ABSTRACT
G protein-coupled receptors (GPCRs) have long been shown to exist as oligomers with functional properties distinct from those of the monomeric counterparts, but the driving factors of GPCR oligomerization remain relatively unexplored. In this study, we focus on the human adenosine A2A receptor (A2AR), a model GPCR that forms oligomers both in vitro and in vivo. Combining experimental and computational approaches, we discover that the intrinsically disordered C-terminus of A2AR drives the homo-oligomerization of the receptor. The formation of A2AR oligomers declines progressively and systematically with the shortening of the C-terminus. Multiple interaction sites and types are responsible for A2AR oligomerization, including disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. These interactions are enhanced by depletion interactions along the C-terminus, forming a tunable network of bonds that allow A2AR oligomers to adopt multiple interfaces. This study uncovers the disordered C-terminus as a prominent driving factor for the oligomerization of a GPCR, offering important guidance for structure-function studies of A2AR and other GPCRs.
INTRODUCTION
G protein-coupled receptors (GPCRs) have long been studied as monomeric units, but accumulating evidence demonstrates that these receptors can also form homo- and hetero-oligomers with far-reaching functional implications. The properties emerging from these oligomers can be distinct from those of the monomeric protomers in ligand binding(1–4), G protein coupling(5–9), downstream signaling(10–13), and receptor internalization/desensitization(14–16). With the vast number of genes identified in the human genome(17), GPCRs are able to form a daunting number of combinations with unprecedented functional consequences. The existence of this intricate network of interactions among GPCRs presents major challenges and opportunities for the development of novel therapeutic approaches(18–23). Hence, it is crucial to identify the driving factors that govern the oligomerization of GPCRs, such that the properties of GPCR oligomers can be understood.
GPCR oligomers with multiple interfaces(24–28) can give rise to myriad ways by which these complexes can be formed and their functions modulated. In the crystal structure of the turkey β1-adrenergic receptor (β1AR), the receptor appears to dimerize via two different interfaces, one formed via TM4/TM5 (transmembrane domains 4/5) and the other via TM1/TM2/H8 (helix 8) contacts(29). Similarly, in the crystal structure of the antagonist-bound μ-opioid receptor (μ-OR), the protomers also dimerize via two interfaces; however, only one of them is predicted to induce a steric hindrance that prevents activation of both protomers(30), hinting at interface-specific functional consequences. A recent computational study predicted that the adenosine A2A receptor (A2AR) forms homodimers via three different interfaces and that the resulting dimeric architectures can modulate receptor function in different or even opposite ways(27). All of the above-mentioned interfaces are symmetric, meaning that the two protomers are in face-to-face orientations, hence forming strictly dimers. Asymmetric interfaces, reported in M3 muscarinic receptor(31), rhodopsin(32–34), and opsin(34), are in contrast formed with the protomers positioning face-to-back, possibly enabling the association of higher-order oligomers.
Not only do GPCRs adopt multiple oligomeric interfaces, but various studies also suggest that these interfaces may dynamically rearrange to activate receptor function(35). According to a recent computational study, A2AR oligomers can adopt eight different interfaces that interconvert when the receptor is activated or when there are changes in the local membrane environment(24). Similarly, a recent study that combined experimental and computational data proposed that neurotensin receptor 1 (NTS1R) dimer is formed by “rolling” interfaces that co-exist and interconvert when the receptor is activated(36). Clearly, meaningful functional studies of GPCRs require exploring their dynamic, heterogeneous oligomeric interfaces.
The variable nature of GPCR oligomeric interfaces suggests that protomers of GPCR oligomers may be connected by tunable interactions. In this study, we explore the role of an intrinsically disordered region (IDR) of a model GPCR that could engage in diverse non-covalent interactions, such as electrostatic interactions, hydrogen bonds, or hydrophobic interactions. These non-covalent interactions are readily tunable by external factors, such as pH, salts, and solutes, and further can be entropically stabilized by depletion interactions(37–39), leading to structure formation and assembly(40–47). In a system where large protein molecules and small solute particles typically coexist in solution, assembly of the protein molecules causes their excluded volumes to overlap and the solvent volume accessible to the solutes to increase, raising the entropy of the system. The type and concentration of solutes or ions can also remove water from the hydration shell around the proteins, further enhancing entropy-driven protein-protein association in what is known as the hydrophobic effect(48). This phenomenon is applied in the precipitation of proteins upon addition of so-called salting-out ions according to the Hofmeister series(49). The ability of IDRs to readily engage in these non-covalent interactions motivates our focus on the potential role of IDRs in driving GPCR oligomerization.
