28.18: Signal Transduction - Taste (Gustation)

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Taste Receptor Cell Types, GPCR Classes, and Shared Signal Transduction

• Describe the organization of taste buds into three TRC subtypes—Type I (salty), Type II (sweet, umami, bitter, and kokumi via GPCRs), and Type III (sour)—and trace the common downstream signaling cascade shared by sweet, umami, and bitter receptors: tastant → heterotrimeric G protein (Gα-gustducin/Gβ3/Gγ13) → PLCβ2 activation → IP3 + DAG production → IP3R-mediated Ca²⁺ release from ER → TRPM4/5 channel opening → membrane depolarization → ATP release through CALHM1/3 channels → P2X2/P2X3 purinergic receptor activation on afferent cranial nerves → action potential to gustatory cortex.
• Distinguish between the structural classes of taste GPCRs: explain how Type 1 receptors (TAS1Rs) are obligatory heterodimers belonging to class C GPCRs with a large N-terminal Venus flytrap domain (VFT), cysteine-rich domain (CRD), and seven-transmembrane domain (TMD) that together relay ligand-induced conformational changes from the extracellular VFT closure through the CRD to activate the TMD and couple to G proteins; and contrast this with Type 2 bitter receptors (TAS2Rs), which are class A GPCRs with a single broadly tuned TMD binding pocket (except TAS2R7) lacking a large extracellular ligand-binding domain.

Sweet, Umami, and Bitter Receptor Mechanisms and Allosteric Modulation

• Explain how the TAS1R2/TAS1R3 sweet receptor achieves its extraordinarily broad ligand recognition by utilizing multiple structurally distinct binding sites across its two subunits—orthosteric agonists (e.g., sucrose, aspartame) binding in VFT of TAS1R2; cyclamate and dulcin binding in the TMD of TAS1R3; sweet proteins (brazzein, thaumatin) binding in the CRD of TAS1R3—and describe how VFT closure in TAS1R2 is relayed through the CRD3 to activate TMD3 of TAS1R3 for G protein coupling, as demonstrated by the A537P CRD mutation abolishing all sweet responses.
• Explain how the TAS1R1/TAS1R3 umami receptor detects L-glutamate through VFT closure in TAS1R1 (requiring residues S172, D192, Y220, E301), how IMP and GMP act as positive allosteric modulators (PAMs) by binding adjacent to glutamate in the VFT of TAS1R1 to stabilize the closed conformation through electrostatic pincer interactions—and contrast this VFT-acting PAM mechanism with the unusual compound methional, whose PAM versus NAM activity is determined by four residues in the TAS1R1 TMD in a species-dependent manner.
• Describe how bitter TAS2Rs achieve broad agonist recognition through a single flexible TMD binding pocket formed by TMD2, 3, 5, 6, and 7 residues (with a vestibular secondary site in TAS2R46), explain why the broadly tuned receptors (TAS2R14, TAS2R46) bind hundreds of structurally diverse bitter ligands with low micromolar affinity while narrowly tuned receptors (TAS2R7) use specific TMD3 His and TMD7 Glu residues for selective metal ion coordination—and describe the pharmacological rationale for developing bitter taste blockers (antagonists like GIV3727 and probenecid, NAMs, and inverse agonists identified through constitutively active mutants) for food and pharmaceutical applications.

Kokumi (CaSR) Sensation and Allosteric Complexity

• Explain how the calcium-sensing receptor (CaSR)—a class C homodimeric GPCR—mediates kokumi sensation: describe how Ca²⁺ and aromatic L-amino acids (Trp, Phe) act as co-agonists through positive heterotropic cooperativity at four VFT Ca²⁺-binding sites and the adjacent amino acid binding cleft (requiring S147, S170, D190, Y218, E297), how kokumi γ-glutamyl peptides (GSH, γ-EVG) act as PAMs at overlapping VFT sites to enhance the Ca²⁺ response and produce the mouthfulness/kokumi sensation—and distinguish between type I calcimimetics (inorganic polycations activating the ECD) and type II calcimimetics (synthetic drugs like cinacalcet binding to the TMD to allosterically activate the receptor).

