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G Protein-Coupled Receptor (GPCR) Dimers
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This entry is adapted from the peer-reviewed paper 10.3390/ijms22063241

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. 

G protein-coupled receptor (GPCR) dimerization allosteric modulation receptor–receptor interaction PPI prediction protein dynamics peptide design

1. Introduction

G protein-coupled receptors (GPCRs) belong to a large family of seven-transmembrane (TM) proteins with structural topologies defined by the general presence of the extracellular (EC) domain, the intracellular (IC) domain, and a TM domain comprising of seven helices that connects the EC and IC domains of the receptors. The TM domain serves as a conduit for the flow of information initiated by the binding of endogenous orthosteric ligands from the cell’s exterior and triggering the binding of cytosolic proteins such as the heterotrimeric guanine nucleotide-binding protein (G protein), GPCR kinases (GRKs), and β-arrestin within the cell. This process, being allosterically driven, spurred studies that aimed to understand the process of allosteric modulation in driving GPCR activation [1][2][3][4][5][6][7].

Structural studies have revealed the significance of conformational plasticity in the allosteric regulation of GPCR activity. The structural flexibility of GPCRs empowers the receptor family to cascade a variety of extracellular signals—spanning from photons to neurotransmitters and hormones—across the membrane, hence equipping GPCRs with the capacity to affect multiple signaling pathways. Depending on the G protein subtypes (Gs, Gi/o, Gq, and G12/13) binding at the intracellular binding site, specific physiological functions ranging from taste, vision, and synaptic transmission are set in motion [8][9]. This versatile nature of GPCRs rendered them attractive as drug targets and opened numerous possibilities in the development of novel therapeutics for the treatment of a wide range of diseases and conditions [1][2][3][4][5]. While numerous experimental and computational studies have been conducted to examine the structural architecture and dynamics of GPCRs as monomers, these studies lead to a riveting question regarding the possibility of synergistic interactions between GPCRs to prompt specific signaling pathways. The growing number of studies investigating the role of dimerization and oligomerization in steering GPCR functions demonstrated the increasing interest in this topic despite its controversial status in the GPCR community [10][11][12][13].

Studies are emerging in support of GPCR homo/heterodimers and higher order oligomers, indicating the possibility of GPCRs to operate beyond the more congenial postulation of functional monomers [10][12][14][15][16][17][18][19][20][21][22]. The earliest allusive indication of GPCR oligomerization arose from kinetic binding assays performed by Limbird et al. for β-adrenergic receptors (β-ARs) on frog erythrocyte membranes [21]. In this study, the negative cooperativity between β-AR monomers on the membrane was inferred based on the different dissociation rates of 3H (-)alprenolol observed in two different conditions set apart by the surplus of unlabeled (-)alprenolol in one. Henceforth, the collection of indirect data from various traditional pharmacological and biochemical experiments such as binding assay, gel electrophoresis, immunoaffinity chromatography, chemical cross-linking, and co-immunoprecipitation studies further substantiated this phenomenon [22]. Recent explicit evidence reported the observation of various classes of GPCRs existing as homodimers, heterodimers, and/or higher-order oligomers through a variety of biophysical studies—single-molecule fluorescence-based approaches, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryogenic electron microscopy (cryo-EM)—as well as computational studies. These have garnered more interests for the study of GPCR oligomerization, particularly for the potential implications to drug design and discovery [12][14][15][16][17][18].

In this review, we will focus on the structural aspect of the allosteric modulation of GPCR dimers, specifically for two well-characterized receptors, namely metabotropic glutamate receptor (mGluR) and γ-aminobutyric acid B receptor (GABABR), both of which have their full-length structures recently solved. This review will also highlight studies that proposed the alteration of GPCR activity and allosteric modulation mechanism through dimerization—an interesting phenomenon that can be exploited to further boost the potential of GPCRs as a therapeutic target for new disease indications [23]. As available three-dimensional structures of GPCR dimers are limited in comparison to the number of dimers validated through experiments, the biophysical characterization of receptor–receptor interactions via computational methods have gained ground as a potential tool for the mapping of intra- and inter-subunit interactions at the receptor–receptor interface. Therefore, we will also highlight some current computational methods that have been or could be applied to investigate the protein–protein interface. Figure 1 illustrates an overview of the topics discussed in this review.

