Subtype-Dependent Regulation of Gβγ Signalling

Abstract

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gβγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gβγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gβγ signalling has recently been attributed to Gβ and Gγ subtype diversity, comprising 5 Gβ and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gβγ dimer, numerous studies have identified preferences of distinct Gβγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gβ and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gβ and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gβγ signalling and associated diseases.

1. Introduction

1.1. GPCRs and G proteins

G protein-coupled receptors (GPCRs) transduce extracellular signals to the cell interior across the plasma membrane (PM) by activating heterotrimeric G proteins that consist of Gα, Gβ, and Gγ subunits. Structural conservation suggests that eukaryotic GPCRs are evolved from prokaryotic channelrhodopsins, and nearly 800 GPCRs are present in the human genome [1–3]. These receptors respond to a wide variety of extracellular ligands, including hormones, local mediators, neurotransmitters, odorants, photons (light), etc. Ligand binding-induced conformational changes in receptors trigger changes in the interacting heterotrimer at the cytosolic face of the receptor. Conformational changes in the heterotrimer promote GDP to GTP exchange in the Gα subunit, resulting in partial or complete dissociation of GαGTP from Gβγ where ligand-bound activated receptor stabilizes an ‘open’ conformation of Gα to release GDP, facilitating GDP to GTP exchange (Fig. 1). When the ligand is no longer bound to the GPCR, an inactive G protein heterotrimer is restored [4, 5]. While signalling of Gα isoforms has been studied extensively, despite the possible availability of 60 different combinations of Gβγ heterodimers, “Gβγ” remains primarily treated as an eponymous unitary signal transducer.

Figure 1. Activation of heterotrimeric G proteins by the activated G protein-coupled receptor (GPCR) upon ligand binding.

Adapted from the Protein Data Bank Identifier (PDB ID) 1GP2, 6R3Q and 6OY9. Ligand (blue) binding induces conformational changes in GPCR (brown), and promotes GDP to GTP exchange on the Gα (purple) subunit in the ‘open’ conformation along with structural changes in the binding site of Gβ (cyan blue). The heterotrimer complex dissociation into GαGTP and free Gβγ allows them to interact with respective downstream effectors.

1.2. Gβγ complexes

The human genome encodes 5 Gβ and 12 Gγ genes, resulting in significant potential structural and functional diversity in G protein heterotrimers. Among mammalian Gβ isoforms, Gβ1 to Gβ4 share more sequence homology than Gβ5 [6, 7]. Gβ1–4 are 36 kDa proteins while Gβ5 is a 40 kDa protein, with only 50% sequence similarity to other Gβ subunits. Comparatively smaller, Gγ subtypes are between 7–8.5 kDa in size [8]. Gγ subunits show greater sequence diversity than Gβ, indicating their possible roles in generating functional diversity of Gβγ signalling. Variation in amino acid sequence is prominent among the 12 Gγ subtypes, ranging from 20% to 80% [7]. Some Gγ subunits can undergo a number of post-translational modifications. Isoprenylation of the Cys residue in the CAAX motif at the carboxyl-terminal is one of the primary post-translational modifications in Gγ subunits. Prenylation allows membrane localization and likely controls the mobility of Gβγ subunits [9]. Many Gγ subunits contain a Leu residue in the CAAX sequence as X, suggested to promote geranylgeranylation of Gγ through a thioether linkage formation [10]. In several other Gγ types, X is a Ser residue, facilitating farnesylation of the protein [11, 12].

The Gβγ dimer that forms a stable structural unit is illustrated in Fig. 2. The Gβ subunit contains seven WD 40 repeats [13–15]. Comparatively shorter Gγ folds into two α helices. The C-terminal α-helix makes extensive contacts with the base of the Gβ propeller. Gβ and Gγ subunits are tightly associated with each other through hydrophobic interactions. As shown in Fig. 2, some interaction sites include Asp258 in Gβ that interacts with two residues linking the coiled-coil to the Gβ1 propeller; Arg22 in Gβ1 and Arg30 in Gγ1. Residues Phe40 and Phe64 in Gγ form further hydrophobic interactions with Gβ [16]. Unlike Gα, the Gβγ dimer does not undergo modification during activation of the G protein heterotrimer. Further, it is believed that association of Gβγ with the GDP-bound form of Gα generally prevents Gβγ from constitutively activating its effectors.

Figure 2. Assembly of Gβ1γ1 dimer crystal structure adapted from the PDB ID 1TBG.

Gβ and Gγ subunits are shown in cyan, blue and green colors respectively. Black numerals indicate seven blades of the Gβ propeller. A ribbon representation of the dimer indicates some of the interaction sites between the subunits. The magnified diagram shows interaction of Gβ-Asp258 with residues on both the subunits. Hydrophobic interactions between Gγ residues including Phe40 and Phe64 with Gβ are also labeled.

1.3. Gβγ effectors

Active GαGTP and free Gβγ at the inner leaflet of the PM interact with numerous effector molecules. Gβγ dimers regulate a large cohort of effectors, including phospholipases, adenylyl cyclases (AC), G protein-coupled receptor kinases (GRKs), and ion channels [17]. The Gβγ dimer can activate phospholipase Cβ (PLCβ) isoforms, including PLCβ2 and PLCβ3. Although a crystal structure is not available, evidence suggests binding of the β-propeller of Gβ with the pleckstrin homology (PH) domain of PLCβ [18]. Further, Gβγ can regulate the activity of AC isoforms that generate the second messenger cAMP upon receptor activation. The effects of Gβγ on production of cAMP depend on the particular isoform of AC. Unlike with other effectors, Gβγ interacts with AC5 and AC6 through multiple interaction sites, reflecting the complex regulation of cAMP production by Gβγ [19]. Gβγ also interacts with ion channel proteins such as GIRK1 (Kir 3.1) and directly binds to both the N-terminal hydrophilic and C-terminal domains of GIRK1, and voltage-gated calcium channels [11] regulating neuronal and cardiovascular excitability [20, 21].

2. Evolution and subtype diversity of heterotrimeric G proteins

2.1. Gβ subtypes and their diversity across evolution

GPCR and G protein-mediated signalling controls numerous cellular functions in eukaryotes. This occurs at the level of the receptor, G protein, or downstream effectors to fine-tune signalling output [22–24]. Previously, it has been shown that receptors, G proteins, and downstream regulators are expanded through lineage-specific modifications, recurrent domain shuffling, gene duplication, and selection, offering additional levels of expression regulation [23]. Also, tissue-specific expression patterns of G protein subunits have been shown to control designated functions at specific locations in the human body. The functional diversity of G proteins is acquired through evolution and modulated by the particular location both within the cell and the tissue in question.

Based on the phylogenetic analysis performed using protein sequence data obtained from invertebrates, mammals, and plants, Gβ subunits are classified into five groups [25]. Except for G5β isoform, Gβ1–4 are highly conserved, sharing more than 80% sequence identity and form functional Gγβ heterodimers with Gγ subunits (Table 1) [26]. G5β is divergent from the rest of the G βsubunits, exhibiting only 50% sequence similarity [25, 26]. Therefore, it has been suggested that Gβ evolved from a common ancestor and then diverged into two super-families. While one superfamily contains Gβ1–4, the other subfamily consists of only Gβ5 [26], and distinct expression patterns of these subtypes can be seen in different organisms.

Table 1:

Identity matrix of mammalian Gβs

	β1	β2	β3	β3S	β4	β5	β5L
β1	100
β2	87	100
β3	80	81	100
β3S	74	73	88	100
β4	86	88	78	71	100
β5	51	52	52	44	53	100
β5L	46	45	45	40	45	89	100

Gβ subunits in lower eukaryotes

Holozoan family animals and their closest single-cell relatives show an ancient duplication in their genomes, which may have given rise to Gβ1–4 and Gβ5 [1]. However, unicellular holozoans such as C. owczarzaki express only two Gβ subtypes, where one subtype is clustered with Gβ1–4 [27]. Data mining also shows that many pre-vertebrate metazoan genomes possessed Gβ subunits with properties resembling Gβ1–4 and Gβ5 before expanding into vertebrates [27]. Gβ subunits are well characterized in budding yeast, Saccharomyces cerevisiae, which is classified under kingdom fungi and domain Eukaryota. Both of their Gβγ types, beta subunit 1 (Gpb1) and 2 (Gpb2), exhibit sequence homology to vertebrate Gβ1 and Gβ3, respectively. Although it does not form a dimer with Gγ a noncanonical Gβ subunit Vsp5 in S. cerevisiae interacts with PI3K[28]. The fission yeast, Schizosaccharomyces pombe, also expresses a Gβ subunit, Gnr1, that shows sequence homology to the mammalian Gβ1 subunit but does not form a Gβγ dimer [29]. Most filamentous fungi and Dictyostelium sp. only express a single Gβ subunit [30], exhibiting a highly conserved sequence homology [31]. However, their Gβ subunit exhibits lower sequence homology to S. cerevisiae (38%) and S. pombe (45%) Gβ respectively [32]. The genome of another filamentous fungus, Neurospora crassa, also encodes a single Gβ subunit, which exhibits a 65% sequence identity to human Gβ1[32].

Gβ subunits in invertebrates

The Drosophila genome encodes three Gβ subunits, Gβ5, Gβ13F, and Gβ76C [33, 34]. The Gβ76C subunit in D. melanogaster is homologous to vertebrate Gβ1–4 [25]. Comparative analysis of invertebrate Gβ isoforms displays a common trait of Gβ subunit evolution between C. elegans and D. melanogaster [25]. GPB-2 from C. elegans and Gβ5 from D. melanogaster are homologous to vertebrate Gβ5. Additionally, this Gβ5 also dimerizes with RGS (R7) family proteins, thereby controlling the expression and stability of Gβ5 [35]. In the sea squirt, Ciona intestinalis, a similar evolutionary pattern of Gβ subunits to Drosophila is evident. The sea squirt expresses three Gβ subunits that are distinct from the Gβ subunits of other species. A cross genome phylogenetic analysis to identify the Gβ subunits in invertebrates showed that, other than Gβ1–4 and Gβ5, a separate Gβ cluster was found in arthropods, which is known as Gβe. Gβe was identified in all the insects and the crustacean D. pulex [27]. However, Gβe has not been found in protostomes, such as annelids and mollusks [27].

Gβ subunits in plants

Compared to the five different Gβ subtypes in vertebrates, plants are evolutionarily limited to one Gβ subunit [36, 37]. Based on the sequence analysis, Gβ type from AGB1 (Arabidopsis), NGB1 (Nicotiana), and RGB1 (O. sativa) are significantly homologous to each other while also showing similarity to vertebrate Gβ2 counterparts [25]. Maize Gβ, ZGB1 is homologous to Arabidopsis AGB1, with 76%. Their sequence homology to animal Gβ subunits is ~41% [37]. Based on the sequence alignment and secondary structure predictions, both ZGB1 and AGB1 consist of 7 WD repeats [37]. An important phylogenetic relationship exists between Gβ subunit in eudicots, monocots, and lower pants [38]. All the monocots exhibit more than 90% sequence similarity to RGB1. RGB1 also contains the canonical seven-β propeller architecture with six identified WD repeats [38]. N-myristoylation signals help RGB1 association with PM, thereby regulating signal transduction such as plant adaptation to high salt stress [39].

Gβ subunits in vertebrates

Using a cross genome phylogenetic analysis, it was found that in vertebrates, the Gβ1–4 cluster is common to all vertebrates except extant vertebrates such as lamprey [27]. The lamprey expresses two genes belonging to the Gβ1–4 cluster. Also, the Gβ5 cluster among mammals showed high sequence homology. Human Gβ5 only shows 50% homology to other Gβ subunits. On the other hand, human Gβ5 showed a 99% sequence similarity to mouse Gβ5. In mammals, Gβ subunits are tightly conserved regardless of their species type. Mammalian species such as mice (Mus musculus) and rats (Rattus norvegicus) have been widely used to study human GPCR, G protein functions since their G protein subtypes are conserved across species [25].

Structural differences in mammalian Gβ types

As discussed earlier, mammals express five different Gβ subunits. There are five different genes to encode Gβ subunits and their splice variants (β1, β2, β3, β3S, β4, β5, β5L) in the human genome [6, 40]. Splice variants in Gβ1, Gβ2, and Gβ4 subtypes have not been detected [41]. Instead of limiting to transcript variants, Gβ subtype-specific functions may have evolved through gene duplication followed by selection. GNB1 (Gβ1) is on chromosome 1 of the human genome. This transcript produces 12 exons, with 9 of them considered coding exons. The first two exons and the last exon of Gβ1 are noncoding. It has been shown that Ser at the second position of the Gβ1 can be N-acetylated or phosphorylated. Phosphorylation at His266 in Gβ1 also contributes to G protein activation. The information encoded in exons 6 and 7 includes the protein region of Gβ1 that forms the Gβγ interaction interface with Gα [17]. Based on the crystal structure of GIRK2 (Kir3.2) and Gβ1γ2, Arg52 in Gβ1 is involved in interactions between Gβ1 and GIRK [42]. GNB2 (Gβ2) is located on chromosome 7 in the human genome, and its transcript has 10 exons with 9 coding exons. Post-translational modifications such as N-acetylation and phosphorylation are predicted at Ser2 and Thr239, respectively. Earlier, it has been suggested that Gβ3 may be linked to either Gβ1 or Gβ2 since a protein corresponding to the Gβ3 gene was not identified. Discordancy analysis and in situ chromosome hybridization revealed that chromosomal localization of GNB3 is different from the locations of genes, GNB1 and GNB2 [43]. GNB3 was discovered on chromosome 12, and its transcription gives rise to 11 exons. The homology model of GNB3 generated after fitting GNB1 crystal structure data demonstrates several amino acid residues of Gβ3 are essential for the folding of β propeller. The Trp339 residue would affect the proper folding of β-propeller, and Ser67 would participate in hydrogen bonding to keep the top of the β barrel in the proper orientation as required for protein-protein interactions. A splice variant of GNB3 (GNB3S) is a result of an alternative splicing event taken place to remove exon 9 (123bp) of GNB3 [44]. Therefore, Gβ3S lacks one WD repeat domain (Fig. 3). Phosphorylation, acetylation, and ubiquitination signals were predicted in Gβ3. GNB4 (Gβ4) encodes 10 exons and shows molecular signatures for N-acetylation and phosphorylation. Gβ4 shares high sequence similarity to Gβ1 and Gβ2, at almost 86%. GNB5 is on chromosome 15 in the human genome with 12 exons. Gβ5 shows approximately 50% sequence homology to Gβ1–4, indicating likely functional differences from other Gβ subunits [45]. It has been shown that Gβ5 is functionally distinct from Gβ1–4, with comparably weak interactions with Gγ subunits [46]. Instead of irreversible binding to Gγ, Gβ5 interacts with R7 RGS family proteins. Gβ5L is identical to Gβ5, but Gβ5L has additional 126 bps due to an additional codon at the 5´ but lacks 5´ portions of its third exon, generating a 44-kDa protein (Fig. 4) [47]. This 126 bp-extension was not derived from the 5´ untranslated region of GNB5 but arose from retina-specific usage of a 5´ exon [40]. Gβ1–5 subunits are approximately 36 kDa in size, while Gβ5L is approximately 44 kDa. Free Gβγ subunits initiate canonical signalling pathways by activating multiple effectors [48–50]. Gβ subtypes (Gβ1–4) can dimerize with 12 different Gγ subtypes encoded in the human genome, resulting in 48 possible Gβγ combinations. A region of 14 amino acids in Gγ (36–49) was identified to control association with Gβ [51]. Gβ1 can form heterodimers with all Gγ subunits while Gβ2 fails to form heterodimers with Gγ1, showing restrictions in its Gγ partners [52, 53]. However, Gγ1 and 2 are unable to form heterodimers with Gβ3 [53]. Gβ3S was first identified as a gain of function mutant leading to enhanced activity of Gα and Gβγ [54]. Gβ3S showed less stability than other Gβ subunits and was incapable of forming functional heterodimers with different Gγ isoforms [55, 56]. However, a more sensitive yeast two-hybrid assay was used to assess Gβ and Gγ interactions. Here, the data showed that all Gγ isoforms could interact with Gβ1 and Gβ2 but poorly with Gβ3 and Gβ4 [57]. However, co-immunoprecipitation analysis of Gβ4 showed dimer formation with all known Gγ types [58]. A similar study also demonstrated that Gγ5 has more robust interactions with Gβ4 compared to Gγ12, 2, and 3[59].