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. In addition to mediating the sensory modalities of olfaction and vision, GPCRs transduce signals for three basic taste qualities: sweet, umami (savory), and bitter, as well as the flavor sensation of 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, including 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. Taste binding to the receptor activates downstream secondary messenger pathways, leading to depolarization and increased intracellular calcium in TRCs, which innervate 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 insights into agonist binding site locations and the molecular mechanisms underlying orthosteric and allosteric modulation. In addition, studies using transgenic mice with 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 the 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 the sensory functions of smell, taste, and vision, as well as a wide variety of physiological processes, including secretion, neurotransmission, metabolism, cellular differentiation, inflammation, and immune responses. Taste is one of the most important sensations in human life, enabling us to perceive the diverse range of flavors in the 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 the papillae of the tongue and palate epithelium. 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 regions of the tongue and palate, as well as the palate papillae, contain taste receptor cells (TRCs) that express taste G protein-coupled receptors (GPCRs). The left side shows how afferent nerves transmit signals to the gustatory cortex in the brain via the 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, which recruits a specific G protein, thereby inducing 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 activates 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, thereby 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, the 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 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 across the different taste modalities, their molecular structures, and the conformational changes that follow 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, together 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-trisphosphate (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, shares 80% identity with the retinal protein α-transducin, and is a key contributor to signal transduction in 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 for the CALHM1/3 complex as an ATP-release channel for 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 the 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 of this interaction would provide further insight into 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; consequently, the calcium-sensing receptor (CaSR) is not yet included in the nomenclature for any subtypes of taste GPCRs, although it would best fit 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 in this class of GPCRs, such as the metabotropic glutamate receptors (mGluRs) or γ-aminobutyric acid type B receptors (GABABRs), which function as homo- or heterodimers, respectively, the TAS1Rs function as obligatory heterodimers. The distinct expression patterns of TAS1R1 and TAS1R2 across different subsets of murine cells suggested that they could detect two distinct taste profiles. However, following the discovery of the TAS1R3 subtype, it became clear that when TAS1R1 heterodimerizes with TAS1R2, the receptor detects sweet-tasting substances. On the other hand, when heterodimerized with TAS1R3 (TAS1R1/TAS1R3), it detects umami (amino acid) tastes. 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 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 and knockout mice for the TAS1R2 and/or TAS1R3 subtypes, which 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 the heterotrimeric Gα-gustducin complex, which includes Gβ3 and Gγ13, as mice lacking Gα-gustducin show a reduced response to sweet substances, whether 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, blocking 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 that involve distinct second 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 the Gαs protein, increasing cAMP levels and initiating cation influx through ion channels. Alternatively, cAMP activates protein kinase A, leading to TRC cell depolarization, calcium influx, 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 neurotransmitter release.  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 the AlphaFold Server (beta) to dock the two proteins after inputting their amino acid sequences (an extraordinary feat possible since 2023, 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 structure of the sweetener-binding site and its 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 using homology and computational modeling based on the crystal structures of mGluRs and GABABRs have predicted the structural and functional features of the sweet receptor's orthosteric and allosteric binding sites. 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 in sweet taste transduction. Molecular modeling with mutagenesis scanning revealed specific regions in the ECD (site II; at the tip of the CRD) and TMD (site IV; including 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 co-expression and trafficking. This reflects the difficulty of consistently expressing these receptors 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 for the food industry, and non-caloric sweeteners are highly sought after to tap into 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 adopt a binding mode similar to that of the 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 occur 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 biaryl core were identified as potential sweet enhancers through high-throughput chemical screening and heterologous expression of the TAS1R2/TAS1R3 heteromer. This study divided the potential molecule into three parts: the "head and linker,” which are essential for its sweet-enhancer activity, and the “tail,” which determines the activity level. This approach provided useful insights into 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 increased the molecule's allosteric activity. Lastly, the distance between the head and the linker, and the insertion of an amide bond, are crucial for its synthesis. Although their binding characteristics and allosteric mechanisms remain unknown, these observations provide a starting point for identifying and synthesizing new sweet PAMs.

Unlike agonists, which bind to the extracellular domain, small-molecule PAMs can also bind to the transmembrane domain of 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 17 critical residues in the TMD 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, the 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 underlying receptor activation. Furthermore, these residues are conserved across mammalian species.

Negative Allosteric Modulation of Sweet Receptor

Like PAMs, negative allosteric modulators (NAMs), 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 the 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-inhibitory effect might be mediated by its binding to TMD3, TMD5, and TMD6 of TAS1R3 and by inducing a conformational 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, in which 2-(2,4-dichlorophenoxy)propionic acid (2,4-DP) was found to be a more potent antagonist and to utilize the same residues, as well as four additional residues (H6413.37, H7345.43, F7786.53, and Q7947.32), 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 on the relevance of structural modifications in NAM compounds that could affect their interactions 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 the 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 markedly diminished response to glutamate and sweet stimuli, and taste cells isolated from these mice respond to IMP and glutamate, a response that 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 either protein show 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 membrane depolarization, and, finally, the release of ATP, which activates ionotropic purinergic receptors in sensory fibers. This pathway was confirmed when mice lacking TRPM5, TRPM4, PLCβ2, and IP3R3 showed reduced umami taste perception in response to glutamate stimulation.