The cytosolic carboxy (C-)terminus of GPCRs is usually an IDR(50, 51). Varying in length among different GPCRs, the C-terminus is commonly removed in structural studies of GPCRs to enhance receptor stability and conformational homogeneity. A striking example is A2AR, a model GPCR with a particularly long, 122-residue, C-terminus that is truncated in all published structural biology studies(24, 27, 52–59). However, evidence is accumulating that such truncations—shown to affect GPCR downstream signaling(60–62)—may abolish receptor oligomerization(63, 64). A study using immunofluorescence has demonstrated that C-terminally truncated A2AR does not show protein aggregation or clustering on the cell surface, a process readily observed in the wild-type form(65). Our recent study employing a tandem three-step chromatography approach uncovered the impact of a single residue substitution of a C-terminal cysteine, C394S, in reducing the receptor homo-oligomerization in vitro(63). In the context of heteromerization, mass spectrometry and pull-down experiments have demonstrated that A2AR-D2R dimerization occurs via direct electrostatic interactions between the C-terminus of A2AR and the third intracellular loop of D2R(66). These results all suggest that the C-terminus may participate in A2AR oligomer formation. However, no studies to date have directly and systematically investigated the role of the C-terminus, or any IDRs, in GPCR oligomerization.
This study focuses on the homooligomerization of the human adenosine A2AR, a model GPCR, and seeks to address: (i) whether the C-terminus engages in A2AR oligomerization, and if so, (ii) whether the C-terminus forms multiple oligomeric interfaces. We use size-exclusion chromatography (SEC) to assess the oligomerization levels of A2AR variants with strategic C-terminal modifications: mutations of a cysteine residue C394 and a cluster of charged residues 355ERR357, as well as systematic truncations at eight different sites along its length. We complemented our experimental study with an independent molecular dynamics study of A2AR dimers of five C-terminally truncated A2AR variants designed to mirror the experimental constructs. We furthermore examined the oligomerization level of select C-terminally modified A2AR variants under conditions of ionic strength ranging from 0.15 to 0.95 M. To test whether the C-termini directly and independently promote A2AR oligomerization, we recombinantly expressed the entire A2AR C-terminal segment sans the transmembrane portion of the receptor and investigated its solubility and assembly properties with increasing ion concentration and temperature. This is the first study designed to uncover the role of the intrinsically disordered C-terminus on the oligomerization of a GPCR.
RESULTS
This study systematically investigates the role of the C-terminus on A2AR oligomerization and the nature of the interactions involved through strategic mutations and truncations at the C-terminus as well as modulation of the ionic strength of solvent. The experimental assessment of A2AR oligomerization relies on size-exclusion chromatography (SEC) analysis.
Size Exclusion Chromatography Quantifies A2AR Oligomerization
We performed SEC analysis on a mixture of ligand-active A2AR purified from a custom synthesized antagonist affinity column (Fig. S1A). Distinct oligomeric species were separated and eluted in the following order: high-molecular-weight (HMW) oligomer, dimer, and monomer (Fig. 1 and Fig. S1B). The population of each oligomeric species was quantified as the integral of each Gaussian from a multiple-Gaussian curve fit of the SEC signal. The reported standard errors were calculated from the variance of the fit that do not correspond to experimental errors (see Table S1 and Fig. S2 for SEC data corresponding to all A2AR variants in this study). As this study sought to identify the factors that promote A2AR oligomerization, the populations with oligomeric interfaces (i.e., dimer and HMW oligomer) were compared with those without such interfaces (i.e., monomer). Hence, the populations of the HMW oligomer and dimer were expressed relative to the monomer population in arbitrary units as monomer-equivalent concentration ratios, henceforth referred to as population levels (Fig. 1).