 
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Figure 1. Summary of topics covered regarding receptor–receptor interactions in G protein-coupled receptor (GPCR) oligomers. Structural changes afforded through the binding of an agonist (L-quisqualate) to mGlu5 portrayed through X-ray crystal structures of mGlu5 in apo (PDB id: 6N52) and active (PDB id: 6N51) states. Helices B and C, which are involved in the stabilization of the dimer, are labelled. (VFT: Venus flytrap domain; CR: cysteine-rich domain).

2. Role of Receptor–Receptor Interactions in the Allosteric Modulation of GPCR Activation

The comprehensive scrutinization of class A receptors has continuously supplied us with information on the structures and dynamics of the proteins, albeit the disproportionate distribution between inactive and active states solved. Nevertheless, advances in protein engineering and biophysical characterization techniques have propelled accessibility to the less solved active state configuration, allowing studies examining the structural disparity between the two states. The juxtaposition of the active and inactive configurations revealed compelling differences in highly conserved motifs known as the molecular switches that are conveyed to be important for allosteric communication between the distal ends of the TM domain, namely the orthosteric and intracellular protein binding sites. This forms the main cognizance of TM domain activation in the GPCR family. However, recent studies have established the presence of GPCR dimers across different classes of GPCRs. This discovery opens the possibility of TM domain activation being governed not just by long-range allosteric communication between the orthosteric and intracellular binding sites within a single receptor (cis-activation) but also through previously unprecedented pathways involving receptor–receptor interactions (trans-activation) [24]. This section will discuss the structural aspect of the mechanism governing the allosteric modulation of the trans-activation of two widely accepted GPCR dimers, namely mGluR homodimer and GABABR heterodimer. 

2.1. Class C GPCRs: A Potential Model for GPCR Trans-Activation

The concept of dimerization has been widely accepted for class C GPCRs, and cooperativity between protomers of this family of receptors—both positive and negative—has been proposed to be vital for signal transduction [25][26][27][28]. Several studies have been conducted to understand the mechanism governing the activation of class C GPCRs, specifically mGluR and GABABR dimers. These studies inevitably led to insights pertaining to the allosteric regulation of signal transduction in GPCR dimers. Class C GPCRs have been proposed to be a potential model for the comprehension of allosteric regulation and cooperativity for other classes of GPCRs, albeit a tendentious comparison since their sequences and overall structures differ from other classes. Nonetheless, several structural similarities with class A GPCRs have been drawn that uphold this comparison.

The most significant similarity lies in the TM domains of class A and class C GPCRs. The similar topologies of the seven TM helices lead to a shared “ionic lock” feature that occurs between the intracellular regions of TM3 and TM6—a conserved “molecular switch” that when formed maintains the inactive conformation of class A GPCRs [26][29][30]. While a salt bridge between a conserved Arg3.50 and Glu(Asp)6.30 defines the ionic lock present in class A, this feature occurs via Lys3.50 and Glu6.35 in class C [31][32]. The numbers in superscript represent the Ballesteros–Weinstein numbering system in which the first digit indicates the TM helices 1 to 7 and the digits following the decimal (a separator) denote the residue position relative to a highly conserved residue within a single TM helix, which is assigned as residue 50 [33]. Site-directed mutagenesis performed at the aforementioned residues and a neighboring Ser613, in IC loop 1 (interacts with Lys3.50), to either stabilize or destabilize the ionic lock in class C GPCRs afforded a decrease or increase in the constitutive activation of their TM domains compared to wild type, respectively. This corroborated the analogous behavior of this motif in both GPCR classes [32]. Residues Lys3.50, Glu6.35, and Ser613 are also highly conserved in mGluR, GABABR, calcium-sensing receptor, and T1R taste receptor, and mutations of these residues or others near the ionic lock reportedly altered the signaling pathways of class C GPCRs [31][32][34]. For instance, the point mutation of Glu6.35 to Lys in mGlu6 was reported to be the cause of congenital night blindness. This phenotype was expressed due to altered G protein signaling, causing the receptor to prefer Gi coupling over the native Go coupling [32][35]. The comparable TM topologies of the two classes of GPCRs was further evinced through homology models of class C GPCRs generated using the crystal structure of bovine rhodopsin [30][36][37]. These studies conducted afforded reliable observations that provided insights on the allosteric modulations of class C receptors [30][36][37][38].