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. Glutamate binding and stabilization of the closed conformation of TAS1R1 activate the downstream signaling pathway, whereas 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

Due to significant advancements in understanding and the application of umami taste in the food industry, its allosteric modulators are being 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 those for glutamate binding. Mutating 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 (rather than the TMD) represents a unique mechanism of positive allosteric regulation in 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 an NAM in the mouse counterpart. This unusual phenomenon provided an opportunity to simultaneously study the mechanisms of both positive and negative modulation in TAS1R1.

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. Given that methional is an important part of food seasoning globally, these observations could help maximize its use to enhance flavor, amino acid, and nucleotide profiles.

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 of glutamate by binding to TAS1R1/TAS1R3 with similar affinity. 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 explored 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

Bitter taste is the most complex of 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 a wide range of agonists among 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 found in most vegetables, would limit the availability of food resources that appear safe for consumption and could therefore 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 elicit bitterness and activate human bitter receptors across various 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, shared among bitter and umami receptors, includes 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 stores, leading to 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.  Some are broadly tuned (TAS2R46, TAS2R14, TAS2R10, and TAS2R43) and 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 for TAS2R7, which resides on the extracellular surface. TAS2Rs are also distinct in that they contain highly conserved TMD regions, with 13 key residues and 2 motifs (LXXXR in TMD2 and LXXSL in TMD5) that are absent in class A GPCRs and may reflect their distinct 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, mutations at conserved residues within the LXXSL and LXXXR motifs result in protein misfolding and reduced surface expression.

The initial study that highlighted the structure–activity relationship of bitter taste receptors was conducted using receptors from 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 within the transmembrane region of the investigated receptors, the extracellular loops appear to be important for agonist selectivity. This empirical finding contrasts with earlier computational studies, which predicted that the agonist binding site would lie within the helical bundle of TAS2Rs, with no 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 interactions with numerous agonists arise from multiple 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 site and thus might help select 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) share high amino acid sequence homology, their agonist profiles only slightly overlap, suggesting 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 is consistent with other studies on the TAS2R16, TAS2R14, and TAS2R7 receptors.

Recent studies used homology modeling and mutagenesis to elucidate the ligand-binding pocket in the 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 apparently flexible, spacious binding site, future work is required to elucidate the contact points between TAS2R binding-site residues and their agonists, including additional binding locations. Most TAS2R studies rely on molecular modeling, mutagenesis, and heterologous expression systems, suggesting that the ligand-binding pocket is formed by several key residues across most TMDs (TMD1, TMD2, TMD3, TMD5, TMD6, and TMD7), except for TMD4.

In 2026, a cyro-EM of the tastant receptor TAS2R43 in complex with a heterotrimeric G_gust protein and the ligand, aristolochic acid I, has been determined.  Figure \(\PageIndex{x}\) below shows an interactive iCn3D model showing G_gust-coupled TAS2R43 with aristolochic acid I (9OXB)

Figure \(\PageIndex{x}\):  G_gust-coupled TAS2R43 with aristolochic acid I (9OXB). (Copyright; author via source). Click the image for a popup or use this external link:  https://www.ncbi.nlm.nih.gov/Structu...d20909b004389d

The ligand is a bitter alkaloid that is also a nephrotoxin and carcinogen.  In the above structure, it binds in the likely orthosteric site to which other bitterants, including caffeine and derivatives found in coffee, such as cafestol, likely bind.  Other proximal sites or different poses within the aristolochic acid I site might also participate in caffeine and other alkaloid binding.

potent nephrotoxin and carcinogen aristolochic acid I. Both structures revealed that aristolochic acid I binds in a presumed orthosteric pocket shared with other bitter taste receptor. Further structural, functional and computational studies revealed potential modes for coffee’s constituents including caffeine and cafestol, which are bitter tastants from coffee. previous studies suggest that several TAS2Rs (TAS2R14, TAS2R31, TAS2R40, TAS2R41 and TAS2R43) are potently activated by (EC₅₀ ranging from nanomolar to micromolar range) aristolochic acid I (AAI), a highly toxic and bitter alkaloid identified as a nephrotoxin and carcinogen extracted from members of the Aristolochiaceae family¹⁰. Among these, TAS2R43, which is responsible for sensing the bitter taste of coffee ingredients including caffeine^11,12, exhibited the highest affinity for AAI, a heterocyclic aromatic compound structurally similar to purines¹⁰

 

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 specifically to residues lining the binding pocket, and, interestingly, the presence of calcium in the assay solution appears to affect TAS2R7's response to metal ions. It is unclear how calcium affects metal-ion binding to TAS2R7, but it might interact 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 spaced one helical turn apart.