Figure 3. Proposed structure of Gβ3 generated using coordinates obtained from Gβ1 crystal structure (PDB id: 3AH8).

The homology model of Gβ3 (green) is superimposed with Gβ3S (blue) homology model generated using Phyre web portal (http://www.sbg.bio.ic.ac.uk) employing Gβ3 as the template. The yellow color indicates the 4^th WD repeat of Gβ3 that absent in its splice variant, Gβ3S.

Figure 4. The molecular structure of Gβ5.

Gβ5 (Green) was modelled using coordinates obtained from PDB file 6N9G is superimposed with Gβ5L (Blue), homology model generated from Phyre web portal (http://www.sbg.bio.ic.ac.uk) using Gβ5 as template. The yellow colored region was encoded by the additional codon at the 5′ end.

Compared to Gβ1–4, due to its significant sequence differences, distinct tissue and even subcellular distributions, and the limited evidence for its ability form dimers with Gγ, Gβ5 has been characterized as unique. Gβ5 subunit dimerizes with R7 subfamily of RGS family proteins that contains a Gγ-like (GGL) domain. The R7 family consists of RGS6,7,9, and 11, and their structures are evolutionarily conserved in all animals from worm to human [60]. The Gβ5-R7 complex is widely expressed in the brain and nervous system. Comparison of Gβ5 and R7 family protein expression in brain is illustrated in Fig. 5. Gβ5-RGS7 is associated with motor control, reward behavior and nociception in mammals [61, 62]. In mammalian nervous systems, the R7 family proteins regulate key physiological functions such as synaptic transmission, memory formation, and light perception [62, 63]. The GGL domain of the R7 family is highly selective for Gβ5 and does not show association with other Gβ subunits. Evidence also shows that Gβ5-R7 complexes rapidly degrade in the absence of the other, indicating the mutual stabilization [64]. Though R7 mRNA levels remained intact, knocking out Gβ5 resulted in a reduction of the R7 family protein concentrations [65]. Interestingly, in vivo or cellular evidence for Gβ5-Gγ interactions is still not available [62]. Gβ5-R7 complexes form heterotrimers with the membrane anchoring R7 subunit, R7BP, which helps the expression and localization [63, 66]. There is also no in vivo evidence for Gα - Gβ5γ heterotrimers. However, recent evidence suggests that Gβ5-RGS7 can associate with several GPCRs, including the M3-muscarinic receptor [67] and orphan GPCRs [68]. Deletion of the third cytosolic loop of M3R reduced the sensitivity of mutant M3R to Gβ5-RGS7 complex. In reconstituted systems, Gβ5-RGS7 has been shown to attenuate Gi- and Gq-mediated signalling [67, 69], however, underlying molecular mechanisms are yet to be understood. Interestingly, a recent in vitro study showed that Gβ5 could associate with Gγ2, 3, 4, 5, 7, 8, and 12 subunits, independently of the Gα subunit [70]. Gβ5 and Gγ complexes also have been shown to activate effectors such as PLCβ2 [45] and AC [35], while they fail to activate ERK or JNK MAP kinases [71]. Gβ5L and Gγ2 overexpression in COS-7 cells showed PLCβ2 activation, indicating Gβ5L-Gγ2 interactions are possible [40]. However, no direct evidence has been shown to confirm Gβ5L-γ2 dimer formation in vivo. Further, interactions of Gβ5L with the other Gγ subtypes have not been reported.

Figure 5. Comparison of Gβ5 and R7 family protein expression in the brain.

Consensus normalized RNA expression of Gβ5 and R7 family proteins in brain. RNA expression data were obtained from the FANTOM5 repository in the human protein atlas database.

The first crystal structure of Gβγ dimer was solved in 1996 [16]. Several crystal structures of Gβγ were solved thereafter, as complexes with different effectors [50, 72–74]. Based on the crystal structure of the transducin bound Gβγ dimer, the β subunit is primarily a seven-bladed β-propeller, and each propeller blade contains small four antiparallel strands spreading outwards from a central axis to generate the seven-fold symmetry [16]. Such symmetry has been found as the most favored arrangement in other proteins, including methylamine dehydrogenase and galactose oxidase [75]. This seven-fold symmetry of β strands also reflects in their amino acid sequence, which consists of seven structurally similar repeats and each contains approximately 40 amino acids with conserved core amino acids bounded by Trp-Asp (WD) [16] and Gly-His (GH) [76] and separated by a variable region. WD repeats are common to β subunits found in all organisms, including invertebrates [26]. The variable-length region is highly conserved within the family while differences have been noted in individual Gβ subunits. For example, the variable region between repeat two and three is different from repeat three and four while showing significant similarities in the same regions in evolutionarily distinct organisms [76]. Crystal structure data also indicates that WD repeats in the Gβ subunit initiate from the outermost strands of the β-sheet and terminate at similar positions in the adjacent β-sheet [16]. The outer β-stands of each blade in Gβ are made up of these variable regions in WD repeats. Therefore, each WD repeat generates four anti-parallel β-sheets, denoted by a-d [16]. Strand “a” is placed in the center of the tunnel, while strand “d” is found outside on the surface of the Gβ. This highly conserved Asp residue is also found in the loop, which connects strands “b” and “c” [16] (Fig. 6). However, the associated functions of these regions are still unknown.

Figure 6. Secondary structure prediction of Gβ2.

Gβ2 amino acid sequence (using Phyre2 web portal (http://www.sbg.bio.ic.ac.uk- Query sequence) is scanned through nr20 protein sequence database. The resultant multiple-sequence alignment is used to predict the secondary structure of the protein using PSIPRED (protein secondary structure prediction software). Gβ2 secondary structure was predicted using Gβ1 and other WD repeat containing proteins.

Furthermore, the Gβ subunit contains an N-terminal coiled-coil structure in the first 30 amino acids to help form three-stranded coiled-coil domains of G protein heterotrimers [77, 78]. Interactions between adjacent β sheets of Gβ are well characterized. Trp is a common residue found in WD repeats that interacts within the same repeat and Asp-His-Ser/Thr, resembling the catalytic triad of serine proteases buried in a non-polar environment between β-sheets [16]. The carboxylate group of Asp makes hydrogen bonds with main-chain amides in the positions of tight turns and adjacent conserved His in the loop, which connects the first and second strands of the same repeat. Consequently, such hydrogen bond arrangements stabilize the tight turn of one β-sheet to the outer strand of the β-sheet it follows [16].

Gβ subunits interact with Gα through two non-overlapping regions defined in the crystal structure of the G protein heterotrimer [17]. The Gβ residues in the switch interface (57, 59, 98, 99, 101, 117, 119, 143, 186, 228, and 332) and the N-terminal interface (55, 78, 80, and 89) interact with Gα. Ala mutants to replace the amino acids of Gβ interacting Gα were tested for their ability to form heterotrimers. Interestingly, Gβ mutants I80A, K89A, L117A, and W332A were defective in forming heterotrimers among all the mutants in this study, illustrating that these residues are critical determinants of Gα binding [17]. Gβ shares a common interface to interact with both Gα subunits and downstream effectors [17]. In the alanine scan, the N-terminal interface of Gβ1 exhibited a decreased ability to activate AC2. The Gβ1 mutants L117A, and N143A resulted in decreased association with GRK2 while W99A and D228A mutants could no longer activate PLCβ2 [17]. Ala mutations of Gβ1 residues 55, 78, 80, 89, 99, and 228 disrupted K⁺ currents via GIRK1/GIRK4 activation.

Tissue and cell type-specific Gβ distribution in mammals

Tissue and cell-type-specific G protein expression in mammals ranges from ubiquitous to restricted based on tissue type. Here, we examine Gβ transcript expression levels in 45 human tissues from the FANTOM5 repository in the human protein atlas database [79]. Consensus normalized expression levels of Gβ subunits (NX) in 45 human tissues are shown in Fig. 7.

Figure 7. Tissue specific distribution of all 5 Gβ subtypes.

Consensus normalized RNA expression of Gβ subtypes in human tissues. RNA expression data for the distribution of Gβ subtypes in human tissues was obtained from the FANTOM5 repository in the human protein atlas database. Though Gβ subtypes exhibited higher sequence homology, they also show tissue type-specific distributions. The coloured-numbers above each tissue type represent the three most abundant Gβ types.

Gβ1 is ubiquitously expressed in all tissues in mammals [80–83]. According to protein atlas data, almost all tested human tissues have a higher expression of Gβ1 compared to other Gβ subunits. The highest expression of Gβ1 is observed in the human putamen and small intestine, whereas in mice, Gβ1 is abundant in the retina. Gβ3 is expressed at higher levels in the human retina along with Gβ1 and Gβ2 [84]. Several other reports also showed that Gβ3 is highly expressed in cone photoreceptors and bipolar cells of the retina [83, 85–88]. Gβ3S protein was found in lymphocytes and platelets of patients with essential hypertension [89]. From human atlas data, tissue-specific expression of Gβ4 exhibits a unique pattern in many cells. Compared to Gβ1 and Gβ2, Gβ4 expression widely varies in different tissues. Independent studies showed that Gβ4 is highly expressed in skin fibroblasts [90], brain, eye, heart, testis [54], lung, and placenta, while it is less abundant in the brain, spleen, and heart [91, 92]. Mouse and human Gβ5 are predominantly expressed in the brain [45], whereas human Gβ5 is also abundant in the pancreas, kidney, and heart [47]. In the human brain, Gβ5 is detectable in all regions but less abundant in the corpus callosum and spinal cord. Compared to other Gβ subunits, Gβ5 has a more restricted tissue expression pattern [45].

2.2. Gγ subtypes and their diversity in the animal kingdom

Gγ subtypes across evolution

Gβγ proteins play a crucial role in the pheromone response pathway in S. cerevisiae [93, 94]. This pathway promotes cell fusion and the generation of the diploid state [95]. Multiple types of Gγ subtypes are expressed in other fungal types [96]. S. pombe expresses a single Gβγ dimer with the Gγ protein, git11, which is involved in pheromone responses [97]. N. crassa expresses the GNG-1 subunit, which plays a role in fertility and asexual development and has a gene structure similar to the Gγ genes of mammals [98].

Like mammals, invertebrates express more than one Gβγ isoform, suggesting subunit evolution over time. For instance, C. elegans expresses two types of Gγ proteins, GPC-1 (guanine nucleotide-binding protein, gamma polypeptide-1) and GPC-2 (guanine nucleotide-binding protein, gamma polypeptide-2), which are similar to the vertebrate Gγ 1/9/11 subfamily and Gγ 13 respectively [25]. Gβγ in C. elegans controls spindle orientation during the early embryonic development stage [99–101]. The D. melanogaster genome encodes two Gγ proteins, Gγ1 and Gγ30, and these are again similar to vertebrate Gγ 1/9/11 subfamily and Gγ 13, respectively. Gγ proteins of D. melanogaster control cell division in neuronal and sensory organs along with three Gβ counterparts [102]. Additionally, Gβγ signalling controls wing expansion in these animals [103].

Fish and mammals have more Gγ subunits than fungi and invertebrates, indicating evolution of a broader signalling footprint. Studies also suggest that some fish species have more Gγ subunits than mammals [104]. Humans and mice have 12 Gγ subunit isoforms [7]. Genomic analyses have identified that there are at least 17 Gγ subunits in zebrafish. Most of these Gγ subtypes are orthologues of human Gγ subtypes. Four Gγ subunits in zebrafish, gng14, gng15, gng16, and gng17, do not have mammalian Gγ orthologues. An orthologue for human Gγ11 was not found in zebrafish; however, two Gγ paralogues, known as gngt2b and gng12b, have been identified. Their high sequence similarities showed that Gγ subunits found in fish species must have evolved from the same ancestor as in mammalian species [25]. This also suggests that the additional Gβγ subunits found in fish species might have evolved after divergence from the common ancestor or been lost in the evolution from fish to mammals [104]. Genomic data of the Atlantic cod, Gadus morhua, showed that Gγ1 and Gγ11 are redundant. It is not yet known whether these subunits are functionally redundant at the protein level as well [25].

Structural differences in mammalian Gγ types

The first Gγ subunit was identified by John Hildebrandt in 1983 in a study of human erythrocyte stimulatory and inhibitory regulatory G proteins [105]. The identified protein had a molecular weight of 5 kDa and complexed with the Gβ subunit [105]. All Gγ subunits are small proteins comprising two α-helical segments. Gγ subunits have no obvious tertiary structure. The N-terminal helix forms a coiled-coil structure with the N-terminal helix of Gβ, while the remainder interacts with the stem of the β-propeller domain of Gβ (Fig. 8). These interactions provide stability to the Gβγ dimer [16]. Gγ subunits are products of two exons that encode two domains of the protein, the N-terminal and C-terminal helices. The first exon encodes ~27–32 amino acids of the N-terminal helix. The second exon encodes ~40 amino acids that form the C-terminal helix. The first residue encoded by exon 2 is almost always a Val, which forms a hinge between the protein components formed by the two domains of the protein, and it is a primary site of Gβγ interaction. This Val residue associates with the N-terminal α-helix and the propeller domain of the Gβ subunit [106]. This residue is shown inside the red square in Fig. 9.

Figure 8. Molecular model structure of Gβγ dimer of Gβ1 and Gγ1, using coordinates obtained from PDB file 1TBG.

Gβ1 and Gγ1 are shown in green and blue respectively. The N-terminal helix of Gγ1 interacts with the N-terminal helix of Gβ1 to form a coiled-coiled structure. The C-terminal helix of Gγ1 shows interactions with the β-propeller domain of Gβ1.

Figure 9. Sequence alignment of N-terminal helices of Gγ subtypes.

Primary point of interaction between Gγ and Gβ protein chains is a Val residue shown here in red. Amino acid sequences of Gγ subunits were obtained from the National Center for Biotechnology Information (NCBI) database for proteins. Sequence alignments were performed using the MUSCLE sequence alignment tool by EMBL-EBI.

Compared to Gβ subunits, Gγ subunits show significantly higher sequence diversity [107, 108]. Investigations into sequence diversity among Gγ subunits and their implications in cellular signalling remain at an early stage. However, recent findings show that the diversity among Gγ proteins in Gβγ dimer could be responsible for distinct signalling outcomes since cells can express distinct subsets of Gγ subtypes [48, 109]. Mammals, including humans, express 12 Gγ subunit subtypes, Gγ1 to Gγ13 (no Gγ6) [109]. Complete sequence analysis of the 12 human Gγ subtypes is shown in Table 2. Obtained sequence similarity scores show wide and discrete series of similarity scores ranging from 20–80 %. This range was much narrow for Gβ subunits, which indicate that the Gγ subunit is likely an important contributor to Gβγ signalling diversity

Table 2:

Sequence similarity scores of complete sequence analysis of human Gγ subunits.

	Gγ1	Gγ2	Gγ3	Gγ4	Gγ5	Gγ7	Gγ8	Gγ9	Gγ10	Gγ11	Gγ12	Gγ13
Gγ1	100
Gγ2	35.1	100
Gγ3	32.1	70.7	100
Gγ4	30.7	70.7	68.0	100
Gγ5	24.7	41.6	44.2	42.9	100
Gγ7	37.8	65.2	58.0	58.0	48.5	100
Gγ8	31.4	68.6	55.7	58.6	42.9	51.4	100
Gγ9	62.9	35.7	32.0	32.0	24.7	40.6	31.4	100
Gγ10	30.4	50.7	50.7	46.4	54.4	51.5	50.0	36.2	100
Gγ11	75.7	33.8	32.4	29.7	27.6	41.1	29.7	60.3	28.8	100
Gγ12	34.2	58.9	57.5	52.1	44.4	70.8	50.7	36.1	43.1	36.1	100
Gγ13	27.0	28.2	25.6	28.3	22.9	28.2	28.2	28.2	23.1	33.3	20.5	100

Since Gγ sequences show a wide range of similarities, a more extensive analysis of the similarity of the Gγ domains was undertaken. Tables 3 and 4 show sequence comparison of N-terminal and C-terminal helices of the 12 human Gγ subunits. As shown in Table 3, sequence similarity scores of N-terminal helices of human Gγ subunits are much lower than the whole sequence similarity scores. This shows that the N-terminal amino acid residues are less conserved among Gγ subunits. This could also suggest that the basis of differences among Gγ subtypes reflects N-terminal sequence differences.