MGluRs have also exhibited similar activation activity as rhodopsin-like receptors. Goudet et al. demonstrated this characteristic by examining the activity of the TM domain of a truncated mGlu5 (no Venus flytrap (VFT) and cysteine-rich (CR) domains) in the presence of a negative allosteric modulator (NAM) (MPEP; 2-methyl-6-(phenylethynyl)-pyridine hydrochloride) and a positive allosteric modulator (PAM) (DFB; 3,3′-difluorobenzaldazine) [39]. The binding of MPEP to the TM domain of the truncated mGlu5 led to the inhibition of the constitutive activity of the receptor relative to wild type. On the other hand, DFB binding resulted in the direct activation of the TM domain. While DFB has been classified as selective PAM with no agonistic effect on wild-type mGlu5, the absence of the VFT and CR domains permitted the ligand to behave as a full agonist, thus enabling receptor activation through a signaling pathway akin to that of a rhodopsin-like receptor [39][40]. A comprehensive analysis of the binding site of MPEP through site-directed mutagenesis and the homology modeling of mGlu5 also discerned a binding pocket at the TM domain that coincides with the orthosteric binding site of rhodopsin [39]. Analysis of three-dimensional structures of class C GPCRs that were solved in the presence of allosteric ligands further highlighted this similarity [18][25][41]. These studies assert the similarities in the structural build of the TM domains of class A and C GPCRs, validating the potential of class C GPCRs to be a model system for the mechanistic study of TM domain activation of GPCR dimers in general.

2.2. Elucidation of Allosteric Modulation via Full-Length Structures of Class C GPCR Dimers

A structural feature that distinguished class C GPCRs from other classes is a large N-terminal EC domain that comprised of approximately 400 to 600 amino acids [26][28]. This domain encompasses a bilobed ligand-binding region that resembles a Venus flytrap; hence, it is also known as the VFT domain. The VFT domain comprises of two lobes, lobe I (N-terminal lobe) and lobe II, with a cleft in between that accommodates an agonist or an antagonist [25][28][42]. This large domain, with the exception of GABABR, is connected to the TM domain via a CR domain [17][26][27][28]. Associations between lobes I of the VFT domains of partnering receptors in both inactive and active states engendered most class C GPCRs as obligate dimers, and this was structurally corroborated through the recently reported full-length apo structures of mGluR homodimer and GABABR heterodimers in the “Roo” (Rest open–open) conformation [28][43][44][45][46] (Figure 2). The type of interactions established at this interface varies across class C GPCRs. Hydrophobic interactions and a nonessential, conserved disulfide bridge that formed between two flexible loops of the protomers are observed in mGluR homodimers, while GABABR heterodimers are mainly stabilized through polar interactions [18][26][47].

 
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Figure 2. Surface representation of two full-length class C GPCR dimers, namely mGlu5 and GABABR. The functional domains of the GPCR dimers, namely the Venus flytrap domains, the cysteine-rich domain in mGluR, the stalk in GABABR, and the transmembrane domains, are labeled accordingly.