Recent mutagenesis studies performed on broadly tuned TAS2R14 with agonists (aristolochic acid, picrotoxinin, thujone) identified several residues in the TMDs 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 W89A mutation resulted in a complete loss of responsiveness to 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, whereas in TAS2R20, they are present in the 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, the 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 residues were identical to those 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 extracellular binding pocket of TAS2R16 and that 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 such as TAS2R7 exhibit two distinct types of critical residues in ligand binding. The first type includes D86, W170, and S181, which are agonist-independent. Their mutation significantly reduces TAS2R7's ability 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, blocking 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 of bitter taste would help elucidate the TAS2R signal transduction mechanism and could also have 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 in both in vitro and in vivo assays. 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 of TAS2R31 and TAS2R43 revealed that residues K2657.39 and R2687.39 in TMD7 are crucial for their 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 significantly suppress probenecid's ability to inhibit salicin activity. 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, acting as a competitive antagonist of TAS2R46, TAS2R30, and TAS2R40, yet activating TAS2R4, TAS2R10, TAS2R14, and TAS2R31 as an agonist.

Similarly, various flavanones were noted as antagonists of 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 focusing 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 offers another avenue for discovering taste inhibitors 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 CAMs) are important tools for investigating new bitter inhibitors. Although constitutive activity has not been observed naturally in TAS2Rs, when induced by mutation, these receptors provide a useful means of investigating the relationship between an active receptor conformation and inverse-agonist pharmacology.

Molecular modeling and functional assays identify five CAMs critical residues for TAS2Rs: one in TMD7 (S2857.47) and four 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 inverse agonists/bitter-taste blockers. However, to better characterize and interpret TAS2Rs, future in vivo studies should be conducted to elucidate the functional relevance of these CAMs. At the same time, the potential presence of endogenous agonists complicates the determination of 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 taste of anything but instead induces a sensation of “mouthfulness,” depth, thickness, and aftertaste. 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 the kokumi flavor. GSH is abundant in food-grade yeast extract and has been used to enhance food flavor.

Kokumi signal transduction was unknown until the expression of calcium-sensing receptors (CaSR) in a subpopulation of taste cells in mice and rats was reported, suggesting they could function as taste receptors 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. Using heterologous expression systems and human sensory analysis, these studies demonstrated that kokumi peptides impart a kokumi sensation to sweet, salty, and umami tastes 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 in mouse lingual tissue that expressed CaSR but not either sweet or umami receptors, further validating this idea. 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 by 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 stems from its ability to activate multiple Gα proteins (Gq/11, Gi/o, G12/13, and Gs), which subsequently modulate multiple signaling pathways implicated in 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 in the ER via IP3 receptor channels. 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 the 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, altering the intracellular loops and the intracellular domain (ICD), thereby 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 by 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 differences in expression systems, crystallization conditions, and analytical 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 Ca²⁺ potency, possibly by altering backbone conformation, thereby weakening Ca²⁺ affinity at 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 dynamics 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 insight into the dynamics of 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, thereby enhancing its activity in the presence of 0.5–1 mM free calcium, acting as positive allosteric modulators. In addition, an ECD crystal structure might help explain the structural and molecular details of the GSH binding pocket, including the nature of critical residues and their binding characteristics. Given recent reports that calcium is emerging as a taste modifier, it would be worth investigating how GSH and Ca²⁺ operate in human kokumi 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 using molecular modeling and mutagenesis identify 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, Ca²⁺, and amino acids engage in an intricate interplay that drives CaSR activation and maintains 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 of the GSH binding site and investigating how GSH binding synergizes 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

