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. Author manuscript; available in PMC: 2025 Jul 1.
Published in final edited form as: FEBS J. 2024 Jan 8;291(13):2784–2791. doi: 10.1111/febs.17049

GPCR-G protein selectivity revealed by structural pharmacology

Sarah M Bernhard 1,2,#, Jianming Han 1,2,#, Tao Che 1,2,*
PMCID: PMC11209754  NIHMSID: NIHMS1955067  PMID: 38151714

Abstract

Receptor-G protein promiscuity is frequently observed in class A G protein-coupled receptors (GPCRs). In particular, GPCRs can couple with G proteins from different families (Gαs, Gαq/11, Gαi/o, and Gα12/13) or the same family subtypes. The molecular basis underlying the selectivity/promiscuity is not fully revealed. We recently reported the structures of kappa opioid receptor (KOR) in complex with the Gi/o family subtypes (Gαi1, GαoA, Gαz, and Gustducin (Gαg)) determined by cryo-electron microscopy (cryo-EM). The structural analysis, in combination with pharmacological studies, provides insights into Gi/o subtype selectivity. Given the conserved sequence identity and activation mechanism between different G protein families, the findings within Gi/o subtypes could be likely extended to other families. Understanding the KOR-Gi/o or GPCR-G protein selectivity will facilitate the development of more precise therapeutics targeting a specific G protein subtype.

Keywords: GPCR, G protein, Selectivity, Bias signaling, structural pharmacology

Graphical Abstract

graphic file with name nihms-1955067-f0001.jpg

A mammalian cell can express up to sixteen Gα protein subtypes. How a receptor determines its signaling effector remains unclear. Recent work from Han et al. revealed structural and pharmacological evidence for G protein selectivity. Here, via a comparative analysis of several GCPR-G protein interactions, we highlight both conserved and non-conserved features that support the observation that G protein subtypes within the Gi/o family are structurally similar but functionally different. Image is credited to Dr. Reid H.J. Olsen.

Introduction

Currently, G protein-coupled receptors (GPCRs) are the largest class of druggable targets. Around 700 approved drugs (~35% of all approved drugs) target GPCRs, indicating the vast potential of GPCRs for therapeutics [1]. Obviously, GPCR signaling is tightly associated with drug development, better understanding of which can aid in developing safer drugs with a bias toward desired signaling pathways [2]. However, despite the broad use of GPCRs, much about intracellular GPCRs signaling is not fully understood. GPCR signaling initiates from G proteins and/or β-arrestins upon agonist stimulation. There are four main classes of G proteins, Gαi/o, Gαs, Gα12/13, and Gαq. GPCRs, particularly the class A receptors, can non-selectively couple to more than one class or different subtypes within a specific class [3]. Many studies indicate key residues and steric clashes to be the reason behind G protein class selectivity [4–6]. Even more unknown is the selectivity of G protein subtypes within the same class. How these signaling transducers within the same class bind and promiscuously signal through a large range of GPCRs has not been extensively studied.

Opioid receptors are Gαi/o GPCRs, mainly including mu-, kappa-, delta-opioid receptors, which have been found to be therapeutically advantageous for their pain-relieving effects. However, most clinically approved drugs targeting mu-opioid receptors (MORs) are associated with severe negative side effects like addiction and respiratory depression [7]. New therapeutics need to be developed to treat pain with reduced side effects [8]. KOR agonists are non-addictive analgesics and offer an alternative way to treat pain [9]. However, they have been associated with dysphoria and psychotomimesis, limiting their therapeutic potential [10].

To better understand KOR signaling, several cryo-EM structures have been solved of KOR bound to four Gαi/o subtypes including, Gαi1 (PDB: 8DZP), GαoA (PDB: 8DZQ), Gαz (PDB: 8DZS), and Gαg (PDB: 8DZR) [11] in presence of different KOR agonists. To gain insights into the structural basis of a same G protein coupled to various GPCRs, here we presented the key features in the KOR-Gαz structure and compared the KOR-Gαi1, -GαoA, and -Gαg structures with other receptor structures. These comparisons bring new viewpoints that may play roles in determining GPCR-G protein selectivity.