A recent study by Koehl et al. combined data from X-ray crystallography, cryo-EM, and biochemical assays to examine the activation pathway of mGlu5 [28]. This study provided the first complete, three-dimensional structures of mGluR in both active and inactive states, thus allowing the scrutinization of the conformational plasticity of mGluR during activation. Ligand binding at both the VFT domains of the mGluR homodimer prompted a configurational change dictated by a less compact packing of two helices—named helices B and C (Figure 1)—in comparison to the apo structure. These helices bordered the interface between adjacent protomers at lobe I, and hydrophobic interactions are mainly established between conserved residues of these helices [26][28]. The more relaxed lobe I–lobe I interface promoted the formation of polar interactions near the apices of helices B leading to the stabilization of the “Acc” (Active close–close) conformation [28]. The concurrent activation of both VFT domains of mGluR homodimers is noted to be essential for optimal receptor activity, although the binding of an agonist at one of the VFT domains has been shown to partially activate mGlu5 receptor via an “Aco” (Active close–open) conformation [18][47].

The comparison of the active and inactive states of mGlu5 revealed that the TM domains moved closer together and undergo a 20° rotation to adopt an active conformation characterized by a TM6–TM6 interface [28][43]. This maneuver, mediated by interactions established between the CR domain and EC loop 2 (ECL2) of the TM domain, was speculated to be vital, as it aids in the translation and rotation of the TM domains that enabled the formation of specific inter-subunit interactions that could ameliorate the activity of mGluR [28][29][48][49]. Observations revealed through the three-dimensional structure of mGlu5 were also congruent with earlier experimental studies, all of which emphasized the importance of both intra- and inter-subunit interactions in modulating allosteric communication between the VFT and the TM domains [25][29][49][50][51].

In addition to the mGlu5 homodimer, several structures of the full-length metabotropic GABABR heterodimer have also been solved [43][44][45][46]. Shaye et al. reported the structures of four full-length GABABR in the active and inactive states as well as two intermediate states. In this study, they have combined the use of cryo-EM as well as molecular dynamics (MD) simulations to elucidate the intricate dynamics of GABABR activation [45]. GABABR forms an obligate heterodimer comprising of two different subunits, namely the GABAB1R (GB1) and the GABAB2R (GB2). In addition to association at the VFT domains, stabilization of the heterodimeric apo form was also assisted through polar interactions established at the intracellular segments of TM3 and TM5 of GB2 and GB1, respectively. Similar to mGluR, allosteric modulation originates from the orthosteric binding site of the VFT domain and engenders a cascade of conformational changes leading to the activation of the TM domain. In other respects, the GABABR heterodimer follows a distinctive signal transduction mechanism in which the agonist only binds to the VFT domain of GB1, and G protein activation proceeds via the activation of the TM domain of GB2 [45][52][53][54][55][56].

The contrasting ligand-binding competence of the subunits rendered GABABR an attractive model for the study of asymmetric trans-activation [53][57]. With the availability of the three-dimensional structures of the intermediate states of GABABR, the allosteric pathway leading to the initiation of downstream signaling via GB2 could be harnessed. The two intermediate states solved for GABABR also evinced the dynamic nature of receptor activation. Ligand-binding at GB1 was proposed to have created an equilibrium between the partially (Int-1) and fully closed (Int-2) conformations of its VFT domain [45]. The partially closed conformation of the VFT domain of GB1 induced the rotation of both GB1 and GB2, which brings the two protomers closer together, while keeping lobes II of GB1 and GB2 far apart. In this state, the TM domains were oriented in the inactive TM5–TM5 topology, albeit no interaction was established between the two helices. As GB1 transitioned to the fully closed configuration at the VFT domain, lobes II of the GB1 and the GB2 subunits gravitated toward each other. This conformational change induced signals that descend a connecting “stalk” (Figure 2), leading to the characteristic active TM6–TM6 topology necessary for class C GPCR activation [26][28][43][45][58]. With available crystal structures, further computational studies of GABABR could furnish us with insights related to the dynamics of negative cooperativity in driving asymmetric G protein signaling, which is a characteristic that has been commonly reported in GPCR dimers [59][60][61].