Table 3:

Sequence similarity scores of the N-terminal helices of human Gγ subunits.

	Gγ1	Gγ2	Gγ3	Gγ4	Gγ5	Gγ7	Gγ8	Gγ9	Gγ10	Gγ11	Gγ12	Gγ13
Gγ1	100
Gγ2	31.2	100
Gγ3	27.8	54.5	100
Gγ4	22.6	61.3	61.3	100
Gγ5	24.2	27.3	33.3	33.3	100
Gγ7	31.2	66.7	59.3	48.1	44.4	100
Gγ8	28.6	71.4	46.4	46.4	28.6	46.4	100
Gγ9	60.7	28.6	27.3	27.3	19.4	28.6	32.1	100
Gγ10	29.6	48.1	44.4	40.7	48.1	51.9	42.9	39.3	100
Gγ11	65.6	28.1	28.1	18.8	28.6	34.4	21.9	53.1	21.9	100
Gγ12	19.4	51.6	51.6	41.9	35.5	58.8	51.6	22.6	41.9	19.4	100
Gγ13	23.3	29.2	29.2	29.0	27.3	25.0	25.0	29.2	25.0	33.3	16.7	100

Table 4:

Sequence similarity scores of the C-terminal helices of human Gγ subunits.

	Gγ1	Gγ2	Gγ3	Gγ4	Gγ5	Gγ7	Gγ8	Gγ9	Gγ10	Gγ11	Gγ12	Gγ13
Gγ1	100
Gγ2	38.1	100
Gγ3	35.7	83.3	100
Gγ4	36.4	75.0	70.5	100
Gγ5	25.0	50.0	50.0	50.0	100
Gγ7	42.9	64.3	57.1	61.9	41.2	100
Gγ8	33.3	66.7	61.9	64.3	50.0	54.8	100
Gγ9	64.3	40.5	35.7	35.7	29.3	48.8	31.0	100
Gγ10	31.0	52.4	54.8	47.6	56.1	51.2	54.8	34.1	100
Gγ11	83.3	38.1	35.7	38.1	26.8	46.3	35.7	65.9	34.1	100
Gγ12	45.2	64.3	61.9	57.1	48.8	80.5	50.0	46.3	43.9	48.8	100
Gγ13	29.5	31.8	27.3	34.1	20.9	32.6	29.5	34.9	25.6	32.6	30.2	100

As shown in Table 4, the C-terminal helices of human Gγ subtypes show higher sequence similarity than the N-terminal helices, congruent with previous studies [48, 106, 110]. This could indicate that functionally important core amino acid residues in Gγ subunits could be in the C-terminal helix. Sequence variation in the N-terminal domain might have functional consequences for distinct Gβγ signalling events or modes of regulation.

Except for Gγ13, all Gγ subtypes possess a conserved Phe residue in the C-terminal region (Fig. 10). This residue acts as the final contact point of Gγ with Gβ. It has also been shown that the amino acids from the above Phe to the prenylated Cys, termed hereafter pre-CAAX region, plays a prominent role in regulating the PM affinity of Gβγ [48]. The sequence comparison of pre-CAAX of the 12 Gγ subtypes is shown in Table 5. Sequence similarity scores of the pre-CAAX and CAAX regions of Gγ subunits are higher than the similarity scores obtained for the complete C-terminal helix. This shows that the conserved pre-CAAX and CAAX regions of Gγ again reflect the high sequence similarity of the C-terminal helices. This also indicates that the functionally conserved amino acid residues of Gγ are located in the pre-CAAX and CAAX regions (Fig. 11).

Figure 10. Sequence alignment of pre-CAAX and CAAX regions of Gγ subtypes.

The point of prenylation, a Cys residue is shown in green. A Phe residue is conserved in the pre-CAAX region of all Gγ subtypes, except in Gγ13. The conserved Phe residue is shown in red. Sequence alignments were performed using the MUSCLE sequence alignment tool by EMBL-EBI.

Table 5:

Sequence similarity scores of the pre-CAAX and CAAX regions of human Gγ subunits.

	Gγ1	Gγ2	Gγ3	Gγ4	Gγ5	Gγ7	Gγ8	Gγ9	Gγ10	Gγ11	Gγ12	Gγ13
Gγ1	100
Gγ2	45.5	100
Gγ3	36.4	90.9	100
Gγ4	45.5	90.9	81.8	100
Gγ5	27.3	45.5	45.5	45.5	100
Gγ7	45.5	54.5	45.5	54.5	40.0	100
Gγ8	27.3	54.5	63.6	54.5	36.4	45.5	100
Gγ9	81.8	45.5	36.4	45.5	20.0	50.0	27.3	100
Gγ10	27.3	54.5	63.6	45.5	40.0	30.0	54.5	30.0	100
Gγ11	81.8	45.5	36.4	45.5	20.0	50.0	36.4	80.0	40.0	100
Gγ12	45.5	54.5	45.5	54.5	40.0	90.0	45.5	50.0	30.0	50.0	100
Gγ13	45.5	45.5	36.4	54.5	20.0	40.0	27.3	50.0	30.0	50.0	40.0	100

Figure 11. Sequence alignment of C-terminal helices of Gγ subtypes.

Pre-CAAX and CAAX regions of Gγ primarily govern Gβγ-PM interactions. Gγ types can be grouped based on their Pre-CAAX similarities. Sequence alignments were performed using the MUSCLE sequence alignment tool by EMBL-EBI.

Tissue and cell type-specific Gγ distribution in humans

mRNA profiling of human Gγ proteins generally shows ubiquitous distribution. However, each Gγ also shows distinct cell and tissue type-specific distribution patterns (Fig. 12), further suggesting that Gγ is a contributor to the functional diversity of Gβγ signalling. Transcript profiling data from the Human Protein Atlas (Fig. 12) shows that Gγ1 (GNG1) is hardly present in any tissue other than the retina. GNG1 expression in the retina showed the highest expression for any Gγ subtype out of all the tissues. Besides GNG5, GNG10, GNG11, and GNG12, other Gγ subtypes were present in trace amounts in most tissues. GNG3 was found to be abundant in the cerebellum and cerebral cortex. However, GNG3 is present only in trace amounts in the midbrain region. GNG5 was found to be the most abundant Gγ subtype in the human body. GNG5 shows exceptionally high amounts of expression in muscle tissues. GNG5 and GNG12 show high amounts of expression in organs in the digestive tract. GNG8 does not show significant expression in any of the tissues in the human body. GNG13 is also another Gγ subtype with reduced expression in most tissues. It only shows significant expression in the cerebellum, cerebral cortex, and retina.

Figure 12. Consensus normalized RNA expression levels of Gγ subtypes in human tissues.

RNA expression data for the distribution of Gγ subtypes in human tissues was obtained from the FANTOM5 repository in the human protein atlas database. Three Gγ subtypes that are shows the highest expression in each tissue are given. At least at the RNA-level, Gγ shows a tissue type-specific distribution. The coloured-numbers labelled above each tissue type represent Gγ subtypes.

3. Gβ-subtype-specific regulation of signalling

GPCR and G protein activation initiate subsequent downstream cellular signalling events resulting in diverse physiological functions [111]. In this section, the determinants of signalling specificity by G protein subunits and specificity of heterotrimer combinations towards specific GPCRs are discussed.

GPCR activation regulates many signalling pathways, generally aligned with coupling to a specific Gα subunit in the heterotrimer including Gi/o, Gs, Gq, G12/13 and Gt, while the associated Gβγ subunits also regulate a large number of effectors [112]. G protein heterotrimers are comprised of 16 Gα, 7 Gβ (including known variants), and 12 Gγ have been identified in mammalian species that interact with GPCRs (Table 6) [41, 113, 114].

Table 6.

Isoforms of G proteins

Subunit	Isoforms
α	s, olf, i1, i2, i3, oa, t1, t2, gus, z, q, 11, 14, 15, 12, 13
β	1, 2, 3, 3S, 4, 5, 5L
γ	1, c (9), 11, 2, 3, 4, 5, 8, 10, 7, 12, 13

Numerous combinations of Gαβγ can form heterotrimers (Fig. 13 A, PDB Id: 1GOT[15]), and heterotrimer activation upon stimulation of GPCRs drives distinct cell- and tissue type-dependent signalling outcomes [41, 114]. Even though hundreds of (~960) heterotrimeric Gαβγ combinations (16Gα x 5Gβ x 12Gγ) and 60 heterodimeric Gβγ (5Gβ x 12Gγ) are theoretically possible, formation of all these different combinations is unlikely, and unique affinities among specific subunit types have been reported. Some examples include the affinity of all Gγ subunits to dimerize with Gβ1 and Gβ4, while Gβ2 and Gβ3 have show attenuated interactions with Gγ1/Gγ11[8, 115]. Additionally, weak affinities of Gβ5 for Gγ subunits have also been reported [8, 115]. In part because of specificity in dimer formation, Gβγ can interact with specific GPCRs [8]. Detailed discussion for different and specific heterotrimer interactions are in the subsequent sections below.

Figure 13. Structure of the Gαβγ transducin heterotrimer (PDB ID: 1GOT).

(A) Here, we indicate the G protein subunit interaction sites such as the helix at the N-terminus of Gα and the first blade of Gβ defined as the switch interface and the N-terminal interface, respectively. Determinants of polar interactions (red dashed lines) such as hydrogen bonds and ion pairs, and hydrophobic interactions (yellow dashed lines) between amino acids of Gα and Gβ subunits in the switch interface (B) and N-terminal interface (C) are shown. Hydrophobic residues such as Iso180, Phe195, Trp207, His209, Cys210, and Phe211 from Gα, interact with Tyr59, Trp99, Met101 and Leu117 residues of Gβ, in the switch interface. The N-terminal interface comprises of the Gα helix containing Ser12, Glu16, Leu19, Asp22, and Ala23, interacting with Leu55, Lys78, Iso80, and Lys89 on Gβ. In Addition, Lys 89 from Gβ is recognized as a conserved residue to facilitate dynamic ion-pair interactions with Gα.

3.1. The specificity of Gβ signalling

Changes in Gβ subunit signalling have been linked to pathophysiological effects such as abnormalities in brain morphology, eye electrophysiology, cardiac functions, vision, and embryonic defects in various animal models[116]. The crystal structure of heterotrimeric G protein transducin complex (Gαtβγ) shows that the β-propeller domain interacts with both Gα and Gγ subunits[15] (Fig. 1A). Key interactions occur between the switch I and II regions of Gα and Gβ (Fig. 1A, B). This region is important for conformational changes upon GDP to GTP exchange on Gα. Other interactions are identified between the helix at the N-terminus of Gα and the first blade of Gβ (Fig. 13C). These two regions are defined as the switch interface and the N-terminal interface [15] (Fig. 13). The switch interface includes hydrophobic residues from both Gα and Gβ; thus, it generates a network of hydrophobic interactions stabilized by hydrophilic interactions such as hydrogen bonds and ion pairs (Fig. 13B and Table 7). In the switch interface, hydrophobic residues such as Iso 180, Phe 195, Trp 207, His 209, Cys 210, and Phe 211 from Gα, interact with Tyr 59, Trp 99, Met 101 and Leu 117 residues of Gβ [15]. The N-terminal interface consists of the Gα helix containing Ser 12, Glu 16, Leu 19, Asp 22, and Ala 23, interacting with Leu 55, Lys 78, Iso 80, and Lys 89 on Gβ (Fig. 13C and Table 7). Additionally, Lys 89 from Gβ was identified as a conserved residue for facilitating dynamic ion-pair interactions with Gα [15]. Though these interacting residues are from the transducin heterotrimer, most residues are conserved across other Gα and β subunits as well. However, careful consideration should be given to other specific interactions between different Gαβ combinations in individual heterotrimers [117].

Table 7.

Interactions between G protein α and β subunits [15]

G protein	Residues involved (PDB ID: 1GOT)
	Switch interface	N-terminal interface
Gα	Iso180, Phe195, Trp207, His209, Cys210, Phe211	Ser12, Glu16, Leu19, Asp22, Ala23
Gβ	Tyr59, Trp99, Met101, Leu117	Leu55, Lys78, Iso80, Lys89

3.2. Gβ-subtype-dependent Gβγ combinations and their control of cellular signalling

Specific roles for Gβ subunits in diverse signalling processes have been identified [25]. For example, Gβ subunit specificity in inhibiting voltage-gated N-type calcium channels has been shown previously [118]. Gβγ-mediated inhibition of N-type calcium channels by α₂-adrenergic receptor (α₂AR) activation was measured in rat superior cervical ganglion (SCG) neurons. Electrophysiological measurement of calcium currents via voltage-dependent calcium channels was performed. The injection of Gβ DNA into rat SCG neurons was performed to generate Gβ subtype levels in these cells. Among different Gβ subtypes, Gβ1 and Gβ2 exhibited greater inhibition of calcium currents. Additionally, compared to Gβ1 and Gβ2, Gβ5 showed a diminished effect while Gβ3 and Gβ4 were unable to regulate calcium currents [118]. In the same study, yeast two-hybrid screening was utilized to identify specific protein-protein interactions with Gβ subtypes and calcium channels. The interactions of a consensus sequence motif QXXER in calcium channels with Gβ has been suggested for specific regulatory effects on calcium currents [118]. For example, a QXXER peptide was identified from AC2 that inhibited the interactions between Gβγ and effectors, including PLCβ3, AC2, GIRK channels, and GRK2 [119]. Gβ subtype specificity was also investigated in other expression systems that incorporated effectors interacting with Gβ. Co-expression of Gβ1, together with AC5 or AC6, showed reduced cAMP generation in COS-7 cells, while co-expression of Gβ5 had no effect [120]. Gβ1, Gβ2, and Gβ4 showed enhanced activation of PLCε, while Gβ3 had a weaker effect [121]. Specific Gβγ combinations were shown to activate PI3K using purified recombinant proteins. There, Gβ1γ2, Gβ2γ2, and Gβ3γ2 induced comparable PI3K activity while Gβ5γ2 had no effect [122]. Another study demonstrated Gβ1γ2 and Gβ3γ2 induced activation of PKD and subsequent control of PKC-mediated cellular processes [123].

Different Gβ subtypes have been shown to involved in various physiological events. For example, Gβ2 has been shown to regulate GIRK channel function and is implicated in regulating heart rate and cardiac function [42]. Gβ3 is highly expressed in cone photoreceptors of the mammalian retina [86] and involved in light-induced photoreceptor activation and subsequent transducin signalling [85]. Additionally, Gβ3 also regulates signalling in bipolar cells in the mammalian retina. Gβ4 is involved in cardiac signalling through M2-muscarinic receptor activation, modulating GIRK channel activity and regulating heart rhythm [91]. Regulation of cardiac and neuronal signalling in mouse and zebrafish by Gβ5 has also been demonstrated [116, 124, 125].

3.3. Evidence for specific Gαβγ heterotrimer combinations and their interaction with preferred GPCRs

Preferential heterotrimer-GPCR interactions by specific Gβγ subtypes have been reported [126]. In this section, different experimental approaches and systems designed to investigate such interactions and corresponding evidence for specific Gβγ subtypes-GPCR interactions are described. Combinations of in vitro and in vivo biochemical assay systems including live cell imaging, electrophysiological measurements of ion currents in cultured cells, studies with purified Gβγ subtypes reconstituted with GPCRs/effectors, co-immunoprecipitation coupled with proteomic analysis have been used to probe heterotrimer-GPCR interactions and associated Gβγ subtypes specificity. In order to regulate expression of Gβγ subtypes in endogenous systems, techniques such as ribozyme suppression, injection of Gβγ subtype specific DNA into cells, use of Gβγ subtype-specific antisense suppression by injecting antisense oligomers into cells, and siRNA screening approaches have been used in conjunction with the earlier described experimental platforms. Thus, evidence from these experiments details the interaction of Gβγ subunits with receptor and the influence of Gβ subtypes in defining specific GPCR-heterotrimer preferences.