Comparison of Gαi1 bound GPCR structures

Gαi1 is broadly expressed in the body and is needed for many behaviors. Humans with variants in the Gαi1 gene exhibit neurodevelopmental disorders including autism spectrum disorder and delay in development [12]. Gαi1 gene variants emphasize the importance of functional Gαi1 signaling within the body. The momSalB-bound structure of KOR with Gαi1 has been solved at 2.71 Å resolution [11]. Gαi1 interacts with KOR mainly through the αN, α4, and α5 helix (Figure 1A), which are conserved in other receptor-Gαi1 complexes. Interestingly, mutagenesis screening of KOR-Gαi1 interface residues showed that KOR residues have much larger effects on Gαi1 coupling than Gαi1 residues. While several intracellular KOR residues significantly reduces or abolished KOR-Gαi1 coupling, there are no Gαi1 residues that can affect KOR-Gαi1 interaction as much as KOR residues [11]. This supports that the structural determinants for GPCR-G protein interactions also reside in the receptor. The resilience of Gαi1 coupling even after Gαi1 mutations might be an adaptive strategy in evolution: in order to allow up to sixteen G proteins to be able to interact with more than 800 GPCRs, G proteins would need to be highly adaptive to be able to bind to many different receptors which have varied intracellular space residues.

Figure 1.

Figure 1.

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Comparison of Gi1 and GoA structures. (A-C) Comparison of Gi1 structures (KOR-Gi1: Orange; 5-HT5A-Gi1: Blue; α2BAR-Gi1: Cyan; CCR5-Gi1: Purple). (A) Gi1 subunit (orange) hydrogen bond interactions with KOR (grey). (B) Overlay of Gi1 subunit. (C) Movement of 5-HT5A and α2BAR α5 and TM6 respective to KOR. (D-G) Comparison of GoA structures (KOR-GoA: Green; 5-HT5A-GoA: Blue; α2BAR-GoA: Cyan; CCR6-GoA: Purple). (D) GoA subunit (green) hydrogen bond interactions with KOR (grey). (E) Overlay of GoA subunit. (F) Movement of 5-HT5A and α2BAR α5 and TM6 respective to KOR. (G) Conserved hydrogen bond interactions with residues Y354H5.26 and N347H5.19 across structures. Structural figures were generated in PyMOL.

Comparing the KOR-Gi1 structure with other Gi1-bound structures provides insights into how Gαi1 can bind to different receptors with varied intracellular sequences. Here we compared KOR-Gαi1 with three other high-resolution structures: α2B adrenergic receptor (α2BAR) [13] (PDB: 6K42), serotonin receptor 5A (5-HT5AR) [14] (PDB: 7X5H), and chemokine receptor 5 (CCR5) [15] (PDB: 7F1R) (Figure 1B). From these structures, it is apparent that the αN position of Gαi1 is varied for each structure. Differences in the αN helix position indicates that different receptors interact with Gαi1 differently. When comparing the Gαi1 α5 position, Gαi1 in KOR and CCR5 structures adopt a similar position in the intracellular receptor pocket while α5 of 5-HT5AR and α2BAR structures is shifted around 5 Å. TM6 of 5-HT5A and α2BAR is also shifted outward around 5 Å, likely due to steric clashes with α5 (Figure 1C). Interestingly, this further shift in TM6 of 5-HT5AR and α2BAR is not reflected in a larger receptor-Gαi1 interface with KOR having the largest receptor interface at 1201 Å2 (5-HT5A: 890 Å2, α2BAR: 1035 Å2, CCR5: 1090 Å2). When looking at specific residue interaction across these structures, many interactions are not conserved across all structures. One hydrogen bond interaction seen in all Gαi1 structures is N347H5.19. N347H5.19 is a common interaction across many G protein subtypes, indicating the interaction is not specific for Gαi1. D350H5.22 interacts with KOR, 5-HT5AR, and α2BAR (potentially also CCR5 but the residue was not resolved, preventing analysis of interaction). D350H5.22 is only present on Gαi subtypes and Gαg. However, mutating D350H5.22A had no effect on KOR recruitment of Gαi1 [11], indicating this interaction might be involved in KOR-Gαi1 stability rather than recruitment. Hydrogen bond interactions with E318h4s6.12 and I319s6.01 are present in KOR and CCR5 structures. 5-HT5A and α2BAR may also form these interactions but no resolution of ICL3 residues prevents analysis of these interactions. No mutational analysis has been thoroughly studied so the importance of these interactions is not well known. However, E318h4s6.12 or I319s6.01 have been found to form interactions in other G proteins subtypes indicating that they are not specific to Gαi1. As previously mentioned, mutations of Gαi1 residues had little effect of Gαi1 recruitment. This finding, along with the lack of specific residue interactions which are present in all Gαi1 structures indicates that G protein interaction is dynamic and not conserved across structures.

It is worth pointing out that Gαi1 has been shown to have the highest allosteric and binding affinity with KOR using the radioactive ligand binding assays when treating different Gαi/o proteins as positive allosteric modulators [11]. This property cannot be explained by the static complex structure yet. It will be interesting to see if the interface size has anything to do with the binding kinetics of G proteins. The presence of Gβ and Gγ subtypes could be other factors contributing to the different activity [16].