C-Terminal Amino Acid Residue C394 Contributes to A2AR Oligomerization
To investigate whether the C-terminus of A2AR is involved in receptor oligomerization, we first examined the role of residue C394, as a previous study demonstrated that the mutation C394S dramatically reduced A2AR oligomer levels(63). The C394S mutation was replicated in our experiments, alongside other amino acid substitutions, namely alanine, leucine, methionine or valine, generating five A2AR-C394X variants. The HMW oligomer and dimer levels of A2AR wild-type (WT) were compared with those of the A2AR-C394X variants. We found that the dimer level of A2AR-WT was significantly higher than that of the A2AR-C394X variants (WT: 1.14; C394X: 0.24–0.57; Fig. 2A). A similar result, though less pronounced, was observed when the HMW oligomer and dimer levels were considered together (WT: 1.34; C394X: 0.59–1.21; Fig. 2A). This suggests that residue C394 plays a role in A2AR oligomerization and more so in A2AR dimers.
To test whether residue C394 stabilizes A2AR dimerization by forming disulfide linkages, we incubated SEC-separated A2AR dimer with 5 mM of the reducing agent TCEP, followed by SDS-PAGE and Western Blotting. The population of each species was determined as the area under the densitometric trace. The dimer level was then expressed as monomer-equivalent concentration ratios in a manner similar to that of the SEC experiment described above. Upon incubation with TCEP, the dimer level of the sample decreased from 1.14 to 0.51 (Fig. 2B). This indicates that disulfide bond formation via residue C394 is one possible mechanism for A2AR dimerization. However, a significant population of A2AR dimer remained resistant to TCEP and C394X mutations (Fig. 2), suggesting that disulfide linkages are not the only driving factor of A2AR oligomer formation. This finding agrees with a previous study showing that residue C394 in A2AR dimer is still available for nitroxide spin labeling,(63) suggesting that additional interfacial sites help drive A2AR dimer/oligomerization.
C-Terminus Truncation Systematically Reduces A2AR Oligomerization
To determine which interfacial sites in the C-terminus other than C394 drive A2AR dimer/oligomerization, we carried out systematic truncations at eight sites along the C-terminus (A316, V334, G344, G349, P354, N359, Q372, and P395), generating eight A2AR-ΔC variants (Fig. 3A). The A2AR-A316ΔC variant corresponds to the removal of the entire disordered C-terminal region as previously performed in all published structural studies(24, 27, 52–59). Using the SEC analysis described earlier (Fig. 1) we evaluated the HMW oligomer and dimer levels of the A2AR-ΔC variants relative to that of the A2AR full-length-wild-type (FL-WT) control. Both the dimer and the total oligomer levels of A2AR decreased progressively with the shortening of the C-terminus, with almost no oligomerization detected upon complete truncation of the C-terminus at site A316 (Fig. 3B). This result shows that the C-terminus drives A2AR oligomerization, with multiple potential interaction sites positioned along much of its length.
Interestingly, there occurred a dramatic decrease in the dimer level between the N359 and P354 truncation sites, from a value of 0.81 to 0.19, respectively (Fig. 3B). A similar result, though less pronounced, was observed on the total oligomer level, with a decrease from 1.09 to 0.62 for the N359 and P354 truncation sites, respectively (Fig. 3B). Clearly, the C-terminal segment encompassing residues 354–359 (highlighted in black in Fig. 3A) is a key constituent of the A2AR oligomeric interface.