Combined in vivo and in vitro studies have been conducted in cultured cells to investigate specific GPCR-heterotrimer interactions. Specific Gβγ combinations have preferred interactions with α₂AR to regulate signalling in in vitro systems such as cultured cells and in synapses in the central nervous system [115, 127, 128]. For instance, synaptosomes from transgenic mouse brain tissue have been isolated, followed by co-immunoprecipitation (co-IP) studies and proteomic analysis have demonstrated Gβ2γ2, Gβ2γ3, Gβ2γ4, and Gβ4γ12 are preferred heterotrimer partners for α₂AR[115]. Plasma membrane preparations of Sf9 cells reconstituted with purified G protein subunits were used to examine the role of Gβγ dimers in signalling specificity at receptor level. When radioligand binding assays using the agonist [³H]-UK14304 was performed, the α₂AR preferred heterodimers containing Gβ3 either with Gγ4, Gγ10, and Gγ11 and subsequent heterotrimer formation with Gαi [127]. This evidence suggests that specific G protein combinations in mice are involved in neuronal signalling and possibly implicated in neurological diseases.

Live cell imaging assays in cultured cells using transiently expressing fluorescently-tagged G protein subunits has been used to examine the selectivity of α₂AR to activate Gαi and Gβ in HeLa cells [130]. Using FRET assays, proximity of proteins within 100 nm is regarded as a sensitive assay for measuring protein-protein interactions. FRET was measured between YFP-tagged Gαi and CFP-tagged Gβ upon the activation of α₂AR [130]. Gαi1, Gαi2, and Gαi3 showed specific interactions with Gβ1, Gβ2, and Gβ4 upon activation of endogenous as well as overexpressed α₂ARs in HeLa cells. Another live cell imaging assay was designed to probe β₂-adrenergic receptor (β₂AR) coupling using CFP-tagged Gαs and split-YFP tagged functional Gβ1γ7 dimer. Here the YFP-Gβ1γ7 dimer was designed using N-terminal YFP attached to Gβ1 (YFP-N-β1) and C-terminal YFP attached to Gγ7 (YFP-C-γ1), and YFP fluorescence was only observed when both Gβ1 and Gγ7 are co-expressed. Internalization of Gαsβ1γ7 upon β₂AR stimulation was observed, indicating specific trafficking patterns for heterotrimers coupled to the β₂AR [131].

Another approach to understand G protein subunit specificity with GPCRs is the use of antisense oligonucleotide or siRNA suppression system in cultured cells. Gβγ dimers containing Gβ4 and Gγ1 in M3-muscarinic receptor-mediated signalling has been observed in native HEK 293 cells [129]. Using a bioluminescence-based calcium assay combined with siRNA screening to identify potential Gβγ combinations, regulation of intracellular calcium and MAPK signalling by the M3-muscarinic receptor was examined [129]. Knocking down Gβ1 and Gβ4, as well as specific Gγ subunits using their respective siRNAs showed that the Gβ4γ4 combination was a key signalling driver, while knocking down Gβ1 disrupted non-canonical signalling events possibly suggesting a role in regulating gene expression. Antisense oligonucleotide injection into rat pituitary-derived GH3 cells to suppress expression of certain Gβ subtypes was used to assess selective Gβγ coupling with GPCRs in physiological conditions. M4-muscarinic receptors and somatostatin receptors required heterotrimers with specific Gβγ subunits to mediate inhibition of L-type Ca²⁺ channels [132]. Silencing Gβ (with Gβ-specific antisense oligonucleotides) and Gγ (with Gγ-specific antisense oligonucleotides) reduced somatostatin receptor activation-induced Ca²⁺ currents, suggesting a role for Gβγ subtype selectivity in somatostatin and M4 receptor signalling [25, 133]. Using electrophysiological measurements and antibody labeling in GH3 cells, the M4-muscarinic receptor interacted specifically with the heterotrimer Gαo1β3γ4, while somatostatin receptor preferred Gαo2β1γ3, inhibiting receptor-induced calcium currents. Ribozyme suppression has also been considered a useful method to identify physiologically relevant specific G protein heterotrimer-GPCR interactions. Specifically designed G protein subtype targeted ribozymes were used to selectively suppress the G protein expression. When tested in HEK 293 cells, this approach showed strong interactions between Gβ1 and Gγ7 suggesting Gβγ specificity for regulation of βAR and Gαs-mediated adenylyl cyclase signalling [134].

Studies with purified G protein subunits and GPCRs have also been reported. For example, baculoviral expression systems have been used in cells to express and purify G proteins with different radioisotope labels. A recent study conducted in Sf9 and HEK 293 cells showed subtype-specific Gβγ interactions with adenosine 1 and 2A receptors [135]. Here, Gβγ subunits in cells were enriched by stable isotope labeling using ¹³C₆-Arg and ¹³C₆-Lys and purified using receptor-Gα fusion proteins. Protein separations were performed by SDS-PAGE and HPLC, while tandem mass spectrometry was used to identify specific Gβγ dimers. Both fusion proteins, adenosine 1 receptors with Gαi and adenosine 2A receptors with Gαs, showed specific interactions with Gβ4 and Gγ5. Additionally, Gβ4 showed a higher affinity toward Gαs-fused adenosine 2A receptor over the adenosine 1-Gαi fusion [135]. In another study, Gβ was demonstrated as a determining factor for Gαs interaction in adenosine 2A receptor and β₁AR-induced signalling [136]. There, the ability of adenosine 2A receptors and the β₁AR to modulate adenylyl cyclase activity by measuring cAMP concentrations as well as G protein activation using GTP-γS assays in the presence of different combinations of purified Gβγ were demonstrated. Sf9 cells were used in combination with baculoviral expression to purify Gβγ and assays were performed using purified Gβγ reconstituted with Sf9 cell membranes expressing adenosine 2A receptors, β₁AR and adenylyl cyclases; AC1 and AC2. Combinations of Gγ2 heterodimers with Gβ1–5 were tested to measure coupling efficiencies of Gαs with both β₁AR, and adenosine 2A receptor, while AC2 stimulation and AC1 inhibition by G protein subtypes were also tested. Both receptors showed stronger interactions with the Gβ4γ2 dimer, while the Gβ5γ2 dimer showed attenuated interactions and signalling. Additionally, Gβ1γ2 showed a higher affinity towards for the β₁AR [136]. In a recent study, in the presence of neurotensin 1 receptor, a higher than usual number of Gβγ combinations forming functional heterotrimers with Gαi and GαsL was shown [70]. Here, baculoviral expression of all three G protein subunits in a single vector compared to individual baculoviral vectors expressing only one G protein. Sf9 cells were used to express G proteins and GPCRs while co-immunoprecipitation was used to purify HA-tagged Gγ with its interacting G protein subunits and GPCRs. Except for Gβ5, nearly 120 Gαβγ combinations comprising Gβ1–4 and Gγ1–5, Gγ7–13 with either Gαi or GαsL were found to form functional heterotrimers with neurotensin 1 receptor. Additionally, with the exception of Gβ3, specific heterotrimer formation of Gαi with Gβ1, 2, 4 and Gγ1, 11 was noted [70].

Using baculoviral expression-aided purification of Gβγ from Sf9 and CHO-K1 cells, interactions between 5-HT1A, A1-adenosine, α₂-adrenergic, and μ-opioid receptors and distinct Gβγ combinations was demonstrated [137]. In this study, Gβ1γ11 heterodimers showed a unique interaction with 5-HT1A and A1-adenosine receptors, while Gβ1γ7 heterodimer showed robust interactions with α₂-adrenergic and μ-opioid receptors when co-expressed in Gαi in cellular systems [137].

Using co-immunoprecipitation of Gβγ subtypes, specific interactions with Gαq and Gβγ to regulate GqGTP/PLCβ1- or PLCβ2-mediated inositol triphosphate production was demonstrated in HEK293T cells [138]. Except for Gβ5γ2 and Gβ5γ13, Gβ1–4 dimers with γ2 and γ13 showed specific interactions with Gαq, regulating Gαq-induced PLCβ1 activation [138].

Additionally, preferred heterotrimer combinations were reported for rod and cone photoreceptors during phototransduction [88]. Using immunocytochemical labeling in retina sections isolated from dark-adapted monkeys, Gβ3, Gγ3, and Gγ2 were shown to interact with all three blue, green, and red cone photoreceptors. Additionally, Gβ3 showed rod bipolar cell expression. Enhanced localization of Gβ1, Gγ1, and Gγ3 signalling was observed primarily in rod photoreceptors implicating specific heterotrimer interactions for vision signalling [88].

Similarly, in olfactory receptors, Gα(olf) was shown to interact with Gβ1 and Gγ13 while in taste receptors, Gα(gustducin) specifically interacted with Gβ3, Gβ1, and Gγ13 [139, 140]. In those studies, olfactory receptor interactions and signalling with these specific Gαβγ combinations were tested using yeast-two-hybrid screening and cAMP measurements in HEK 293T cells [139]. Taste receptor activity in the presence of specific Gαβγ combinations was characterized using murine taste tissues by measuring IP3 levels induced by PLCβ2 [140]. Additionally, the specific heterotrimer combination Gα (olf), Gβ2 and Gγ7 with adenosine-A2A receptors was shown using Gγ7 knockout mice [141].

Although there are numerous investigations on specific GPCR-heterotrimer interactions and associated coupling, thorough analysis should be conducted to assess in vivo translatability of such experimental approaches since in celllulo and in vitro data do not always mimic physiological conditions found in vivo. Therefore, more physiologically relevant experimental approaches should be encouraged to draw conclusions regarding specific heterotrimer-GPCR interactions. Table 8 and Fig. 14 summarize what we know about Gαβγ combinations, their interacting GPCRs, and associated physiological functions.

Table 8.

Gα & Gβγ combinations, interacting GPCRs, and functions

Gα & Gβγ combination	Interacting GPCR	Function
Gαi1 with Gβ3Gγ4, Gβ3Gγ10, Gβ3Gγ11	α2-adrenergic	Anesthetic sparing, and working memory enhancement, nervous system [127]
Gαi, Gβ4, and Gγ5	Adenosine A1	Cardiac and neonatal physiology [135]
Gαi with Gβ1Gγ1, Gβ1Gγ11, Gβ2Gγ1, Gβ2Gγ11, Gβ4Gγ1, Gβ4Gγ11	Neurotensin 1	Hypotension, hyperglycemia, antinociception, hypothermia [70]
Gαi with Gβ1Gγ7	μ-opioid	Pain and addiction [137]
Gαi with Gβ2Gγ2, Gβ2Gγ3, Gβ2Gγ4	Auto-α2-adrenergic	Anesthetic sparing, and working memory enhancement, nervous system [115]
Gαi with Gβ4Gγ12	Hetero-non-adrenergic	Anesthetic sparing, and working memory enhancement, nervous system [115]
Gαi with Gγ11	5-HT1A	Neurotransmission [137]
Gαi/o with Gγ11	Adenosine A1	Cardiac and neonatal physiology [137]
Gαo1 with Gβ3, and Gγ4	M4-muscarinic	Inhibit voltage-sensitive Ca2+ channels [132]
Gαo2 with Gβ1, and Gγ3	Somatostatin	Inhibit voltage-sensitive Ca2+ channels [132]
Gαt with Gβ3Gγ3 & Gβ3Gγ2	Cone photoreceptors	Vision [88]
Gαt with Gβ1Gγ1 &Gβ1Gγ3	Rod photoreceptors	Vision [88]
Gα(olf) with Gβ1Gγ13	Olfactory receptors	Olfaction [139]
Gα(olf) with Gβ2Gγ7	Adenosine A2A	Cardiac physiology, Parkinson’s disease [141]
Gα(gustducin) with Gβ3Gγ13 and Gβ1Gγ13	Taste receptors	Taste [140]
Gαs with Gβ1, and Gγ7	β-adrenergic	Cardiac physiology, heart rate [134]
Gαs with Gβ1Gγ2	β1-adrenergic	cAMP signalling [136]
Gαs with Gβ4, and Gγ5	Adenosine A2A	Cardiac physiology, Parkinson’s disease [135]
Gαq with Gβ1, Gβ2, Gβ3, Gβ4 and Gγ2, Gγ13	N-type Ca2+ channels and endogenous GPCRs	Olfaction [138]
Gαq with Gβ4γ1	M3-muscarinic	Calcium signalling, nervous system, muscle contraction [129]

Figure 14: Gα and Gβγ combinations, their interacting GPCRs, and associated physiological functions.

A summary of known combinations of G protein heterotrimers linked to particular GPCRs in different cells and tissues.

The major G protein subunits in individual tissues were identified and categorized according to the human protein atlas database (RNA expression data available from http://www.proteinatlas.org) (Table 9). The expression levels of specific G proteins may contribute to the overall signalling specificities in these tissues. Some tissues show similar expression levels of G protein subunits, and thus the possible preferred heterotrimer formation and their contribution to signalling specificities need examination. Overall, the specificity of heterotrimeric G-proteins towards certain GPCRs and particular signalling events and cell and tissue-specific localization are likely to to be important in physiological and pathophysiological settings.

Table 9.

RNA expression level-based G protein subtype expression in different tissues

Tissue	Gα	Gβ	Gγ	Tissue	Gα	Gβ	Gγ
adipose tissue	αi2	β1	γ5	ovary	αi2	β2	γ11
amygdala	αs	β1	γ2	pancreas	αs	β2	γ5
appendix	αi2	β1	γ5	pituitary gland	αs	β1	γ10
basal ganglia	αL	β1	γ7	placenta	αi2	β2	γ11
breast	αs	β2	γ11	pons and medulla	αs	β1	γ3
cerebellum	αs	β1	γ13	prostate	αi2	β1	γ5
cerebral cortex	αs	β1	γ3	retina	αt1	β3	γt1
cervix, uterine	αi2	β2	γ5	salivary gland	αs	β2	γ5
colon	α11	β1	γ5	seminal vesicle	αi2	β2	γ5
corpus callosum	αi2	β1	γ7	skeletal muscle	αs	β2	γ5
ductus deferens	αi2	β2	γ5	small intestine	α11	β1	γ12
endometrium	αi2	β1	γ12	smooth muscle	αs	β1	γ12
epididymis	αi2	β2	γ5	spinal cord	αs	β1	γ10
esophagus	αi2	β2	γ5	spleen	αi2	β2	γ5
gallbladder	αi2	β2	γ5	testis	αi2	β2	γ5
heart muscle	αs	β2	γ5	thalamus	αi2	β1	γ10
hippocampus formation	αs	β1	γ3	thymus	αi2	β1	γ5
kidney	αs	β1	γ5	thyroid gland	αs	β2	γ5
liver	αi2	β2	γ5	tongue	αs	β2	γ5
lung	αi2	β2	γ5	tonsil	αi2	β1	γ5
lymph node	αi2	β2	γ5	urinary bladder	αi2	β1	γ5
midbrain	αi2	β1	γ5	vagina	αi2	β1	γ5
olfactory region	αs	β1	γ2

4. Gγ subtype-specific regulation of GPCR and G protein signalling

4.1. Isoprenylation of Gγ subunits, PM composition, and Gβγ-PM interactions.

The plasma membrane (PM) is a semipermeable barrier that surrounds the cell interior, including the cytoplasm of the cell. The PM is composed of a lipid bilayer with embedded proteins, polysaccharides and lipids/lipoproteins. The lipid component is mainly composed of phospholipids, which are amphipathic molecules with a polar phosphate head group and non-polar two acyl lipid anchors. They arrange themselves in a bilayer in which lipid anchors are oriented towards the interior while polar head groups face outside. The PM is rich with five major types of phospholipids-phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin, where phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine are abundant in the inner leaflet of the PM [25, 142, 143].