Given their pathophysiological importance, various synthetic PAMs and NAMs for CaSR have been identified and are in clinical use. The allosteric modulation of CaSR by synthetic drugs has been recently reviewed. Since 1990, the terms calcimimetics and calcilytics have been used to refer to drugs that mimic or antagonize the effects of extracellular Ca2+ on CaSR activity, respectively. Pharmacologically, calcimimetics activate the CaSR and include 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 and cinacalcet, respectively, and by NPSR-568. 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 functional heterologous expression of GPCRs, insufficient post-translational modifications, high conformational flexibility, and low detergent stability. However, significant advancements in structural biology technologies, such as 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 elucidate potential synergies in taste signal transduction mechanisms, particularly among bitter, umami, sweet, and kokumi receptors.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

Taste perception is mediated by three morphologically distinct taste receptor cells (TRCs) within taste buds distributed across the tongue and palate. Type II TRCs express GPCR-based receptors for sweet, umami, bitter, and kokumi tastes, all of which converge on a shared downstream signaling cascade: tastant binding activates heterotrimeric G proteins containing Gα-gustducin (or Gαq/11 for kokumi), which release Gβγ subunits that activate PLCβ2 to generate IP3 and DAG. IP3 triggers Ca²⁺ release from the ER via IP3R3, and the resulting Ca²⁺ rise activates TRPM4/5 channels, depolarizing the TRC and driving ATP release through CALHM1/3 channels. ATP then stimulates P2X2/P2X3 purinergic receptors on afferent cranial nerve fibers, generating action potentials that travel to the gustatory cortex. Semaphorin proteins 7A and 3A provide additional specificity by instructively linking sweet and bitter TRCs, respectively, to their dedicated ganglionic neurons.

Sweet and umami receptors are obligate class C GPCR heterodimers. The sweet receptor (TAS1R2/TAS1R3) is unique in that it possesses multiple ligand-binding sites distributed across all three structural modules—VFT, CRD, and TMD—of both subunits, enabling recognition of an extraordinarily diverse array of natural sugars, artificial sweeteners, D-amino acids, and sweet proteins. Agonist binding in the VFT of TAS1R2 drives domain closure that is mechanically relayed through the CRD to activate the TMD of TAS1R3 for G protein coupling, as demonstrated by the A537P CRD mutation that abolishes all sweet responses. The umami receptor (TAS1R1/TAS1R3) detects L-glutamate through closure of the TAS1R1 VFT, with IMP and GMP acting as hallmark positive allosteric modulators (PAMs) by binding adjacent to glutamate to stabilize the closed conformation through electrostatic pincer interactions. The compound methional illustrates the mechanistic complexity of class C GPCR allostery by acting as a PAM at the human TAS1R1 TMD but as a NAM at the mouse receptor, with the switch determined by four specific TMD residues. Both sweet and umami receptors are negatively allosterically modulated by lactisole and gymnemic acid, which bind to TAS1R3's TMD and prevent the conformational changes required for activation.

Bitter taste receptors (TAS2Rs), classified as class A GPCRs, represent 25 subtypes in humans with a wide spectrum of tuning—from narrowly tuned receptors (TAS2R7, sensing metal ions via specific His and Glu TMD residues) to broadly tuned receptors (TAS2R14, TAS2R46) that recognize hundreds of structurally diverse bitter ligands through a single flexible TMD binding pocket. Many TAS2Rs additionally possess a vestibular extracellular site adjacent to the primary orthosteric pocket, expanding recognition capacity. Their activity on bitter compounds—including plant alkaloids, synthetic compounds, bacterial acyl homoserine lactones, and metal ions—serves as a critical aversive signal protecting against ingestion of toxic substances, explaining why TAS2Rs are activated at low micromolar concentrations rather than the nanomolar range typical of other GPCRs. Bitter taste blockers, including competitive antagonists (GIV3727), NAMs (probenecid), and inverse agonists discovered through constitutively active TAS2R mutants, represent a growing pharmacological toolkit with applications in food palatability and drug formulation.

The kokumi receptor (CaSR) is a class C homodimeric GPCR that mediates the mouthfulness and flavor-deepening sensation of kokumi. Unlike the other taste GPCRs, CaSR requires a co-agonist mechanism: Ca²⁺ ions occupy four distinct VFT binding sites while aromatic L-amino acids (Trp, Phe) bind at the interdomain cleft and act through positive heterotropic cooperativity to enhance Ca²⁺-mediated activation and VFT domain closure. Kokumi γ-glutamyl peptides, including glutathione (GSH) and γ-Glu-Val-Gly, act as PAMs at overlapping VFT sites, enhancing the response to Ca²⁺ and underlying the synergistic enhancement of sweet, salty, and umami tastes. CaSR signals through Gαq/11 to activate PLCβ and trigger ER Ca²⁺ release, and is allosterically regulated by calcimimetics (cinacalcet, phenylalkylamine PAMs binding to the TMD) and calcilytics (NAMs including NPS2143) that have important therapeutic applications in parathyroid and renal disease in addition to their emerging role in taste biology.