Since segment 354–359 contains three consecutive charged residues (355ERR357; Fig. 3A), which could be involved in electrostatic interactions, we hypothesized that this 355ERR357 cluster could strengthen inter-protomer A2AR-A2AR association. To test this hypothesis, residues 355ERR357 were substituted by 355AAA357 on A2AR-FL-WT and A2AR-N359ΔC to generate A2AR-ERR:AAA variants (Fig. 3C). We then compared the HMW oligomer and dimer levels of the resulting variants with controls (same A2AR variants but without the ERR:AAA mutations). We found that the ERR:AAA mutations had varied effects on the dimer level: decreasing for A2AR-FL-WT (ctrl: 0.49; ERR:AAA: 0.29) but increasing for A2AR-N359ΔC (ctrl: 0.33; ERR:AAA: 0.48) (Fig. 3C). In contrast, the ERR:AAA mutations reduced the HMW oligomer level of both A2AR-FL-WT (ctrl: 0.88; ERR:AAA: 0.66) and A2AR-N359ΔC (ctrl: 0.68; ERR:AAA: 0.38) (Fig. 3C). Consistently, the ERR:AAA mutation lowered the total oligomer level of both A2AR-FL-WT (ctrl: 1.37; ERR:AAA: 0.94) and A2AR-N359ΔC (ctrl: 1.01; ERR:AAA: 0.85) (Fig. 3C). These results suggest that the charged residues 355ERR357 participate in A2AR oligomerization, with a greater effect in the context of a longer C-terminus and for higher-order oligomer formation. The question then arises as to what types of interactions are formed along the C-terminus that help stabilize A2AR oligomerization.
C-Terminus Truncation Disrupts Complex Network of Non-Bonded Interactions Necessary for A2AR Dimerization
Given that the structure of A2AR dimers or oligomers are unknown, we next used molecular dynamics (MD) simulations to seek molecular-level insights into the role of the C-terminus in driving A2AR dimerization and to determine the specific interaction types and sites involved in this process. First, to explore A2AR dimeric interface, we performed coarse-grained (CG) MD simulations, which can access the length and time scales relevant to membrane protein oligomerization, albeit at the expense of atomic-level details. We carried out a series of CGMD simulations on five A2AR-ΔC variants designed to mirror the experiments by systematic truncation at five sites along the C-terminus (A316, V334, P354, N359, and C394). Our results revealed that A2AR dimers were formed with multiple interfaces, all involving the C-terminus (Fig. 4A and S3A).
The vast majority of A2AR dimers were symmetric, with the C-termini of the protomers directly interacting with each other. A smaller fraction of the dimers had asymmetric orientations, with the C-terminus of one protomer interacting with other parts of the other protomer, such as ICL2 (the second intracellular loop), ICL3, and ECL2 (the second extracellular loop) (Fig. 4A).
Our observation of multiple A2AR oligomeric interfaces, consistent with previous studies(24, 27), suggests that tunable, non-covalent intermolecular interactions are involved in receptor dimerization. We dissected two key non-covalent interaction types: electrostatic and hydrogen bonding interactions. (The criteria for designating inter-A2AR contacts as electrostatic interactions or hydrogen bonds are described in detail in Materials and Methods.) Electrostatic interactions were calculated from CGMD simulations. Hydrogen bonds were quantified from atomistic MD simulation, given that the CG model merges all hydrogens into a coarse-grained bead and hence cannot report on hydrogen bonds. This analysis was performed on the symmetric dimers as they constituted the majority of the assemblies. With the least truncated A2AR variant containing the longest C-terminus, A2AR-C394ΔC, we observed an average of 15.9 electrostatic contacts (Fig. 4B) and 26.7 hydrogen bonds (Fig. 4C) between the C-termini of the protomers. This result shows that both electrostatic interactions and hydrogen bonds play important roles in A2AR dimer formation.
Upon further C-terminus truncation, the average number of both electrostatic contacts and hydrogen bonds involving C-terminal residues progressively declined, respectively reaching 5.4 and 6.0 for A2AR-A316ΔC (in which the disordered region of the C-terminus is removed) (Fig. 4B and 4C). This result is consistent with the experimental result, which demonstrated a progressive decrease of A2AR oligomerization with the shortening of the C-terminus (Fig. 3B). Interestingly, upon systematic truncation of the C-terminal segment 335–394, we observed in segment 291–334 a steady decrease in the average number of electrostatic contacts, from 10.4 to 7.4 (Fig. 4B). This trend was even more pronounced with hydrogen bonding contacts involving segment 291–334 decreasing drastically from 21.0 to 7.0 as segment 335–394 was gradually removed (Fig. 4C). This observation, namely that truncation of a C-terminal segment reduces inter-A2AR contacts elsewhere along the C-terminus, indicates that a cooperative mechanism of dimerization exists, in which an extended C-terminus of A2AR stabilizes inter-A2AR interactions near the heptahelical bundles of the dimeric complex. Besides the intermolecular interactions, we also identified a network of intramolecular salt bridges involving residues on the C-termini, including cluster 355ERR357 (Fig. 7A). These results demonstrate that A2AR dimers can be formed via multiple interfaces predominantly in symmetric orientations, facilitated a cooperative network of electrostatic interactions and hydrogen bonds along much of its C-terminus.
