is required for a Ca²⁺ induced receptor response. Notably, extracellular Ca2+ above a threshold level is required for amino-acid-mediated CaSR activation, and amino acids increase the receptor's sensitivity toward Ca²⁺. Taken together, amino acids and Ca²⁺ ions act jointly to trigger CaSR activation.

Knowing that aromatic L-amino acids (Trp, Phe, His) are important tastants in kokumi flavor, CaSR becomes more relevant for taste biology. Moreover, the kokumi tripeptide, glutathione (GSH), and glutamyl peptide are suggested to bind allosterically to CaSR at the same site as L-amino acids and enhance its activity in the presence of 0.5–1 mM free calcium, thereby acting as a positive allosteric modulator. In addition, an ECD crystal structure might help to explain the structural and molecular details of the GSH binding pocket, such as the nature of critical residues and their binding characteristics. Given recent reports of calcium emerging as a taste modifier, it would be worth investigating how GSH and Ca²⁺ operate in kokumi human perception.

To summarize, the CaSR is a classic GPCR with seven transmembrane helices and is in subfamily class C along with taste receptors T1R1 and T1R3. As its name implies, it's activated by Ca²⁺ and kokumi peptides.  It's expressed in taste cells. The peptide γ-EVG acts as a positive allosteric modulator (PAM)  Figure \(\PageIndex{8}\) shows the Cryo-EM map of the human CaSR/γ-EVG complex.

Figure \(\PageIndex{8}\):  Cryo-EM map of the CaSR/γ-EVG complex.  Yamaguchi, H., Kitajima, S., Suzuki, H. et al. Cryo-EM structure of the calcium-sensing receptor complexed with the kokumi substance γ-glutamyl-valyl-glycine. Sci Rep 15, 3894 (2025). https://doi.org/10.1038/s41598-025-87999-1.  Creative Commons Attribution 4.0 International License.  http://creativecommons.org/licenses/by/4.0/.

Panel (a) Overall density map of CaSR (left) and cartoon of the model of CaSR in an active form (PDB: 7m3G) (right). CaSR is composed of a VFT domain, a CRD, and a 7TM domain. (b) Focus-refined map of the VFT domain of CaSR. Stick model of γ-EVG (orange) is overlaid with the cryo-EM density. (c) Chemical structure of γ-EVG. Cryo-EM cryo-electron microscopy, CaSR calcium-sensing receptor, γ-EVG γ-glutamyl-valyl-glycine, VFT Venus flytrap, CRD cysteine-rich domain, 7TM seven-transmembrane.

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of Cryo-EM Structure of calcium sensing receptor domain of CaSR in complex gamma-glutamyl-valyl-glycine as a kokumi substance γ-glutamyl-valyl-glycine or γ-EVG (9J7I).

Figure \(\PageIndex{9}\): Taste receptor type 1 member 2 (TAS1R2) AlphaFold model (uniprot Q8TE23). (Copyright; author via source). Click the image for a popup or use this external link:  https://www.ncbi.nlm.nih.gov/Structu...QVzVoftzAz3337.

The protein is showin as a homodimer.  Peptides (spacefill) are bound to the orthosteric site.  The grey subunit contains a bound γ-EVG.  Key residues in binding the γ-EVG (Pro39, Phe42, Arg66, Ser147, and Glu297) are labeled as sticks. The brown subunit has a GVG peptide bound.  Peptides are shown as colored spheres.   

Allosteric Modulation of Calcium-Sensing Receptor

CaSR is classically known to be involved in the pathophysiology of parathyroid and renal-related diseases by sensing calcium ions in the extracellular fluid. Research on related therapeutic applications has identified several classes of PAMs and NAMs that modulate CaSR agonist sensitivity. More recently, this has been applied to kokumi taste signal transduction.

Endogenous Modulators (L-amino Acids, Anions, and Glutathione Analogs)

Several studies based on molecular modeling and mutagenesis report L-amino acids (L-Phe, L-Tyr, L-His, and L-Trp) as PAMs because they enhance the Ca²⁺-induced response of CaSR. Aromatic L-amino acids bind in the VFT domain and require a highly conserved five-residue binding motif (S147, S170, D190, Y218, and E297). Among these residues, E297 was identified through the natural mutation E297K as essential for structural and functional activity, as summarized in Table \(\PageIndex{8}\).