Comparison of GoA GPCR structures

GαoA is the most abundant G protein subtype in the central nervous system and is implicated in various behaviors. Gαo knockout mice experience an extensive list of behavioral effects including motor deficits, hyperalgesia, hyperlocomotion, and altered sexual behaviors [17, 18]. Mutations in Gαo in humans can show many of the same symptoms [19]. Gαo knockout mice were also found to reduce MOR agonist antinociception, indicating an interaction with Gαo and analgesia in the opioid system [20]. The cryo-EM structure of KOR with GαoA has been solved with momSalB bound at 2.82 Å resolution [11]. When comparing the KOR-GαoA structure with other Gα structures, it is apparent that GαoA-receptor interactions display unique features. In the canonical state, G proteins-receptor interactions involve several regions: α5, αN, αN-β1 loop, β2-β3 loop, α4-β6 loop. When these GαoA structures are overlayed, the αN of GαoA appears to not involve in the interaction (Figure 1D), although the molecular dynamics simulations still observe KOR-αN interactions [11].

Comparing the KOR-GαoA structure with other GαoA-bound structures could provide insights into how GαoA can bind to different receptors which have varied intracellular sequences. Comparison of GαoA structures was done with three other high-resolution structures: α2BAR [13] (PDB: 6K41), 5-HT5AR [21] (PDB: 7UM5), and chemokine receptor 6 (CCR6) [22] (PDB: 6WWZ) (Figure 1E). Similar to Gαi1 structures, KOR and CCR6 show similar GαoA-α5 orientation in the receptor intracellular space, while α2BAR and 5-HT5AR have α5 shifted around 5 Å. These shifts push TM6 further outward 6–7 Å due to steric clashes (Figure 1F). This conserved displacement of α5 and TM6 across Gαi1 and GαoA highlights differences in G protein interaction across receptors. Like Gαi1 structures, this shift in TM6 is not reflected in a larger receptor-GoA interface with CCR6 having the largest interface at 1241 Å2, followed by KOR (1062 Å2), α2BAR (942 Å2), and 5-HT5AR (723 Å2). The reason α2BAR and 5-HT5AR have smaller binding pockets varies. α2BAR has ICL2, TM4, and TM5 oriented further inward compared to CCR6 and KOR, resulting in a smaller binding pocket. In the case of 5-HT5AR, most helices are further outward compared to CCR6, KOR, and α2BAR. Differences in the receptor-GαoA interface suggests that these receptors could be interacting with GαoA differently. KOR, 5-HT5AR, and α2BAR all had at least ~100 Å2 larger receptor-G protein interfaces with Gαi1 compared to GαoA. The larger interface is due to slight differences in TM and α5 orientation, though these differences vary for each receptor. The larger receptor-Gαi1 interface could is possibly due to more bulky side chains compared to GαoA. For example, at H5.22, Gαi1 has an aspartic acid while GαoA has a glycine. At H5.17, Gαi1 has a lysine while GαoA has an alanine. These more bulky side chains occupy more space and could lead to a larger receptor-G protein interface. Gαi1’s larger side chains and receptor interface could mean there are limitations of Gαi1 coupling if the receptor is unable to accommodate these side chains. More work needs to be done to validate this hypothesis.

The distal portion of the α5 helix is not incased in the intracellular receptor region and exhibits a highly diverse orientation in all these structures. Although there are some conformational differences of α5 in the intracellular space, it is limited by needing to fit into this region, however, the distal portion of α5 does not interact with the receptor and allows for highly diverse orientations.

Overall, KOR forms the most hydrogen bond interactions under 4 Å with GαoA compared to CCR6, α2BAR, and 5-HT5AR. Most of the hydrogen bond interactions occur on α5 with isolated interactions on β2-β3 loop, and α4-β6 loop depending on the structure. There are two GαoA residues on α5 which form hydrogen bond interactions in all structures, N347H5.19 and Y354H5.26 (Figure 1G). These two residues commonly interact with the receptor in other Gαi/o structures, suggesting they are not specific for GαoA. Additionally, alanine mutation of Y354H5.26 to prevent hydrogen bonding has no effect on GαoA recruitment to KOR. In fact, most mutations done on GαoA have little effect on GαoA recruitment to KOR. This lack of effect suggests that GαoA-receptor interaction is resilient, as observed in Gαi1. Resiliency of G protein signaling makes sense considering their high promiscuity with an extensive number of GPCRs. While intracellular portions of receptors are more conserved, G proteins still need to adapt to differences amongst receptors.