3. Altered GPCR Activities Induced through Heterodimerization

Even though the homodimerization of mGluR has been widely acknowledged to regulate neuronal function, the existence of mGluR heterodimers is still as debatable as the concept of dimerization for other GPCR families. Even so, the presence of several mGluR heterodimers has been alluded through experimental studies [51][62]. The formation of the heterodimeric complex between Group I mGluRs, namely mGlu1 and mGlu5, at the hippocampal neurons has been verified by Pandya et al. through a series of immunoprecipitation experiments [62]. The tendency of this dimer to exist as a functional heterodimer and contribute to signal transduction was subsequently verified by Werthmann et al. through functional complementation experiments in HEK293 cells [51]. Additionally, the mGlu1/5 dimer was also proposed to afford a distinct allosteric modulation pathway in comparison to their homodimeric counterparts. The MGlu1/5 dimer follows the symmetric signaling (equal probability for both protomers to engage G protein) exhibited by their respective homodimers. However, the receptor’s response to G protein coupling is dependent on the protomer that the intracellular protein engages, and the activation of both protomers is necessary for G protein activation. This observation contradicts the activation pathway observed in their respective homodimers, whereby the inhibition of one protomer did not curtail G protein activation [63].

The mGlu2/4 dimer has also been identified in vivo and is one of the most studied mGluR heterodimers [59][64]. Unlike their respective homodimers and mGlu1/5 dimer, the mGlu2/4 heterodimer follows an asymmetric activation pathway, which entails selective G protein binding to mGlu4 [59]. However, when mGlu4 is stabilized in its inactive state via NAM binding or when a PAM is bound to the TM domain of mGlu2, the mGlu2/4 dimer adopted an alternative activation profile via G protein coupling at mGlu2. Asymmetric cooperativity has also been found to be ubiquitous for heterodimeric pairs comprising of mGlu2 and other Group II (mGlu3) and Group III (mGlu4, mGlu6-8) mGluRs [59][60][61]. The binding of G protein to only one protomer is also a mechanism that has been evidently adopted by most GPCR homodimers and heterodimers despite differences in the allosteric modulation pathway. This observation iterates the importance of negative cooperativity between the TM domains of GPCR dimers through which the activation of one protomer blocks the signaling capability of the other, directing G protein coupling to a single protomer. Positive cooperativity between TM domains was also observed in mGlu2/4 and mGlu1/5 dimers. In this case, the inactive state of one protomer initiated the activation of the other through positive allosteric effects [51][63][65].

While the structures of class A and C GPCRs differ considerably as a whole, the TM domains of these receptors share similar topologies (vide supra) leading to the possibility of class A GPCRs existing as dimers. The acquiescence of class A GPCR dimerization is also stimulated through experimental evidence of their physical interactions with mGluRs and other class A receptors. Numerous studies conducted to understand the physiological aspect of class A/class C GPCR heterodimers have associated heterodimerization to the modification of the receptor’s function, trafficking, and pharmacology [12][19][26][66]. While the mechanism controlling the dimerization process is still unclear and research have afforded diverse explanations for their assemblies, physical interactions between class A and class C GPRCs have been reported, evincing the formation of heterodimers. These heterodimers include mGluR/serotonin 5-HT2A receptor (5-HT2AR), mGlu5/adenosine A2A R (A2AR), Glu5/dopamine D1 receptor (D1R), and mGlu5/mu-opioid receptor (MOR) [26][67][68][69][70][71][72]. Among these heterodimers, mGlu2/5-HT2AR is the most widely investigated and association to the pathophysiology of psychosis in schizophrenia and Parkinson’s disease, as well as dyskinesia in the latter rendered this heterodimer an attractive target for the treatment of these diseases [68][69][70][73].

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Subjects: Biophysics
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Sun Choi
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Raudah Lazim
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