Ionic Strength Modulates Oligomerization of C-Terminally Truncated A2AR Variants
So far, we have demonstrated that the C-terminus clearly plays a role in forming A2AR oligomeric interfaces. However, the driving factors of A2AR oligomerization remain unknown. The variable nature of A2AR oligomeric interfaces suggests that the main driving forces must be non-covalent interactions, such as electrostatic interactions and hydrogen bonds as identified by the above MD simulations. Modulating the solvent ionic strength is an effective method to identify the types of non-covalent interaction(s) at play. Specifically, with increasing ionic strength, electrostatic interactions can be weakened (based on Debye-Hückel theory, most electrostatic bonds at a distance greater than 5 Å are screened out at an ionic strength of 0.34 M at 4°C), depletion interactions are enhanced with salting-out salts, and hydrogen bonds remain relatively impervious. For this reason, we subjected various A2AR variants (FL-WT, FL-ERR:AAA, N359ΔC, and V334ΔC) to ionic strength ranging from 0.15 to 0.95 M by adding NaCl (buffer composition shown in Table 1). The HMW oligomer and dimer levels of the four A2AR variants were determined and plotted as a function of ionic strengths.
The low ionic strength of 0.15 M should not affect hydrogen bonds or electrostatic interactions, if present. We found that the dimer and total oligomer levels of all four variants were near zero (Fig. 5). This is a striking observation, as it already excludes electrostatic and hydrogen-bonding interactions as the dominant force for A2AR association. The question remains whether depletion interactions could be involved.
At higher ionic strengths of 0.45 M and 0.95 M, the dimer and total oligomer levels of A2AR-V334ΔC still remained near zero (Fig. 5). In contrast, we observed a progressive and significant increase in the dimer and total oligomer levels of A2AR-FL-WT with increasing ionic strength (Fig. 5). This result indicates A2AR oligomerization must be driven by depletion interactions, which are enhanced with increasing ionic strength, and that these interactions involve the C-terminal segment after residue V334.
Upon closer examination, we recognize that at the very high ionic strength of 0.95 M, the increase in the dimer and total oligomer levels was robust for A2AR-FL-WT, but less pronounced for A2AR-FL-ERR:AAA (Fig. 5). Furthermore, this high ionic strength even had an opposite effect on A2AR-N359ΔC, with both its dimer and total oligomer levels abolished (Fig. 5). These results indicate that the charged cluster 355ERR357 and the C-terminal segment after residue N359 are required for depletion interactions to promote A2AR oligomerization to the full extent.
Taken together, we demonstrated that A2AR oligomerization is more robust when the C-terminus is fully present and the ionic strength is higher, suggesting that depletion interactions via the C-terminus are a strong driving factor of A2AR oligomerization. The question then arises whether such depletion interactions are the result of the C-termini directly interacting with one another, necessitating an experiment that investigates the behavior of A2AR C-terminus sans the transmembrane domains.
The Isolated A2AR C-Terminus Is Prone to Aggregation
To test whether A2AR oligomerization is driven by direct depletion interactions among the C-termini of the protomers, we assayed the solubility and assembly properties of the stand-alone A2AR C-terminus—an intrinsically disordered peptide—sans the upstream transmembrane regions. Since depletion interactions can be manifested via the hydrophobic effect(48), we examined whether this effect can cause A2AR C-terminal peptides to associate.