Due to the net negative charge of the polar head groups of phospholipids, which are predominant in the inner leaflet (i.e., phosphatidylinositol, phosphatidylserine), the inner layer of the membrane has a net negative charge. With this composition (being hydrophobic and negatively charged), the PM plays a role in protein anchoring (i.e., the cytoskeleton), where electrostatic and hydrophobic interactions play a significant role. Anchorage to the PM and the proper localization of G proteins are crucial for their function. Gβγ subunits are anchored to the PM via a prenyl group attached to the C terminus of the Gγ subunit in the Gβγ dimer. Gγ is post-translationally modified with an isoprenyl lipid anchor, and there are two types of prenyl groups; 15 carbon farnesyl and 20 carbon geranylgeranyl[144].

Post-translationally lipidated proteins contain a common amino acid sequence known as the CAAX motif at their carboxy termini [144]. The CAAX motif is composed of a conserved cysteine residue (C) followed by two aliphatic amino acids (aa) and any amino acid (X), which varies depending on the protein [145]. As seen in Gγ9, Gγ1, and Gγ11, where X can be Met, Ser, Gln or Ala, the Gγ subunit is expected to be farnesylated. When the X residue is Leu, as seen in the other nine Gγ subtypes, Gγ geranylgeranylation is expected [146]. Out of the 12 Gγ isoforms, only Gγ1, Gγ9, and Gγ11 are known to be farnesylated, while the rest are geranylgeranylated [147]. Initially, it was believed that the final residue of the CAAX motif, ‘X’, which is variable, determines the type of isopropyl transferase/prenylation involved in Gγ prenylation [148]. However, recent studies have suggested that the other residues of the Gγ C terminus also play a role in this determination [146]. Though farnesylation or geranylgeranylation of most CAAX motif-containing proteins anticipated to follow the prenylation rules described above, there are deviations from this traditional paradigm[144]. Therefore, more studies are required to elucidate how the type of prenylation of Gγ is determined.

However, the prenyl group is attached to the carboxy terminus of Gγ by forming a thioether bond with the Cys residue of the CAAX motif. In farnesylation, the farnesyl group is transferred to the Cys residue of the CAAX motif from farnesyl diphosphate (FPP) by the enzyme farnesyltransferase, and during geranylgeranylation, the geranylgeranyl group is transferred from geranylgeranyl diphosphate (GGPP) by geranylgeranyl transferase (GGT-1)[145]. After prenyl group attachment, the last three amino acid residues (-aaX) are removed/cleaved by the proteolytic enzyme Ras-converting CAAX endopeptidase (RCE1), followed by methyl group addition to the new C terminus (carboxymethylation) by carboxymethyl transferase (Fig. 15) [149]. The hydrophobic nature of the isoprenyl group mediates anchorage of Gβγ with this lipid modification to the PM, which is also hydrophobic due to the presence of fatty acyl chains (Fig. 15). These two prenyl groups (15 C farnesyl and 20 C geranylgeranyl) have different PM affinities, and the geranylgeranyl group has relatively higher membrane affinity than the farnesyl group[48] (Fig. 16).

Figure 15. Prenylation and post prenylation processing at the CAAX motif in Gγ.

Farnesyl or geranylgeranyl moieties are transferred to the carboxy terminus of Gγ from FPP and GGPP with the aid of either farnesyl or geranylgeranyl transferases, respectively. The last three amino acids are subsequently cleaved off from the CAAX motif by RCE1 and is followed by a methyl group addition to the new C terminus. GGPP: Geranylgeranyl pyrophosphate, FPP: Farnesyl pyrophosphate, PPi: Pyrophosphate.

Figure 16. Farnesylated and geranylgeranylated membrane-anchored Gγ.

While prenyl groups (farnesyl and geranylgeranyl group in Gγ9 and Gγ3, respectively) anchor Gγ to the PM, positively charged and hydrophobic residues in the pre-CAAX region further strengthen this anchorage by controlling electrostatic and hydrophobic interactions, respectively with the negatively charged and hydrophobic PM.

4.2. Subtype-specific Gγ prenylation and PM affinity of Gβγ

G protein attachment to the inner leaflet of the PM is critical for G protein-mediated signal transduction since it allows G proteins to interact with their cognate receptors, undergo activation, and transduce signals through activation of PM-localized effector molecules. The crystal structure of the G protein heterotrimer provides information about the spatial orientation of the separate subunits in the heterotrimeric G protein complex[15]. The amino terminus of Gα subunits in G proteins is palmitoylated and/or myristoylated while the carboxy terminus of Gγ in Gβγ dimer is isoprenylated either with a 15C farnesyl or a 20C geranylgeranyl lipid anchor as discussed above [12]. It is suggested that a more hydrophobic geranylgeranyl group is sufficient to stably anchor a protein to membranes [150]. In previous studies, a high affinity, heat- and protease-sensitive binding sites have been identified for prenylated peptides in microsomal membrane preparations[151]. This binding site may play a role in targeting prenylated proteins to a membrane compartment where C-terminal proteolysis and carboxyl methylation occur [151]. Also, this carboxyl methylation neutralizes the negatively charged C terminus. It has been shown to increase the affinity of farnesylated peptides for lipid vesicles by ~10-fold and is considered a contributor to Gβγ membrane association [152].

Further, Gα lipid modifications have been shown to provide additional support required for PM targeting of Gβγ subunits [153]. The need of Gα for PM localization of Gβγ was demonstrated by overexpressing different combinations of Gβ and Gγ, without Gα, which resulted in limited localization of the Gβγ at the PM with the majority of Gβγ accumulating in intracellular structures. In contrast, co-expression of Gα resulted in strong PM localization of Gβγ [154]. This may also suggest that heterotrimer formation is a critical step on the correct folding or maturation of functional G proteins, especially those destined for the PM.

According to the crystal structure of Gβγ (PDB ID: 1TBG), Gγ interacts with the barrel surface of the Gβ propeller, however, without forming covalent interactions. The carboxyl terminus of the Gγ lies within ~18 Å of the amino terminus of the Gα. It is also closer to the carboxyl terminus of Gα. In the assembled Gβγ dimer, the amino terminus of the Gα and the prenylated carboxyl terminus of the Gγ lie parallel. The CAAX motif Cys in Gγ and amino terminus Gly and/or amino terminus Cys of Gα are the two domains in the heterotrimer that contain posttranslational lipid modifications, suggesting that these subunits play a major role in anchoring heterotrimers to the membrane[150]. The amino terminus of the Gα and the carboxyl terminus of the Gγ subunit lie near the inner surface of the PM while it interacts with the PM at the prenylated and carboxymethylated Cys[155].

Upon GPCR activation, heterotrimeric G proteins undergo activation and one fate is that they completely dissociate (there is some evidence that dissociation can also be partial, especially when they regulate the same effector protein) into active GαGTP and free Gβγ. Although Gβγ was initially considered as a PM-bound protein, recent work has shown that free Gβγ can detach from the PM and reversibly travel through the cytosol between the PM to internal membranes (IMs) until an equilibrium is reached, termed Gβγ translocation [48] (Fig. 17). Numerous studies have also shown that Gβγ signalling can occur in the nucleus and other organelles but it remains unclear how they are translocated to these compartments (reviewed in [156, 157]). The differences between the properties of the PM and IMs (i.e., membrane composition) and the higher surface area of IMs compared to the PM support the forward translocation from the PM to IMs as long as GPCRs are active [48].

Figure 17. GPCR-G protein activation and subsequent Gβγ translocation.

G protein α_GDP and βγ subunits are associated at the PM as a heterotrimer and when a ligand activates the GPCR, Gα_GTP and Gβγ can dissociate from each other due to conformational changes in the Gα subunit. Gβγ then translocates from the PM to IMs in a reversible manner to regulate downstream signalling activities.

It has been found that Gβγ translocation is dependent on the particular Gγ subunit in the Gβγ dimer [48]-[11]. The translocation rates of different Gγ isoforms were measured using the time to reach the half-maximum translocation (Tt_1/2). All 12 Gγ subunits showed distinct translocation rates and extents upon GPCR activation, where Gγ9 showed the fastest (Tt_1/2 < 10 s) and Gγ3 showed the slowest Gβγ translocation. Gγ9, which has a farnesyl anchor and other Gγ subtypes, predicted to have farnesyl lipid modification (i.e., Gγ1 and Gγ11) showed faster translocation compared to the Gγ subunits with geranylgeranyl anchors [48]. Also, the rapidly translocating Gγ subunits showed a greater extent of translocation compared to slowly translocating ones [48]. This suggests that the PM affinity of Gβγ is Gγ subtype-dependent as Gγ provides the only PM anchor for Gβγ dimer. However, even though there are only two types of prenyl groups acting as membrane anchors for Gβγ subunits, the presence of 12 distinct translocation rates and differing degrees of translocation suggests that additional regulatory mechanisms also govern the PM affinity of Gβγ.

As shown in Table 2, a broader range of sequence homologies is exhibited by the Gγ isoforms, approximately ranging from 20 to 80% [110]. Sequence alignment of Gγ subunits shows that except for Gγ13, all other 11 Gγ types possess a conserved Phe residue (Phe59) at their C termini (Fig. 8). Based on the conserved domain analysis function at NCBI, the 5 to 6 residues between this conserved Phe, which demarcates the last contact point with Gβ and the CAAX motif (pre-CAAX region), also appear to interact with the PM, providing additional support to the prenyl anchor [158, 159].

A recent study with Gγ mutants comprised of high PM affinity Gγ with a substituted C terminus from a low PM affinity Gγ (i.e., Gγ3 with pre-CAAX + CAAX of Gγ9 (Gγ3-γ9CT) and vice-versa has shown that the mutants display translocation properties of the Gγ in which the introduced C termini are extracted [48]. However, when only the CAAX motifs were switched, the translocation properties did not entirely resemble that of the parental Gγ subtype, although a significant change was observed [48]. Also, a complete loss of PM localization was observed in Gγ9 upon Cys removal from the CAAX motif, and complete elimination of translocation was observed with incorporation of an additional Cys to Gγ3 CAAX motif.

Overall, these observations imply that the Gγ prenylation is essential for Gβγ anchorage to the PM, and that the pre-CAAX region fine-tunes Gβγ-PM interactions by controlling electrostatic and hydrophobic interactions, respectively [48]. This notion has been further supported by examining the amino acid composition of pre-CAAX regions of different Gγ types. As shown in Table 10, pre-CAAX regions of slowly translocating Gγ3 and Gγ2 (with high PM affinity) are composed of ∼80% positively charged and hydrophobic residues, which can form strong interactions with the negatively charged and hydrophobic PM. In comparison, only ∼50% hydrophobic residues are present in farnesylated Gγ subunits with low PM affinity (rapidly translocating) such as Gγ9 and Gγ1 [48].

Table 10:

Pre-CAAX regions of all 12 Gγ subunits, their translocation Tt_½ times and PM affinities [48].

Gγ	Pre-CAAX	Tt½ (s)	PM affinity
Gγ9	NPFKE-KGGC-far	5 ± 1	Low affinity
Gγ1	NPFKELKGGC-far	13 ± 2
Gγ11	NPFKE-KGSC-far	38 ± 2
Gγ7	NPFKDKKP-C-gerger	41 ± 2
Gγ5	NPFRPQKV-C-gerger	71 ± 3	Moderate affinity
Gγ12	NPFKDKKT-C-gerger	80 ± 2
Gγ10	NPFREPRS-C-gerger	97 ± 4
Gγ13	NPWVE-KGKC-gerger	100 ± 3
Gγ4	NPFREKKFFC-gerger	116 ± 2
Gγ8	NPFRDKRLFC-gerger	124 ± 1
Gγ2	NPFREKKFFC-gerger	181 ± 4	High affinity
Gγ3	NPFREKKFFC-gerger	270 ± 4

4.3. Gγ subtype-dependent receptor selectivity of G protein heterotrimers

Since the general classification of GPCRs is based on their coupling to different Gα subtypes, selective coupling of Gα subunits to specific GPCRs has been the primary point of receptor definition [160, 161]. Nevertheless, emerging evidence from experiments conducted both in vivo and in vitro reports the interactions of Gγ subunits with the receptor and their influence in defining selectivity of heterotrimers for specific GPCRs [6]. Moreover, the Gβγ dimer collectively plays a vital role in facilitating Gα binding to receptors [162]. Therefore, in addition to Gα, Gβγ is also likely to be required for heterotrimer-GPCR interactions and subsequent signalling [163].

One study conducted in superior cervical ganglion (SCG) neurons using C-terminal prenylated peptides specific for different Gγ subtypes indicated the attenuation of signalling by M2- and M4-muscarinic receptors in the presence of Gγ5 peptide, while no significant effect was reported with Gγ7 or Gγ12 peptides [164]. This implied more favorable binding of Gγ5 heterotrimers to M2/M4-muscarinic receptors. The reported differential selectivity of Gγ5 and Gγ7 to interact with M2-muscarinic receptors led to another study to test ability to activate different G protein combinations of Gαo and Gαi with Gγ5 and Gγ7 [165]. This was achieved by examining the influence of different subunit compositions of heterotrimer on M2-muscarinic receptor interaction by using possible combinations of Gα and Gγ subtypes; αoβ1γ5, αoβ1γ7, αi2β1γ5, and αi2β1γ7. A significant difference between the receptor’s ability to activate αoβ1γ5 and αoβ1γ7 was observed, indicating a prominent interaction of αoβ1γ5 heterotrimer with the receptor. Further, selective interaction of different Gγ subtypes with the receptor was found to be due to the differential coupling of the specific Gγ subtypes to the M2 receptor, regardless of the type of Gβ isomer present in the Gβγ dimer [165]. A separate study conducted in HEK 293 cells also showed perturbation of β-adrenergic receptor signalling upon ribozyme-mediated suppression of Gγ7 subtype expression, indicating the selectivity of the β-adrenergic receptor for Gγ7 containing heterotrimers [166].

The C-terminus of Gγ has been shown to be predominantly involved in the Gβγ dimer-receptor interaction [167]. In addition to the highest amino acid sequence variability at the C-termini of Gγ subunits, the C-terminal CAAX sequence also determines the type of prenylation in a specific Gγ subtype. Therefore, the C-terminal sequence, together with the type of prenylation, can be important determinants of receptor selectivity for differential Gγ coupling [168, 169]. In an early study on identifying the effect of Gγ prenylation on Gβγ-GPCR interactions, specifically Gγ1 and Gγ2, which are post-translationally modified respectively with farnesyl and geranylgeranyl prenyl anchors [147]. In this study, the formation of the high-affinity agonist binding conformations of the A1 adenosine receptor and following exchange of GDP to GTP on the Gα subunit was used to measure the effectiveness of different Gβγ dimers on receptor interactions. In both assays, farnesylated Gβ1γ1 dimers were significantly less effective compared to geranylgeranylated Gβ1γ2 dimers. In order to ascertain the observed prenylation type-dependent affinity of Gβγ subtypes for A1 adenosine receptor, mutated versions of Gβγ dimers; Gγ1 with geranylgeranyl anchor (Gβ1γ1-S74L) and Gγ2 with a farnesyl anchor (Gβ1γ2-L71S) were used. Compared to WT Gβ1γ2, the Gβ1γ2 mutant was less effective while the Gβ1γ1 mutant was more effective in transducing receptor activity, indicating that the type of prenylation of Gγ subunits is an important determinant of its interaction with the receptor [147]. Consistent with these observations, a 10-fold difference between Gγ1 and Gγ2 subtypes in their affinities for bovine rhodopsin was reported [170, 171]. Investigation of the contribution of the primary structure and isoprenoid modification of Gγ on interaction with the receptor showed a differential affinity of farnesylated Gβ1γ1 and geranylgeranylated Gβ1γ2 for rhodopsin [172]. Also, the mutated version of Gγ2 with farnesylated lipid anchor exhibited a 2-fold decrease in affinity for the receptor compared to Gγ2 WT, while the geranylgeranylated Gγ1 exhibited an increased affinity for receptor compared to Gγ1 WT. Overall, the study suggested that C-terminal isoprenoid modification on Gγ subtypes contributes to their differential receptor selectivity.