Taste perception is mediated by three morphologically distinct taste receptor cells (TRCs) within taste buds distributed across the tongue and palate. Type II TRCs express GPCR-based receptors for sweet, umami, bitter, and kokumi tastes, all of which converge on a shared downstream signaling cascade: tastant binding activates heterotrimeric G proteins containing Gα-gustducin (or Gαq/11 for kokumi), which release Gβγ subunits that activate PLCβ2 to generate IP3 and DAG. IP3 triggers Ca²⁺ release from the ER via IP3R3, and the resulting Ca²⁺ rise activates TRPM4/5 channels, depolarizing the TRC and driving ATP release through CALHM1/3 channels. ATP then stimulates P2X2/P2X3 purinergic receptors on afferent cranial nerve fibers, generating action potentials that travel to the gustatory cortex. Semaphorin proteins 7A and 3A provide additional specificity by instructively linking sweet and bitter TRCs, respectively, to their dedicated ganglionic neurons.

Sweet and umami receptors are obligate class C GPCR heterodimers. The sweet receptor (TAS1R2/TAS1R3) is unique in that it possesses multiple ligand-binding sites distributed across all three structural modules—VFT, CRD, and TMD—of both subunits, enabling recognition of an extraordinarily diverse array of natural sugars, artificial sweeteners, D-amino acids, and sweet proteins. Agonist binding in the VFT of TAS1R2 drives domain closure that is mechanically relayed through the CRD to activate the TMD of TAS1R3 for G protein coupling, as demonstrated by the A537P CRD mutation that abolishes all sweet responses. The umami receptor (TAS1R1/TAS1R3) detects L-glutamate through closure of the TAS1R1 VFT, with IMP and GMP acting as hallmark positive allosteric modulators (PAMs) by binding adjacent to glutamate to stabilize the closed conformation through electrostatic pincer interactions. The compound methional illustrates the mechanistic complexity of class C GPCR allostery by acting as a PAM at the human TAS1R1 TMD but as a NAM at the mouse receptor, with the switch determined by four specific TMD residues. Both sweet and umami receptors are negatively allosterically modulated by lactisole and gymnemic acid, which bind to TAS1R3's TMD and prevent the conformational changes required for activation.

Bitter taste receptors (TAS2Rs), classified as class A GPCRs, represent 25 subtypes in humans with a wide spectrum of tuning—from narrowly tuned receptors (TAS2R7, sensing metal ions via specific His and Glu TMD residues) to broadly tuned receptors (TAS2R14, TAS2R46) that recognize hundreds of structurally diverse bitter ligands through a single flexible TMD binding pocket. Many TAS2Rs additionally possess a vestibular extracellular site adjacent to the primary orthosteric pocket, expanding recognition capacity. Their activity on bitter compounds—including plant alkaloids, synthetic compounds, bacterial acyl homoserine lactones, and metal ions—serves as a critical aversive signal protecting against ingestion of toxic substances, explaining why TAS2Rs are activated at low micromolar concentrations rather than the nanomolar range typical of other GPCRs. Bitter taste blockers, including competitive antagonists (GIV3727), NAMs (probenecid), and inverse agonists discovered through constitutively active TAS2R mutants, represent a growing pharmacological toolkit with applications in food palatability and drug formulation.

The kokumi receptor (CaSR) is a class C homodimeric GPCR that mediates the mouthfulness and flavor-deepening sensation of kokumi. Unlike the other taste GPCRs, CaSR requires a co-agonist mechanism: Ca²⁺ ions occupy four distinct VFT binding sites while aromatic L-amino acids (Trp, Phe) bind at the interdomain cleft and act through positive heterotropic cooperativity to enhance Ca²⁺-mediated activation and VFT domain closure. Kokumi γ-glutamyl peptides, including glutathione (GSH) and γ-Glu-Val-Gly, act as PAMs at overlapping VFT sites, enhancing the response to Ca²⁺ and underlying the synergistic enhancement of sweet, salty, and umami tastes. CaSR signals through Gαq/11 to activate PLCβ and trigger ER Ca²⁺ release, and is allosterically regulated by calcimimetics (cinacalcet, phenylalkylamine PAMs binding to the TMD) and calcilytics (NAMs including NPS2143) that have important therapeutic applications in parathyroid and renal disease in addition to their emerging role in taste biology.