Comparison of Gg GPCR structures

Gαg is expressed in taste cells where it binds to taste receptors. A previous study used engineered KOR expressed in taste cells to determine that KOR can bind and signal through Gαg in vivo [23]. The GR89,696-bound cryo-EM structure of KOR with Gαg has been solved at 2.61 Å resolution [11]. Gαg is evolutionarily closer to Gαi1 in sequence identity, leading to similar binding poses between the two subtypes. Gαg interacts with KOR similar to Gαi1 with interactions mainly present at αN, α4, and α5 helix (Figure 2A).

Figure 2.

Figure 2.

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Comparison of Gg and Gz structures. (A-F) Comparison of Gg structures (KOR-Gg: Pink; TAS2R46-Gg: Purple). (A) Gg subunit (pink) hydrogen bond interactions with KOR (grey). (B) Overlay of Gg subunit. (C) Similar Gg interactions found in both KOR and TAS2R46 structures. (D) Key interaction for KOR Gg recruitment which is not found with TAS2R46. (E) TAS2R46 has TM5 shifted closer to α5, allowing it to form more interactions. (F) ICL2 of TAS2R46 is shifted upward, preventing interaction with α5 and loops seen in KOR structure. (G) Gz subunit (teal) hydrogen bond interactions with KOR (grey). Structural figures were generated in PyMOL.

The only other GPCR Gαg-bound structures are with the class F taste receptor, TAS2R46 [24] (PDB: 7XP6). The orientation of TM in taste receptors is considerably different than class A KOR making it difficult to overlay TAS2R46 and KOR for comparison. Thus, α5 was used for alignment of these two structures. KOR and TAS2R46 share similar Gαg orientation (Figure 2B) and a similar size Gg-receptor interface with KOR at 1192 Å2 and TAS2R46 at 1169 Å2. KOR and TAS2R46 also share many similar interactions with Gαg, despite different TM orientation. Both KOR and TAS2R46 form hydrogen bond interactions with similar α5 interactions, D341H5.13, K345H5.17, N347H5.19, C351H5.23, and F354H5.26 (Figure 2C). These residues are not Gαg specific, as many other G protein subtypes also use these residues to form interactions with receptors. Mutating K345H5.17, C351H5.23, and F354H5.26 to alanine did not significantly change Gαg recruitment, suggesting these residues are not necessary for Gαg recruitment. One mutation which eliminated Gαg signaling was N3368.49 on helix 8 of KOR. When comparing structures, helix 8 of TAS2R46 does not extend down toward Gαg, preventing the same interactions as KOR (Figure 2D). The lack of this interaction at TAS2R46-Gαg suggests that necessary residue interaction is not conserved across receptor subtypes for Gαg signaling.

There are also some differences in KOR and TAS2R46 interaction with Gαg. For example, the TM5 of TAS2R46 is shifted left compared to KOR and extends further down (Figure 2E). The orientation of TM5 in TAS2R46 allows the receptor to form hydrogen bond and hydrophobic interactions with more distal areas of α5 which are not seen in the KOR structure. Additionally, placement of TAS2R46 ICL2 is further outward than KOR ICL2 (Figure 2F), preventing hydrogen bond and hydrophobic interactions present in the KOR structure. However, it is unclear whether this difference is due to the mini-Gαs/Gαg chimera. The TAS2R46 structure is not Gαg but mini-Gαs/Gαg chimera where mini-Gαs has α5 replaced with Gαg. Thus, there may be interactions of Gαg with TAS2R46 which are not seen with this structure, such as with ICL2. Differences in receptor-Gαg interaction at KOR and TAS2R46 suggest that although there may be some conserved binding interactions, these receptors interact with Gαg differently.

KOR-GαZ structures

Gαz is highly expressed throughout the central nervous system. Gαz has the slowest rate of guanidine triphosphate (GTP) hydrolysis and is the only member of the Gi/o family to be insensitive to pertussis toxin [25]. Mice with Gαz knockout exhibit higher anxiety-like behavior and altered effects of serotonergic and dopaminergic systems [26–28]. When measuring momSalB and GR89,696-induced recruitment of Gαi/o subtypes to KOR, Gαz is the most potent of all subtypes [11]. A similar trend is seen in other KOR agonists as well like U50,488, Nalfurafine, and WMS-X600 [29]. The GR89,696-bound structure of KOR and Gαz was solved at 2.65 Å resolution [11].