4.4. Gγ subtype-dependent control of Gβγ-effector signalling

Upon activation of GPCRs in response to extracellular stimuli, signal transduction is initiated by activating Gα(GDP)βγ heterotrimers, converting them to Gα(GTP) and free Gβγ [173]. Activated Gα(GTP) and free Gβγ can independently interact with effectors, and in some cases the same effectors [153]. Supporting their interaction with Gβγ, multiple Gβγ-binding sites have been identified on numerous effectors including phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3Kγ), adenylyl cyclase (AC) isoforms, GIRK channels, PLC isoforms (PLCβ2, β3), GPCR kinases (GRKs), etc. [174–177]. By interacting with these effectors, Gβγ subunits modulate a broad array of cellular and physiological functions, including gene transcription, cellular secretion, contractility, and cell migration [25, 153]. Based on the subcellular localization of the effector, there are two general mechanisms of Gβγ-dependent effector regulation [153]. The first describes the recruitment of cytosolically distributed effector proteins to the PM to interact with their substrates located at the PM by binding to PM-anchored Gβγ. The second mechanism involves the direct interaction of activated Gβγ with PM-localized effectors [17, 153]. Although there are realistically over 50 possible combinations of different Gβγ dimers available [70], Gβγ-mediated effector activation has primarily been considered a single signalling event, considering Gβγ as a monolithic signalling actuator. The individual subtype-specific contributions of Gβ and Gγ subunits in Gβγ-effector interactions are just emerging.

Since the majority of the Gβγ effectors are near PM localized, interactions with effectors primarily occur at the PM, and therefore varying PM affinities of different Gβγ combinations should or likely contribute to differential Gβγ-effector interactions and signalling activation [178]. As previously mentioned, the conventional model of GPCR signalling describes Gα-mediated pathways as the central mediators of GPCR-G protein signal transduction [179–181]. However, the very first discovery of Gβγ mediated activation of acetylcholine-regulated GIRK channel in atrial myocytes, which subsequently govern muscarinic receptor-gated potassium channel activity [182], focused attention on the ability of Gβγ to directly activate downstream effectors. This led to an understanding that Gβγ activated several other effectors responsible for facilitating diverse signalling and cellular processes and cell physiology. The activation of the endogenous GIRK channel was observed in HL-1 cells upon activating endogenous Gi-coupled M2-muscarinic receptors [183]. In a study which investigated differential Gβγ translocation rates of regulated 12 Gγ isoforms, M2-muscarinic receptor activation of GIRK channels was reported to be dependent on the translocation rates of the accompanying Gγ type in Gβγ [184]. Here, the effect of slow translocating (high-PM-affinity) Gγ3 and fast translocating (low-PM-affinity) Gγ9 on the properties of muscarinic receptor-induced GIRK channel activation was measured in HL-1 cardiomyocytes. Interestingly, a higher amplitude of GIRK current was measured in Gγ3 expressing cells while control cells utilizing endogenous Gγ subunits of HL-1 cells and Gγ9 expressing cells reported a relatively lower GIRK current amplitude. This study suggested the dependence of translocation rates and relative abundance of Gγ isoforms on GIRK channel activation.

Among different isoforms of PI3K, PI3Kγ activity is directly induced by Gβγ [176, 185], and it has been clearly shown that Gβγ plays a crucial role in PI3Kγ-mediated migration of neutrophils in response to chemoattractants [186]. PI3Kγ-mediated PIP3 generation at the PM is a major regulator of the migratory activity of cells such as macrophages [48]. The accumulation of PIP3 at the leading edge of a migratory cell is essential for polarization of the cell, which in turn directs itself towards chemoattractants [187, 188]. Additionally, Gα_i/o-coupled GPCR, blue opsin activation also has exhibited the accumulation of PIP3 at the leading edge of mouse macrophage cell line RAW 264.7, as a result of the generation of free Gβγ [189].

To catalyze PIP3 production by interacting with Gβγ, the catalytic subunit (p110) of PI3Kγ must be translocated to PM upon its activation [185, 190]. A recent study clearly demonstrated the inherent ability of GPCR-mediated PIP3 production in RAW 264.7 cells, which show predominant endogenous expression of high-PM-affinity Gγ3 and Gγ4 subunits [48]. However, HeLa cells, which show a significantly less abundant expression of these Gγ types, did not show PIP3 generation upon GPCR activation. Interestingly, the same study also showed basal levels of PIP3 generation, even without GPCR activation and robust production of PIP3 upon GPCR activation when Gγ3 was overexpressed in HeLa cells [48]. However, overexpression of low PM affinity Gγ9 in HeLa cells did not influence the ability of these cells to produce PIP3 upon GPCR activation.

Cytosolic Ca²⁺ plays a crucial role in inducing trailing edge retraction in migratory cells, and it is demonstrated that release of Ca²⁺ from intracellular stores is a Gβγ-mediated process during Gi-coupled GPCR-induced RAW 264.7 cell migration [191]. Accordingly, the Gγ subtype dependency of Ca²⁺ mobilization in RAW 264.7 cells upon endogenous Gi-coupled complement component 5a (C5a) receptor activation was examined [48]. This study showed a higher level of Ca²⁺ mobilization in RAW 264.7 cells utilizing endogenous Gβγ and overexpressed high PM affinity Gγ3. This data further confirmed the native expression of high PM affinity Gγ isoforms in these cells, consequently facilitating their characteristic cellular and physiological activities. Moreover, exogenous introduction of low PM affinity Gγ9 could not induce a significant level of Ca²⁺ mobilization. Interestingly, only a weak Ca²⁺ response was observed in cells expressing moderate PM affinity Gγ subunits such as Gγ4 and 12. Exhibiting the critical role of CT and CAAX motif sequence of Gγ subtypes in determining PM affinity and consequent effector interaction, the same study also showed loss of Ca²⁺ mobilization when mutant Gγ3 subunits wherein either CT or CAAX motif of Gγ3 WT was replaced by the respective Gγ9 sequence were introduced to RAW 264.7 cells [48].

Binding of cytosolic G protein-coupled receptor kinases (GRKs) to activated GPCRs at the PM and subsequent phosphorylation of receptors is a regulatory mechanism to prevent excessive GPCR signalling [192, 193]. Following GRK mediated phosphorylation, β-arrestin binds to GPCRs and facilitates clathrin-mediated receptor endocytosis [194]. Among the seven isoforms of GRKs (GRK1–7), GRK 2, 3, 5, and 6 are ubiquitously expressed. It was shown that Gαq, Gβγ, and phosphatidylinositol 4,5-bisphosphate (PIP2) play significant roles in specifically regulating GRK2 and GRK3 recruitment to active GPCRs [192, 195]. By employing a live cell confocal imaging approach, a recent study was able to identify the Gγ subtype specificity in regulating GRK2 recruitment [196]. A 3-fold higher GRK2 recruitment to activated M3-muscarinic receptor in low PM affinity Gγ9 expressing HeLa cells compared to high PM affinity Gγ3 expressing cells was observed. Considering the contrasting translocation kinetics and PM affinities of these two different Gγ subtypes, the observed differential magnitudes of GRK2 recruitment was explained. Here, it was suggested that heterotrimer dissociation upon GPCR activation facilitates exposure of Gαq-GTP and activated M3-muscarinic receptor for GRK2 interaction, allowing Gβγ to spatially re-orient and sandwich GRK2 between M3 receptor and Gβγ. Low PM affinity Gγ9 favors complete heterotrimer dissociation allowing a more permissive spatial orientation for GRK2 to interact in between the receptor and Gβγ. In contrast, the low mobility of high PM affinity Gγ3 may not sufficiently expose activated Gαq-GTP or GPCR for GRK2 to interact with, suggesting that differential PM affinities of Gγ subtypes play a crucial role in regulating GRK2 recruitment to activated GPCRs.

4.5. Gγ subtype-dependent regulation of cellular physiology

Golgi vesiculation

Even though the initial identification of GPCR-mediated G protein signalling is restricted to the PM, mounting evidence shows that the signalling ability of G proteins is also extended to subcellular locations such as the endoplasmic reticulum, the nucleus, and Golgi as well [11, 197, 198]. In addition to the role of G protein resident in IMs such as Golgi complexes [199, 200], it is also suggested that the GPCR-driven Gβγ dimer translocation from the PM to IMs [201–203] plays a specific role in signalling that occurs in IMs [204]. GPCR activation can stimulate Golgi fragmentation via a protein kinase D (PKD)- and PLCβ-driven pathway [204]. Considering the major role of PKD in Golgi vesiculation and their trafficking from trans-Golgi network to the PM [205, 206], it was later suggested that this is driven by GPCR-mediated Gβγ translocation from the PM to the Golgi complex [204]. Since different Gγ isoforms exhibit varying rates and extents of Gβγ translocation towards IMs based on their affinity for the PM [48, 109, 201, 203], this implies that Gβγ translocation-mediated Golgi vesiculation may be Gγ subtype dependent [204]. In the work mentioned above, M3-muscarinic receptor activation-induced Golgi fragmentation was demonstrated primarily in the presence of Gγ11, one of the rapidly translocating Gγ subtypes. In contrast, high-PM-affinity Gγ3 expressing cells failed to induce Golgi fragmentation upon M3R activation [204]. Consistent with this data, even in the absence of Gγ11 overexpression, M3-muscarinic receptor activation-induced Golgi vesiculation was observed in a cell line derived from lung tissue; A549 (human alveolar epithelial cells) [204], which endogenously expresses Gγ11 as a primary Gγ subtype [160]. The same cell line did not show Golgi fragmentation when slow translocating Gγ3 or Gγ4 were overexpressed [204]. This work established a paradigm where low-PM-affinity Gγ subtypes provide access for Gβγ to internal organelles and regulate effectors and signalling, following external receptor stimuli. This may have far ranging implications for Gβγ signalling inside the cell.

Cellular senescence and autophagy

Cellular senescence is the irreversible cell cycle arrest of proliferating cells. During cellular senescence, upregulation of endogenous Gγ11 expression has been observed compared to other Gγ subtypes [207, 208]. Additionally, cellular senescence can be induced by overexpression of Gγ11 in normal human fibroblasts [208]. As one of the rapidly translocating Gγ subtypes from the PM to IMs upon GPCR-mediated G protein activation [201, 202] given its role in modulating Golgi structure [204], Gγ11 was also observed in cells undergoing senescence after being translocated to IMs [207]. Senescent cells secrete numerous factors such as cytokines, growth factors, metalloproteinases, and extracellular matrix proteins in increased amounts [209], and the Golgi complex facilitates these events. It was also reported that enhanced expression of Gγ11 in senescent cells is caused by reactive oxygen species (ROS) such as H₂O₂, suggesting a possible molecular mechanism linking the effects of ROS in cellular senescence [208, 210]. ERK 1/2 has been identified to promote cellular senescence [211]. Gγ11 mediated activation of ERK1/2 was also observed, further establishing its role in cellular senescence [208]. Reduction in transcription of GNG11 has been reported in several cancer tissues such as medullary thyroid carcinoma [212] and splenic marginal zone lymphoma [213], where cell proliferation is accelerated while cellular senescence is reduced.

Even though the involvement of Gα signalling in autophagy is better studied, the role of Gβγ is not well understood. Knockdown of Gαs, Gαq11, and Gα12/13 has been shown to induce autophagy [214, 215]. However, subsequent work reported Gγ7 as the first Gγ subtype to be identified as an autophagy inducer [216]. The molecular mechanism behind the ability of Gγ7 to induce autophagy my involve interaction of Gγ7 with mTOR, a central regulator for cell proliferation and autophagy. Gγ7 induces cell division inhibition, cell death, and autophagy by perturbing the mTOR pathway. Further, Gγ subtypes are also found to exhibit differential effects on regulation of the actin cytoskeleton. For instance, reduction of actin stress fibers was observed in HeLa and U2OS cell lines upon the overexpression of Gγ7 [216]. Gγ7 results in efficient Gβγ translocation, and molecules that trigger cell death and autophagy are either cytosolic or located in cell organelles.

Insulin secretion

Glucose serves as a major physiologic regulator, ultimately leading to insulin secretion via a cascade of signalling in pancreatic β cells [217]. Several GPCRs have been shown to provide distinct ‘on’ and ‘off’ signals regulating insulin secretion [218]. The M3-muscarinic receptor mediates insulin secretion in pancreatic β cells upon activation [219]. The mechanism that drives M3-stimulated insulin secretion is explained partly as a result of PKD-mediated insulin vesicle formation from the Golgi complex [220]. As discussed earlier, as vesicular formation is a Gβγ translocation-mediated and Gγ subtype dependent process [204], insulin secretion may also be Gγ subtype-dependent. M3 receptor activation in a mouse pancreatic β cell line, NIT-1, exhibited a considerable increase in insulin secretion, confirming direct involvement of M3 receptors in the process and showed endogenous expression of relatively efficient mediators of Gβγ translocation such as Gγ5, Gγ10, and Gγ13 [184, 204]. In agreement with this observation, overexpression of slowly translocating Gγ3 significantly reduced M3-muscarinic receptor-induced insulin secretion [204], suggesting that GPCR-induced insulin secretion is Gγ subtype-dependent.

SNARE regulation and neuromodulation

Neurotransmission is a critical function driven by GPCRs upon stimulation by neurotransmitters to maintain the proper functioning of neuronal circuits [221]. Specifically, the inhibitory activity of pre-synaptic Gi-coupled GPCRs plays a major role in controlling autoreceptors that reduce neurotransmission [222]. This modulation is achieved by inhibiting pre-synaptic exocytosis via three major membrane-delimited mechanisms by; i) inhibiting Gβγ regulation of voltage-dependent Ca²⁺ channels (VDCCs) [223, 224], ii) Gβγ-mediated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels [225], and iii) direct interaction of Gβγ with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins which results in disruption of Ca²⁺ uptake and downstream exocytosis [222, 226]. To understand the molecular requirements for Gβγ-SNARE interaction, differential binding affinities of Gβγ isoforms to SNARE have been examined [227]. Compared to Gβ1γ1, Gβ1γ2 had a ~20 fold higher affinity for target membrane SNARE (t-SNARE) complex binding along with higher efficiency in inhibiting membrane fusion in permeabilized PC12 cells [227]. In peptide competition studies, it was later discovered that the N-terminal 2–7 residues of Gγ2 partly contribute to the tight binding of Gβ1γ2 to the t-SNARE complex [228]. It is conceivable that the enhanced Gβγ and t-SNARE interactions are governed by their mutual subcellular localization.

Cell migration

Cell migration is implicated in several physiological processes such as embryonic development and the immune response, where cell migration defects lead to complications in immune responses, wound healing, tissue repair, and cancer metastasis [229–232]. Although thought of as a Gi-coupled chemokine receptor-governed process, Gβγ plays a major role in modulating macrophage migration by inducing PI3K-PIP3 signalling at the leading edge of a migratory cell [191]. Further, another study also showed the necessity of Gβγ-mediated activation of PLCβ for macrophage migration [48]. As described above, PI3K-mediated PIP3 generation is driven by high PM affinity Gγ isoforms such as Gγ3 and Gγ4. Is cell migration also influenced by high PM affinity Gγ subtypes? Supporting this idea, the migratory mouse macrophage cell line, RAW264.7 showed a higher endogenous expression of high PM affinity Gγ3 and moderate PM affinity Gγ4. Interestingly, endogenous expression of low PM affinity Gγ subunits such as Gγ9, Gγ11, and Gγ1 in this cell line was comparatively lower. A complete loss of migratory ability of RAW264.7 cells was achieved by knocking down endogenous Gγ3, and interestingly this inhibitory effect was rescued by exogenously expressing high PM affinity Gγ2 in Gγ3 knockdown cells [48]. Since the PM affinity of a Gγ subtype is defined by the C terminal CAAX and pre-CAAX sequence of Gγ, which determine the type of prenylation, the influence of CAAX and CT sequences of Gγ isoforms on regulating RAW264.7 migration was also examined. Interestingly, mutant Gγ9 with Gγ3-CT was able to induce migration in RAW264.7 cells while cells expressing Gγ3 mutants containing either Gγ9 CAAX or CT sequences did not support GPCR induced migration [48]. An earlier study conducted using anti-peptide antibodies specific for Gγ isoforms known at the time (Gγ1, Gγ2, Gγ3, Gγ5, Gγ7) indicated that Gγ5 was co-localized with focal adhesions and actin stress fibers in neonatal cardiac fibroblasts [233]. Since focal adhesions are metabolically active regions associated with modulating adhesion, proliferation, and cell motility [234–236], and focal adhesion size also is correlated with predicting cell migration rate [237], Gγ5 may play a role in governing migration of embryonic cells [233].