As recently identified NAMs, anions SO₄²⁻ and PO₄³⁻ are important modulators of the Ca²⁺-induced response. They bind in the VFT region and act as moderate NAMs for CaSR activity. Based on anomalous difference maps, four anion-binding sites were identified in the inactive and active CaSR ECD structures. Sites 1 and 3 are located above the interdomain cleft in LBD1, while site 4 lies in the LBD2 region. Sites 1 and 3 appear to stabilize the inactive conformation.

In contrast, site 2, present in both active and inactive conformations, appears important for receptor function as mutations in its residues (R66H, R69E, and S417L) abolished the Ca²⁺-induced response. In addition, each protomer structure contains one Ca²⁺ ion and three SO₄²⁻ ions, contributing to the receptor's structural integrity. Taken together, anions along with Ca²⁺ and amino acids are involved in an intricate interplay for CaSR activation to maintain conformational equilibrium between inactive and active states.

As positive allosteric modulators, γ glutamyl peptides including glutathione (γGlu-Cys-Gly) and its analogs (see Table \(\PageIndex{8}\)) are predicted to have overlapping binding sites with L-amino acids in the VFT region. Kokumi peptides that activate CaSR resemble amino acids in having free α-amino and free α-carboxylate groups because they contain both amide bond formation between the γ-carboxylate group of L-glutamate and the α-amino group of its neighboring Cys residue. However, compared to amino acids, glutathione analogs have much larger side chains and are more potent activators of CaSR. Nonetheless, free sulfhydryl is not required for CaSR activation.

The crystal structure of ECD enables mapping the GSH binding site and investigating how GSH binding works in synergy with Ca²⁺ to modulate the kokumi sensation. NPS2143, the sole kokumi NAM identified to date, has been reported to inhibit kokumi taste sensation to GSH and its analogs. This provides an opportunity to screen for novel kokumi-enhancing molecules in a cell-based assay.

Synthetic Drugs as Allosteric Ligands of Calcium-Sensing Receptor

Because of its pathophysiological importance, various synthetic PAMs and NAMs of CaSR have been identified and are in clinical use. The allosteric modulation of CaSR by synthetic drugs has been recently reviewed. Since the 1990’s the terms calcimimetics and calcilytics, have been used for drugs that mimic or antagonize the effect of extracellular Ca²⁺ on CaSR activity, respectively. Pharmacologically, a calcimimetic activates the CaSR and includes agonists (type I) and allosteric ligands (type II). Most type I calcimimetics are inorganic or organic polycations (e.g., Mg²⁺, Gd³⁺, neomycin).

In contrast, type II calcimimetics are small naturally occurring molecules (aromatic amino acids or GSH) or synthetic drugs and peptides (NPS R-568, cinacalcet). Type II calcimimetics (like aromatic amino acids) bind in the ECD, while others (e.g., NPS R-568, NPS R-467) bind in the TMD of the CaSR. Calcilytics are thus small organic molecules that appear to act as NAMs and bind in the TMD of the receptor.

Homology modeling and mutational studies show that both PAMs and NAMs have overlapping but non-identical binding sites in TMD and can partially allosterically modulate CaSR activity in the complete absence of the ECD. Still, their potencies vary among structurally different compounds (see Table \(\PageIndex{8}\)). Several residues reportedly critical for allosteric modulation, W818^6.48, F821^6.51 (TMD6) and E837^7.39, I841^7.43 (TMD7), R680^3.28, F684^3.32, F688^3.36 (TMD3) impair calcimimetic and calcilytic induced CaSR signaling. Nevertheless, subtle differences in ligand–receptor interactions drive negative vs. positive modulation of CaSR signaling by NPS2143 or cinacalcet and NPSR-568, respectively. The details of CaSR allosteric modulation by synthetic drugs are out of the scope of the current review; for a comprehensive explanation, refer to these studies.

Conclusion

Taste GPCR research has advanced rapidly over the past two decades, providing a more thorough understanding of receptor molecular pharmacology and signal transduction pathways. Except for the kokumi receptor ECD, high-resolution crystal structures for any taste receptor would be a major step toward designing novel and potent surrogate taste receptor ligands and selective antagonists. This has been a challenge due to low taste GPCR functional heterologous expression, appropriate post-translational modifications, high conformational flexibility, and low detergent stability. However, significant advancements in structural biology technologies of serial femtosecond crystallography using X-ray free-electron lasers and high-resolution cryo-electron microscopy provide promising tools for understanding conformational dynamics and visualizing the receptor activation process with high spatial and temporal resolution. The physiological relevance of taste GPCRs will be further advanced through in vivo studies to help provide information on potential synergies in taste signal transduction mechanisms, particularly among bitter, umami, sweet, and kokumi receptors.