Unfortunately, there are no other receptor-Gαz bound structures to compare the KOR structure to. However, there are insights into Gαz binding from this structure alone. When comparing Gαz binding interface to the other KOR Gαi1, GαoA, and Gαg structures, Gαz has the largest interface (1262 Å2) with the receptor. Most interactions between KOR and Gαz occur in the α5 and αN helices (Figure 2G) of Gαz similar to Gαi1 and Gαg while GαoA only has interactions in the α5 helix. Mutational analysis of residues on KOR and Gαz revealed several key interactions (e.g., I352H5.23) specific to Gαz compared to any other subtypes [11]. The reason for observed higher potency of Gαz at KOR is not apparent in the cryo-EM structure. More analysis of intermediate states could provide more insights into their binding interactions. The property of slower GTP hydrolysis might contribute to Gαz’s higher potency as it will also slow down the dissociation of G protein heterotrimer from the receptor.

Ligand-specific KOR-G protein interactions

The binding of agonists to GPCRs recruit G proteins and form a ternary complex. Specific receptor-G protein interactions cannot fully explain the G protein selectivity as it is also dependent on ligand. For example, R257ICL3 of KOR forms hydrogen bonds with Gαi1, GαoA, Gαz, and Gαg [11]. Specifically, the momSalB-bound KOR structures of Gαi1 and GαoA both show two hydrogen bond interactions between R257ICL3 and G protein. GR89,696-bound KOR structures of Gαz and Gαg show three hydrogen bond interactions. However, recruitment of these G protein subtypes to KOR with an R257ICL3A mutation differentially effects G proteins, dependent on the agonist used. R257ICL3A has no effect on recruitment of Gαi1, GαoA, Gαz, and Gαg when momSalB is used. However, Gαi1, Gαz, and Gαg recruitment is significantly reduced with the mutant when GR89,696 is applied8. Nalfurafine is also affected by the R257ICL3A mutation [29]. Gαi1, GαoA, and Gαz recruitment was significantly decreased by the R257ICL3A with Nalfurafine. Another mutation, R2716.32A, only affected Gαg recruitment with momSalB but affected Gαi1 and Gαg with GR89,696. For Nalfurafine, R2716.32A affected all G protein subtypes except Gαg. L2535.65A reduced recruitment of all subtypes for momSalB and GR89,696. With Nalfurafine, L2535.65A reduced recruitment of Gαi1, GαoA, and Gαz but not Gαg. These effects are not related to potency at the G protein subtypes as momSalB and Nalfurafine have similar Gαi1, GαoA and Gαz potency, yet these subtypes are differentially affected by various mutations. These results indicate that although this is the same receptor and same G protein subtypes, residues responsible for G protein engagement are different based on the ligand. Therefore, G protein interaction may have similarities across different agonists, but binding is not conserved for all agonists even if the potency of recruitment is similar.

Limitations and future prospects

So far, many active-state structures of KOR in complex with Nb39 or different G-protein transducers and high-quality inactive-state structures of KOR in presence of JDTic have been solved. Together with comprehensive biochemical studies, this confluence has significantly enriched researchers’ profound understanding in the signal activation and transduction mechanisms of KOR. However, there are still some limitations. For instance, it is difficult to elucidate the functional disparities between different G proteins or receptor residues in different KOR structures observed from a purely structural perspective. One of the leading causes for this difficulty is that KOR structures activated by multiple agonists display very similar conformations, as do the structures of diverse G proteins engaged with the activated receptors. The similar structural configurations indicate that KOR signaling regulation could rely on a more complicated three-dimensional network interaction. Therefore, determination of structures of KOR in complex with G proteins at intermediate states is one of the most promising ways to explain functional differences observed from biochemical experiments, which facilitates a deeper comprehension of KOR signal activation and transduction for us. Additionally, based on the present structures of KOR activated by small-molecule agonists (MP1104, Nalfurafine, momSalB and GR89,696) and endogenous peptide agonist dynorphinA1–13, drug designs could target more sites on the KOR (e.g., ECLs), subsequently unraveling the functionalities of these sites, thereby laying a robust foundation for the precise design of drugs for KOR in the future.

Acknowledgements

This work was supported by the NIH grant R35 GM143061 to T.C.

Abbreviations

GPCR

G protein-coupled receptor

Cryo-EM

cryo-electron microscopy

MOR

mu opioid receptor

KOR

kappa opioid receptor

DOR

delta opioid receptor

Gg

Gustducin

α2BAR

α2B adrenergic receptor

5-HT5AR

serotonin receptor 5A

CCR5

chemokine receptor 5

CCR6

chemokine receptor 6

TAS2R46

Taste receptor type 2 member 46

ICL

intracellular loop

ECL

extracellular loop

TM

transmembrane

Footnotes

Conflicts of interest

The authors declare no conflict of interest.

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