4.6. Subcellular localization-specific Gβγ signalling and their Gγ subtype dependence

The long-standing norm that GPCR-G protein signalling was limited to the PM has been strongly challenged recently, and continuously emerging evidence reveals GPCR-G proteins also signal at and from IMs, including the endoplasmic reticulum (ER), the Golgi compartments, and the nucleus. After their synthesis, GPCRs and their associated signalling molecules are trafficked to the PM to execute their appropriate signalling functions. Upon prolonged exposure to their agonists, desensitization leads to the internalization of the receptor. The accepted notion was that internalized GPCRs also could activate arrestin-dependent signalling pathways, which may be either or both functionally and structurally independent of G proteins [238, 239]. Additionally, other cognate molecules associated with GPCR signalling such as heterotrimeric G proteins [240, 241], adenylyl cyclase isoforms [242], phospholipase A₂ [243], phospholipase Cβ [244], RGS proteins [245], β-arrestin 1 [246], and GRKs [247] are also reported to be transported to IMs attesting to the importance of their signalling at IMs.

The involvement of G proteins in regulating critical cellular events associated with cell motility, migration, and development is highlighted by their direct interactions with cytoskeleton and cell adhesion elements [233, 248–250]. The interaction of G proteins with the actin cytoskeleton has been shown in several studies [233, 249, 250]. Here, the localization of both Gq/11 and Gβγ dimers with the actin cytoskeleton is highlighted. The localization of Gβγ to the actin cytoskeleton was again noted for dimers containing specific Gγ subtypes. One study reported the localization of Gβγ dimers with Gγ12 on F-actin [249]. As a result of heterologous Gβ1γ12 expression in NIH 3T3 cells, rounding of cells, disruption of stress fibers, and enhanced cell migration was observed [249, 250]. Another study reported prominent localization of Gγ5 containing Gβγ to focal adhesions [233]. However, the relevant physiological and biochemical significance associated with their specific localization remains incompletely characterized.

Many studies have revealed roles for heterotrimeric G proteins in asymmetric cell division in model organisms such as C. elegans and D. melanogaster but in mammals as well [251]. The involvement of Gβγ in regulating heterotrimer activity consequently reduces Gαi interaction with GPR1/2 (G protein regulator 1/2)/Pins/LGN (leucine–glycine–asparagine), which acts as a positive regulator of asymmetrical cell division [252]. According to the reclassification by Human Genome Organization using a standard nomenclature, the analogous vertebrate protein is named G-protein signalling modulator-2 (GPSM2) [253]. Multiple studies have demonstrated that cell division-associated subcellular translocation of GPSM2, which includes movement from the cytoplasm to the midbody [254], the spindle pole [253] or the cortex [255]. During cell division, Gβγ competes with GPSM2 and its related proteins for their common binding partner, Gαi/o. A study using live-cell imaging of fluorescently-tagged Gβ shows the dynamic trafficking of Gβγ between cell cortex and endosomes, wherein the presence of Gβγ is enhanced during mitosis [256]. Even though different relative binding affinities and guanine nucleotide dissociation (GDI) potencies of GPSM2 towards different isoforms of Gα were observed [257], further studies on the relative abilities of different subtypes of Gβ and Gγ to compete with GPSM2 for Gαi/o binding are required.

A growing body of evidence demonstrates that Gβγ subunits can function at the Golgi complex facilitating the trafficking of macromolecules from trans Golgi network (TGN) to PM [258]. It has been shown that Golgi vesiculation is driven by constitutive or inducible targeting of Gβγ to the Golgi while PM-localized Gβγ had no effect [186]. This was further confirmed by inhibition of protein transport in the presence of Gβγ sequestration using a Golgi-targeted GRK2ct [258]. One possible explanation for Gβγ targeting the IMs, including Golgi, is their translocation potential upon GPCR-mediated G protein activation [201, 202]. In that sense, perhaps Gγ subtypes signalling at Golgi are mostly translocation competent. In agreement with this notion, one study showed the ability of GPCR activation-mediated Gβγ translocation to induce Golgi fragmentation and regulate insulin secretion [204]. Golgi fragmentation was achieved by the overexpression of Gγ11, one of the rapidly translocating Gγ subtypes. We have discussed above the enhanced Golgi fragmentation in the A569 cell line, which shows an abundant endogenous expression of Gγ11 [204]. Cellular senescence is another cellular physiological function potentially regulated by Gγ signalling at the Golgi complex, and the increased expression of Gγ11 in senescent cells was reported in another study [207]. Consistent with all the above data, the downregulation of cellular functions driven by signalling at the Golgi complex was observed due to expression of low translocating/dominant-negative Gγ3 [204, 207].

In addition to conventional signalling of GPCR-mediated activation of inositol triphosphate 3 (IP3) receptors on ER by PLCβ, it has been shown that Gβγ also can induce Ca²⁺ release to the cytosol via a PLCβ-independent pathway by directly interacting with IP3 receptors. In this study, introduction of Gβγ into cells restored the ability to release Ca²⁺ in cells where IP3 binding to IP3 receptors was inhibited [259].

An earlier study showed G protein activity in the nucleus by demonstrating the regulatory effects of Gγ5 interactions with the transcriptional repressor adipocyte enhancer-binding protein 1 (AEBP1) [260]. Gγ5 was shown to prevent transcriptional repression activity of AEBP1 by forming a complex with AEBP1 in the nuclei of 3T3-L1 cells. Nevertheless, Gγ7 was not able to form a similar complex [260]. Other studies also reported Gβγ nuclear localization and their subsequent interaction with various transcription factors and mediation of their regulation [261, 262]. By interacting with glucocorticoid receptors (GRs) and co-migrating with it into the nucleus, Gβγ suppresses GR-induced transactivation of the GR, and associated gene expression [261]. Interestingly, it was also shown that the anti-glucocorticoid activity of Gβγ is exhibited only in Gβγ with unprenylated Gγ subunits [262]. This was further confirmed by treatment of cells with a prenylation inhibitor, lovastatin to increase nuclear localization of Gβγ, and increase anti-glucocorticoid activity.

5. Impact of Gβγ subtype-diversity in disease and their potential as therapeutic targets

It is evident that improper signalling by Gβγ is detrimental, and dysregulation of Gβγ-mediated signalling could impact common events in heart failure, inflammation, carcinogenesis, and morphine-dependent antinociception [263]. There are several issues to be dealt with in considering Gβγ as a potential therapeutic target. As Gβγ is ubiquitously expressed and subtype-specific Gβγ-effector interactions remain incompletely understood, blocking all Gβγ functions with the current tools we have available might lead to undesired side effects. Since Gβγ is required for interaction of G protein heterotrimers with GPCRs, genetic deletion of the native Gβγ completely disrupts GPCR-G protein signalling. Further, considering their propensity for translocation, Gβγ heterodimers plays key roles in controlling the activity of effectors at different subcellular locations [178]. This may also have to be considered when developing them as viable therapeutic targets, as potential pharmacokinetic constraints may make this more difficult.

Gβγ subunits control numerous cellular processes ranging from immune system function, visual responses, and heart rate control to cell migration and cancer metastasis [76, 153, 175, 177, 264–266]. Tissue-specific expression of both Gβ and Gγ subunits suggests a possible relationship between particular subunit combinations and designated functions in specific tissues and organs [46, 80, 267]. Abnormal G protein signalling either due to conditions involving dysregulated control of their expression and/or mutations in Gβ and Gγ subtypes, results in altered signalling in disease. To support this notion, here we discuss several mutants and variants of both Gβ and Gγ subunits that have been identified in association with diseases in the visual, nervous, and cardiovascular systems and that impact cancer. Investigation of molecular links between mutations and variants of Gβ and Gγ subtypes and resultant pathological outcomes in light of underlying signalling perturbation will be the first step of identifying and developing therapeutic targets.

Nervous system

Millions of people worldwide are affected by neurological, neurodevelopmental and neurodegenerative disorders (i.e. epilepsy, Alzheimer’s disease, Parkinson’s disease, brain tumors, etc.), which affect both central and peripheral nervous systems. Here, we discuss selected Gβ and Gγ variants, which have been reported to cause neurological disorders. Several pathogenic variants of heterozygous Gβ1 coding gene (GNB1) have been identified in autosomal dominant neurodevelopmental disorder, MRD42 (Mental Retardation, Autosomal Dominant 42, or GNB1 encephalopathy or GNB1 disorder) in humans. Germline mutations in the GNB1 gene causes a rare neurodevelopmental disorder characterized by global developmental delay, intellectual disability, hypotonia, and seizures [268]. Similarly, an individual with GDD, hypotonia, multiple congenital joint contractures, and multiple morbidities caused by a de novo GNB2 variant p.Gly77Arg has been described [269]. Although only one GNB2 variant has been identified, 58 individuals encompassing thirty different GNB1 missense variants have been described to date [268, 270–278]. While the individuals are heterozygous for these GNB1 variants, mosaicism has been noted in a patient with a milder phenotype [274]. Though most reported cases have occurred de novo, the disorder is inherited in an autosomal dominant fashion. To date, four individuals have been reported to have inherited a pathogenic GNB1 missense variant, three with the p.Arg96Leu [272] mutation, and one with the p.Thr243Ala mutation [270]. Most variants are seen in exons 6 and 7 and occur at the interaction surface between Gβ1 and Gα subunits or various downstream effectors. In fact, some of these mutations have been shown to disturb heterotrimer formation and coupling to the dopamine D1 receptor leading to a loss of function phenotype [272]. While the vast majority of GNB1 mutations have been reported to be missense variants, three splice site variants (c.268–1G>T, c.917–1G>T, c.700–1G>T) and three frameshift variants (c.272_275del, c.915_916del, c.987_988delAG) have also been reported [272, 279]. Interestingly, c.700–1G>T and c.987_988delAG have also been shown to impact heterotrimer formation and coupling to the dopamine D2 receptor [279].

Global knockout of Gβ1 in mice results in neural tube defects leading to embryonic or neonatal lethality. In addition, severe brain malformations such as reduced cortical thickness and reduced brain volume have also been observed in these animals. Furthermore, abnormal morphologic changes in neural progenitor cells and impaired neural progenitor cell proliferation were also noted with Gβ1 knockdown, indicating a key role for Gβ1 in neurogenesis in the embryonic state [25, 280]. Gβ1 silencing (with Gβ1-specific antisense oligonucleotides) reduced Ca²⁺ currents with somatostatin receptor activation, suggesting a role for Gβ1 as a mediator of somatostatin receptor activity [25, 133]. Since somatostatin receptors modulate neuronal activity [281], with a wide distribution in the central nervous system in the mammalian brain [282], Gβ1-specific signalling might be considered as a new therapeutic target for neurological disorders.

Mutations in GNB4 have been shown to cause an autosomal dominant hereditary form of Charcot-Marie Tooth disease (CMTDIF), characterized by progressive muscle atrophy and weakness, distal sensory impairment, decreased reflexes, and variable nerve conduction velocities. Eight individuals have been reported to date, the first described being a family of six affected individuals where two were heterozygous for the p.Gly53Asp mutation, and a de novo case where the individual harbored the p.Lys89Glu mutation. Both mutations were shown to impair bradykinin receptor signalling resulting in a reduction of PLCβ2 activation, lower IP3 production, and a reduction in cytosolic calcium [92]. Another de novo mutation, p.Lys57Glu, was seen in an individual presenting with muscle atrophy, pes cavus and scoliosis with an age of onset of 35 years old [283]. The p.Gln220Arg variant was seen in a Japanese male and his father whose disease severity was mild, age of onset was higher, and had axonal and demyelinating neuropathies respectively [284].

Mutations in GNB5 are associated with either intellectual developmental disorder with cardiac arrhythmia (IDDCA) or language delay or the milder attention deficit-hyperactivity disorder/cognitive impairment with or without cardiac arrhythmia (LADCI) [125, 285–288]. Clinical severity correlates with the genetic variant where individuals who are homozygous for the p.Ser81Leu missense variant exhibit the milder phenotype characterized by language delay, mild intellectual disability, ADHD, with or without arrhythmias [125, 288]. In contrast, individuals either homozygous or compound heterozygous for a null allele suffer from the more severe phenotype including severe intellectual disability, epilepsy, hypotonia, retinal disease, bradycardia, sick sinus syndrome, and premature sudden death [125, 285–287].

Gγ2 is the most abundant Gγ subunit in the brain [25, 46, 80], and is involved in nociception mediated through the peripheral and central nervous systems [289, 290]. Gγ2 silencing by specific antisense oligonucleotide injection followed by administration of morphine, DPDPE (δ-opioid receptor agonist), and the non-opioid receptor agonists (WIN 55212–2 and clonidine), resulted in significant reduction of analgesia in mice, compared to control mice with no Gγ2 silencing, indicating a role for Gγ2 in nociception [291, 292]. Also, antisense oligonucleotide-mediated Gγ2 silencing resulted in a reduction in galanin-induced inhibition of voltage-gated Ca²⁺ channels in rat pituitary-derived GH3 cells and RIN5mF rat insulinoma cells, suggesting that Gγ2-containing G protein heterotrimers are coupled to galanin receptors, which are found in the peripheral and central nervous systems as well as the endocrine system [293]. Additionally, this observation further suggests that Gγ2 has a potential role in regulating intracellular Ca² homeostasis. Since Ca²⁺ homeostasis plays an important role in neurotransmitter release in the central nervous system [294], this could be a novel drug target for neurological disorders, including Alzheimeŕs and Parkinsońs diseases, in which the neurotransmission is reduced. Furthermore, Gγ3 knockout mice have been shown to be highly susceptible to seizures signifying a role for Gγ3 in regulating neuronal excitability [295].

Adenylyl cyclase (AC) activity plays an important role in memory, synaptic plasticity, as well as neurodegeneration [296], and altered AC activity has been shown in Alzheimer’s disease, where gradual and long-term memory loss is observed. With the loss of Gγ7 expression, reduced AC activity upon dopamine D1 receptor or adenosine A_2A receptor activation has been observed in HEK 293 cells [141, 166, 297], implying the involvement of Gγ7 in both dopamine D1 and adenosine A_2A receptor signalling. Since the adenosine A_2A receptor plays an important role in regulating CNS neurotransmission [298], this would be a potential target for drug development for neurological disorders.

While human expression of GNG8 seems to be low in all tissues, it was one of many genes lost in 19q13.32 microdeletion syndrome, which presents as intellectual disability, dysmorphic features, and cardiac deficits [299]. In mice, GNG8 is expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN), where knocking out GNG8 caused impairments in spatial and long-term memory, as well as cholinergic signalling [300]. Furthermore, Gγ3 knockout mice have been shown to be highly susceptible to seizures signifying a role for Gγ3 in regulating neuronal excitability. This phenotype was exacerbated in GNG3(−/−)GNG7 (−/−) double knockout mice which displayed a severe seizure phenotype that reduced their median life span to 75 days [301]. GNG13 is known for expression in the cerebellum; however, its expression in cerebellar Purkinje cells was reduced in Alzheimer’s patients compared to healthy control subjects [302], suggesting a possible role for GNG13 expression as a biomarker of cerebellar health. Additionally, a female patient with global developmental delay, cardiac defects, and failure of gonad development was seen to have a gain in copy number of the short arm of chromosome 16, which encompasses the GNG13 gene [303].