Summary

This chapter provides a comprehensive exploration of the molecular and structural underpinnings of taste perception mediated by G protein-coupled receptors (GPCRs). It details how distinct classes of taste receptors—responsible for sweet, umami, bitter, and kokumi sensations—transduce chemical signals from food into neuronal responses, ultimately shaping taste perception.

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Taste Receptor Cell Architecture and GPCR Classification

• Taste Bud Organization:
  Taste receptor cells (TRCs) are organized into taste buds distributed on the tongue and palate. Type II cells, which express GPCRs, are primarily responsible for detecting sweet, umami, and bitter stimuli, while type III cells mediate sour taste and type I cells may detect salt.

• Receptor Families:
  The chapter distinguishes between Type I GPCRs (TAS1Rs) that function as heterodimers—TAS1R2/TAS1R3 for sweet and TAS1R1/TAS1R3 for umami—and Type II GPCRs, which include monomeric bitter receptors (TAS2Rs) and the calcium-sensing receptor (CaSR) associated with kokumi sensation. Structural domains such as the large extracellular Venus flytrap domain (VFT), cysteine-rich domain (CRD), and seven transmembrane domain (TMD) are highlighted as critical for ligand binding and receptor activation.

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Signal Transduction Mechanisms

• Intracellular Pathways:
  Upon binding of tastants, taste GPCRs activate heterotrimeric G proteins (e.g., Gα‑gustducin for sweet, umami, and bitter receptors, and Gα‑q/11 for CaSR in kokumi signaling). The released Gβγ subunits stimulate phospholipase Cβ2 (PLCβ2), which catalyzes the production of IP₃ and DAG. IP₃ triggers Ca²⁺ release from endoplasmic reticulum stores, activating downstream ion channels (e.g., TRPM4/5) that depolarize TRCs and induce the release of ATP—a key neurotransmitter in taste signaling.

• Neurotransmission:
  The ATP released from taste cells activates purinergic receptors on afferent cranial nerves, transmitting signals to the gustatory cortex and culminating in the perception of taste.

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Receptor–Ligand Interactions and Allosteric Modulation

• Orthosteric Binding:
  Sweet and umami receptors bind a variety of natural and synthetic ligands in their VFT domains, leading to conformational changes that facilitate dimerization and G protein coupling. In contrast, bitter receptors are broadly tuned to diverse ligands using a single, often spacious binding pocket located within the TMD and extracellular loops.

• Allosteric Modulation:
  The chapter describes how positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) can enhance or inhibit receptor responses without directly activating the receptor. For example, compounds such as neohesperidin dihydrochalcone (NHDC) enhance sweet taste receptor activity, whereas lactisole acts as a NAM by binding to the TMD of TAS1R3, thereby inhibiting both sweet and umami responses. Similar allosteric interactions are explored for the umami receptor and CaSR (kokumi receptor), where endogenous ligands like 5′-ribonucleotides (IMP, GMP) and glutathione (GSH) modulate receptor activity.

• Molecular Modeling and Mutagenesis:
  Advances in computational modeling (using tools like AlphaFold and AutoDock Vina) and site-directed mutagenesis have been pivotal in identifying key residues involved in ligand binding and receptor activation. These studies have elucidated how both the extracellular and transmembrane domains contribute to receptor conformation, dimerization, and signal transduction.

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Physiological and Pharmacological Implications

• Taste Perception and Food Science:
  The integration of multiple signaling pathways allows the taste system to discern complex flavor profiles, influencing dietary choices and food enjoyment. Understanding these mechanisms offers opportunities to design novel taste modifiers—both enhancers and inhibitors—that can improve food palatability and patient compliance with bitter-tasting medications.

• Therapeutic and Research Challenges:
  Despite significant progress, the crystallization and high-resolution structural determination of taste GPCRs remain challenging due to their low expression levels, conformational flexibility, and stability issues. Emerging technologies such as cryo-electron microscopy and serial femtosecond crystallography are promising tools that may overcome these barriers, leading to the development of more potent and selective taste receptor ligands.

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In summary, this chapter synthesizes current knowledge on the structure and function of taste GPCRs, providing insights into the complex mechanisms by which these receptors detect and transduce taste stimuli. It emphasizes the interplay between receptor architecture, ligand binding, allosteric modulation, and intracellular signaling, highlighting both the physiological relevance and the pharmacological potential of targeting taste receptors in food science and therapeutic applications.