Visual system

Gα transducin (Gt) forms heterotrimers with Gβγ and plays a pivotal role in phototransduction [304–307]. Gγ1 was initially identified as the primary Gγ subtype present in transducin heterotrimer in rod cells [88, 307, 308]. Although Gγ1 shows a broad tissue expression; in the liver, kidney, placenta, uterus, etc., the majority of studies focusing on Gγ1 deal with signalling/phototransduction in the eye [25]. Photoreceptors in the retina undergo degeneration in several eye diseases, including macular degeneration, retinal detachment, and retinitis pigmentosa [309]. Disease is usually characterized by the loss of differentiated cells (i.e., rod cells) in the retina due to progressive cell death [310]. There are many causes of this degeneration, including genetic abnormalities (i.e., Stargardt disease), genetic and mechanistic diversity, so therapeutic development remains challenging [311]. Recent studies have also suggested that photoreceptor cell death may result primarily from non-apoptotic mechanisms, independent of caspase activity [312]. A study conducted in mice noted photoreceptor degeneration upon knock-out of Gγ1, suggesting a role of Gγ1 in photoreceptor function [304]. Since photoreceptors are not regenerated in mammals as in some other species (i.e., zebrafish) [313], scientists have recently tested if mammalian Müller glial cells; a source of retinal stem cells, could be stimulated to develop into rod photoreceptors in mammals [314]. However, since these mechanisms are not as robust as intrinsic repair mechanisms, development of novel methods to promote repair or regeneration of damaged photoreceptors is an essential goal in vision research, and one potential target gene might be Gγ1.

GNB3 has been identified as the cause of congenital stationary night blindness type 1H (CSNB1H), a unique retinal disorder with a dual anomaly in visual processing. It is characterized by varying degrees of dysfunction in the ON-class of bipolar cells and reduced cone sensitivity. CSNB1H was identified in a Lebanese-Armenian family where two brothers were compound heterozygous for the p.Lys57del deletion and the p.Trp339* nonsense mutation, while their aunt was homozygous for the p.Trp339∗ mutation [85]. A fourth sporadic case was identified in a woman homozygous for the p.Ser67Phe mutation [85]. A n additional GNB3 mutation was reported in a patient homozygous for the p.Arg42Ter nonsense mutation, who presented with an inherited retinal disease beginning in childhood characterized by nystagmus, mild disturbance of the central macula, and electroretinographic abnormalities [315]. In ON bipolar cells of the retina, mGluR6 activation leads to the closing of the TRPM1 channels. Gβ3 is part of the heterotrimeric G protein responsible for mGluR6 signalling. GNB3-knock out mice show that the absence of Gβ3 diminished light responses and mislocalized and reduced expression of other cascade partners, thereby altering synaptic organization [316]. In cones, the absence of Gβ3 resulted in reduced expression of G protein heterotrimer components and reduced light sensitivity [317]. In chicken, a three bp deletion (p.D153del) in GNB3 causes a retinopathy globe enlarged (rge) phenotype, categorized by retinal degeneration and embryonic mortality [318, 319].

Vision abnormalities, including vision impairment and abnormal eye movements are also seen in 60% of de novo GNB1 cases [278]. Abnormal eye movements such as nystagmus are most common, but others such as strabismus, and ophthalmoplegia have also been observed. This disease is also associated with cortical visual impairment, which is caused by disturbance of visual pathways such as optic nerve atrophy, retinal disease, etc. [116, 268, 320]. An abnormal morphology of retina with progressive degeneration has been noted in GNB1 heterozygous mice, further supporting the ophthalmic manifestations reported in affected patients with mutations in GNB1 [116].

Deactivation of G proteins induces the termination of light signalling responses in the mammalian eye. This process is catalyzed by a protein complex that includes Gαt, the regulator of G protein signalling 9 (RGS9), and a splice variant of Gβ5 subunit, Gβ5-L [25, 321, 322]. Gβ5-L is exclusively expressed in retinal photoreceptor cells and is involved in this process by interacting with G protein γ like domain (GGL domain) in RGS9–1 (Fig. 18) [25, 40, 45, 323]. With knockout of Gβ5L, G protein deactivation was decelerated in mice, indicating a role for Gβ5L in regulating heterotrimer inactivation [25, 321]. Individuals with mutations in the Gβ5 subunit have been shown to exhibit ophthalmologic abnormalities such as retinal disease and nystagmus [125, 286]. By measuring the optokinetic response, a GNB5-knockout zebrafish model showed that GNB5 is required for normal eye movement control [125]. GNB5-knockout mouse models have also been shown to display defective visual adaptation accompanied by an accelerated recovery of visual responses with altered development and function of retinal bipolar cells [116, 324–326].

Figure 18.

Crystal structure showing the Gβ5 (green) interaction with the GGL domain (maroon) of RGS9 (PDB ID: 2PBI) (light brown). The orientation of Gβ5 with the GGL domain of RGS9 is similar to Gγ1-Gβ1 interaction in the transducin heterotrimer, which is involved in vision transduction. RGS9 in mammalian eyes is involved in the propert termination phototransduction.

Cardiovascular system

Cardiovascular diseases remain a major cause of death and disability throughout the world. Here, we discuss the implication of certain GNB and GNG genes, and their variants as potential targets. An increased heart rate has been detected in GNB2 knockout mice, which reveals the involvement of Gβ2 in the regulation of cardiac muscle contractility [327]. Also, a heterozygous missense variant (Arg52Leu) of GNB2 has been identified in humans with autosomal dominant form of sinus node dysfunction (SND), which displays atrioventricular conduction dysfunction and atrial fibrillation without structural changes of the heart [42, 116]. Following the crystal structure of the mammalian G protein-coupled inwardly rectifying potassium channel 2/G protein complex (GIRK2)-β₁γ₂), reduced Gβ2-GIRK interactions could be anticipated with the Arg52 variant since this residue in Gβ2 is found in the Gβγ binding interface for GIRK [42, 116]. A functional study revealed involvement of this variant on GIRK channel activity alterations, which increases Ach-activated K⁺ currents [42]. Furthermore, a recent study with observations from sinus node dysfunction (SND) patients identified another Gβ2 variant (Trp101Cys), in addition to mutations in the KCNJ5 gene, which encodes the Kir3.4 subunit of the GIRK channel [328]. Since cardiac GIRK channels are directly activated by Gβγ dimers and are involved in controlling heart rate [182, 329], such Gβ variants may suggest strategies in potential drug design. Genome-wide association studies have mapped GNB2 and GNB4 to loci associated with variations in heart rate [330, 331]. While a role for GNB2 in control of heart rate has been investigated, a role for GNB4 has yet to be established. Further, a C825T polymorphism of GNB3 has been linked with increased atrial inward rectifying currents, which may reduce the risk of developing atrial fibrillation, characterized by irregular heartbeat [332, 333]. Furthermore, Gβ3 knockout mice have been shown to display mild bradycardia [56]. This may indicate that Gβ3 also plays a distinct role in regulating cardiac signalling by controlling the heart rate [116]. Although the exact molecular mechanism underlying these observations remains to be investigated further, the C825T polymorphism in Gβ3 is reported to favor alternative splicing resulting in two different splice variants; Gβ3s and Gβ3s225, altering their signalling ability [334–336]. Several studies have reported enhanced coronary vasoconstriction [337] and impaired vasodilation [338] with the C825T polymorphism in Gβ3, supporting this notion [334].

As previously mentioned, LADCI and IDDCA patients also exhibit cardiovascular phenotypes including bradycardia and sick sinus syndrome in addition to the neurological phenotypes described above, suggesting a role for GNB5 in cardiovascular disease. In addition, when GNB5-knockout zebrafish larvae were treated with carbachol, a strong decrease in heart rate was observed suggesting that GNB5 normally plays an inhibitory role in GIRK activity mediated by the parasympathetic nervous system [125]. Conversely, when the sympathetic agonist isoproterenol was used, heart rate did not differ from wild-type animals implying GNB5 is not crucial for sympathetic control [125].

Gene expression profiling analysis indicated that GNG2 was differentially expressed in children with vasovagal syncope type 1 [339], a transient loss of consciousness often presented as a rapid decline in heart rate and a drop in blood pressure. Copy number variations resulting in deletions in the GNB1 or GNG7 genes have been associated with the risk of atrial fibrillation-related thromboembolism in the general Taiwanese population [340]. In contrast, in the Polish population, the rs13093 GNG5 mutation is a risk factor for essential hypertension [341]. A cardiovascular role for GNG5 is further emphasized by the phenotypes observed in GNG5 knockout mice. GNG5(^−/−) mice exhibit severe cardiac defects such as un-looped hearts containing a single ventricle, abnormal headfolds, and hypoplastic pharyngeal arches, ultimately resulting in embryonic lethality [342].

Cancer

Many studies have also linked Gβγ signalling and mutations in individual Gβ and Gγ subunits to progression and outcomes in different types of cancer. Only one patient with a germline GNB1 mutation discussed above has been reported to have cancer in addition to the neurodevelopmental phenotype. The patient was heterozygous for the p.Gly77Ala mutation, developed acute lymphoblastic leukemia (ALL), which was successfully treated at an early age [273]. Somatic mutations in the GNB1 and GNB2 genes have been shown to occur in multiple cancers often conferring cytokine-independent growth. Interestingly, 11 GNB1 K57 mutations were seen in myeloid neoplasms, while I80 mutations were seen in 7 of 8 B cell neoplasms, suggesting lineage-based clustering. As in the de novo GNB1 mutations, these GNB1 mutations have been shown to reduce binding to most Gα subunits but not Gγ subunits. This also led to increased phosphorylation of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and mTOR substrates [343]. GNB1 mutations were shown to occur with other oncogenic alterations such as BCR-ABL in B cell acute lymphoblastic leukemia and JAK2 mutations in myeloid neoplasms, resulting in resistance to their respective inhibitors; similarly, the GNB2 mutation K78E was identified in BRAF-mutated melanoma, also resulting in resistance to BRAF inhibition [343]. The K89M GNB1 mutation, in particular, was identified as the cause of tyrosine kinase inhibitor resistance in ETV6-ABL1-positive leukemic cells by restoring the inhibited PI3K/Akt/mTOR and MAPK signalling pathways [344].

The previously mentioned C825T polymorphism in the GNB3 gene has also been shown to be implicated in the progression of various cancers such as breast cancer [345], bladder cancer [346], thyroid carcinomas [347, 348], head and neck squamous cell carcinoma [349] and glioblastoma multiforme [350]. Additional GNB3 somatic mutations have also been found in malignant melanoma samples [351]. GNB4 was identified as a target silenced by DNA methylation in anti-estrogen-resistant breast cancer cell lines, where its overexpression and knockdown indicated a role for GNB4 in breast cancer growth [352]. GNB4 was also shown to promote gastric cancer cell proliferation and migration, where its expression was associated with the long-term survival rate of gastric cancer patients [353], indicating the possibility of using GNB4 as a predictive marker. Using GNB4 as a predictor has also been suggested in other cancers as two haplotype blocks have been identified in intron 1 of the GNB4 gene, where the first is associated with survival in bladder cancer patients [354], and the second with survival in colorectal cancer patients [355].

While Gβ5 is known to be predominantly expressed in the brain, Gβ5 has also been shown to be implicated in colorectal cancer. Whole-exome sequencing and gene expression analysis have suggested that mutations in GNB5 contribute to lung metastasis in colorectal cancer, possibly through phospholipase C signalling [356]. Gβ5 has been shown to regulate colorectal cancer cell apoptosis induced by TRAIL by reducing the surface expression of the TRAIL-R2 receptor, increasing expression of the anti-apoptotic protein XIAP, and activating the NF-κB signalling pathway [357]. In addition, Gβ5 antagonism has been shown to overcome TRAIL-mediated apoptotic resistance and cetuximab resistance in both wild-type and mutant KRAS cells, indicating a role for Gβ5 in colorectal cancer cell therapy [357, 358].

Malignant melanoma is a highly aggressive melanocytic tumor. Evidence exists encouraging investigation of GNG2 as a potential therapeutic target for the metastasis of malignant melanoma. GNG2 expression is reduced in malignant melanoma [359], leading to increased cell migration and invasion, and augmented proliferation, while GNG2 overexpression inhibits metastasis in human malignant melanoma cells [360]. Gγ10 may also serve a role in melanoma metastasis as somatic mutations in GNG10 were seen in 8.75% of metastases [351]. GNG3 was a key differentially expressed gene in glioblastoma multiforme (GBM) [361, 362]. In addition, GNG4 was shown to be hypermethylated and downregulated in GBM; Gγ4 is a potential tumor suppressor as overexpression in vitro reduced proliferation, colony formation, and transformation of immortalized astrocytes [363]. A tumor suppressor role for GNG4 has been previously suggested in renal cell carcinoma [364]. In colon cancer, GNG4 may serve as a diagnostic indicator since expression was observed to be higher in left-sided colon cancer than in right-sided colon cancer, and a prognostic indicator as high GNG4 expression showed higher disease stage and lower survival rate [365]. GNG5 was identified as one of 10 genes that correlate to lower grade glioma tumor purity and patient prognosis [366]. Gγ5 was shown to be an unfavorable prognostic indicator as glioma patients with GNG5 overexpression had shorter overall survival times [367].

In classical Hodgkin’s lymphoma, screening for homozygous deletions in four cell lines determined one of the genes deleted was GNG7, indicating a potential tumor suppressor role [368]. GNG7 expression levels verified in osteosarcoma, cervical carcinoma, breast cancer, colon cancer, and nasopharyngeal carcinoma cells lines all displayed a reduction in Gγ7 expression [216]. Reduced Gγ7 expression was also observed in clear cell renal cell carcinoma [369], head and neck squamous cell carcinoma (HNSCC) [370], esophageal cancer [371], intrahepatic cholangiocarcinoma [372], and pancreatic cancer [373]. Though the precise reason for differing GNG7 expression levels in cancer have not been fully elucidated, the GNG7 gene promoter was heavily methylated in clear cell renal cell carcinoma tissues [369], in HNSCC [370, 374], and in esophageal cancer [371]. GNG7 promoter hypermethylation, as well as GNG7 gene mutations in clear cell renal cell carcinoma, and loss of heterozygosity in esophageal cancer, may serve as a reason for decreased GNG7 expression observed in various cancers. GNG7 expression correlated with poor overall survival as well as tumor grade, size, and invasion [369–372]. GNG7 was also identified as a differentially expressed and prognosis-related gene in lung adenocarcinoma, proposed to be used for diagnosis and predicting prognosis both on its own and as part of a four-gene panel [375, 376]. Together evidence suggests a tumor suppressor role for GNG7 in multiple types of cancer.

GNG10 expression levels in peripheral blood mononuclear cells correlated with prognosis in head and neck squamous cell carcinoma and radiotherapy response in nasopharyngeal carcinoma [377]. GNG11 was identified as a gene hub in lung squamous cell carcinoma, where it is associated with tumor size, the maximum standardized uptake value, and recurrence-free survival [378]. GNG11 was also shown to act as a hub gene in lung adenocarcinoma where high expression correlated with better survival outcomes, suggesting a potential tumor suppressor role [379, 380]. In female non-smokers with lung adenocarcinoma, low GNG11 expression was observed and associated with poor patient survival rates [381, 382], further highlighting a potential role for Gγ11 and tumor suppression. GNG11 was also shown to be downregulated in acute myeloid leukemia, B-lineage acute lymphoblastic leukemia, and T-lineage acute lymphoblastic leukemia [383]. GNG12 expression was observed to be higher in pancreatic ductal adenocarcinoma (PDAC) patient specimens and was accompanied by a poor prognosis of pancreatic cancer; Elevated GNG12 expression promoted PDAC tumor growth both in vitro and in vivo, suggesting a role for GNG12 in PDAC treatment or as a marker of unfavorable prognosis [384]. In gastrointestinal stromal tumors, high GNG13 expression may also serve as a poor prognosis-related biomarker [385].

Conclusion

As we move away from thinking about Gβγ signalling as if it were mediated by a single protein, we come to see that a comprehension of the impact of subunit diversity is critical to our understanding of their vast functions in health and disease. Targeting these events pharmacologically will be contingent on a clearer understanding of the myriad mechanistic nuances involved.

Highlights.

• Gβγ subunits are major contributors to GPCR-G protein signalling.

• A broad functional array of Gβγ signalling has recently been attributed to Gβ and Gγ subtype diversity,

• We review the literature on the repercussions of Gβ and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes.

• We provide perspective in understanding the roles of subtype-specific roles of Gβγ signalling and associated diseases