It is an active debate(67) whether the hydrophobic effect can be promoted or suppressed by ions with salting-out or salting-in tendency, respectively(68–70). We increased the solvent ionic strength using either sodium (salting-out) or guanidinium (salting-in) ions and assessed the aggregation propensity of the C-terminal peptides using UV-Vis absorption at 450 nm. We first observed the behavior of the C-terminus with increasing salting-out NaCl concentrations. At NaCl concentrations below 1 M, the peptide was dominantly monomeric, despite showing slight aggregation at NaCl concentrations between 250–500 mM (Fig. 6A). At NaCl concentrations above 1 M, A2AR C-terminal peptides strongly associated into insoluble aggregates (Fig. 6A). Consistent with the observations made with the intact receptor (Fig. 5), A2AR C-terminus showed the tendency to progressively precipitate with increasing ionic strengths, suggesting that depletion interactions drive the association and precipitation of the peptides. We next observed the behavior of the C-terminus with increasing concentrations of guanidine hydrochloride (GdnHCl), which contains salting-in cations that do not cause proteins to precipitate and instead facilitate the solubilization of proteins(71, 72). Our results demonstrated that the A2AR C-terminus incubated in 4 M GdnHCl showed no aggregation propensity (Fig. 6A), validating our expectation that depletion interactions are not enhanced by salting-out salts. These observations demonstrate that the C-terminal peptide in and of itself can directly interact with other C-terminal peptides to form self-aggregates in the presence of ions, and presumably solutes, that have salting-out effects.
Attractive hydrophobic interactions among the hydrophobic residues are further enhanced by water solvating the protein having more favorable interactions with other water molecules, ions or solutes than with the protein, here the truncated C-terminus(73–75). We explored the possible contribution of hydrophobic interactions to the aggregation of the C-terminal peptides using differential scanning fluorimetry (DSF). In particular, we gradually increased the temperature to melt the C-terminal peptides, exposing any previously buried hydrophobic residues (Fig. S4A) which then bound to the SYPRO orange fluorophore, resulting in an increase in fluorescence signal. Our results showed that as the temperature increased, a steady rise in fluorescence was observed (Fig. 6B), indicating that multiple hydrophobic residues were gradually exposed to the SYPRO dye. However, at approximately 65°C, the melt peak signal was abruptly quenched (Fig. 6B), indicating that the hydrophobic residues were no longer exposed to the dye. This observation suggests that, at 65°C, enough hydrophobic residues in the C-terminal peptides were exposed such that they collapsed on one another (thus expelling the bound dye molecules), resulting in aggregation. Clearly, the hydrophobic effect can cause A2AR C-terminal peptides to directly associate. These results demonstrate that A2AR oligomer formation can be driven by depletion interactions among the C-termini of the protomers.
DISCUSSION
The key finding of this study is that the C-terminus of A2AR, removed in all previously published structural studies of this receptor, is directly responsible for receptor oligomerization. Using a combination of experimental and computational approaches, we demonstrate that the C-terminus drives A2AR oligomerization via a combination of disulfide linkages, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. This diverse combination of interactions is greatly enhanced by depletion interactions, forming a network of malleable bonds that give rise to the existence of multiple A2AR oligomeric interfaces.
The intermolecular disulfide linkages associated with residue C394 play a role in A2AR oligomerization. However, it is unclear which cysteine on the second protomer is linked to this cysteine. A previous study showed that residue C394 in A2AR dimer is available for nitroxide spin labeling(63), suggesting that some of these disulfide bonds may be between residue C394 and another cysteine in the hydrophobic core of A2AR that do not form intramolecular disulfide bonds(76–78). Many examples exist where disulfide linkages help drive GPCR oligomerization, including the CaR-mGluR1 heterodimer(79), homodimers of mGluR5(80), M3R(81), V2R(82), 5-HT4R(83) and 5-HT1DR(84), and even higher-order oligomers of D2R(85). However, although unconventional cytoplasmic disulfide bonds have been reported(86, 87), no study has shown how such linkages would be formed in vivo, as the cytoplasm lacks the conditions and machinery required for disulfide bond formation(88–91). Nevertheless, residue C394 is highly conserved and a C-terminal cysteine is almost always present among A2AR homologs(92), suggesting that this cysteine cannot be excluded for serving an important role in vivo.
The electrostatic interactions that stabilize A2AR oligomer formation come from multiple sites along the C-terminus. From a representative snapshot of a A2AR-C394ΔC dimer from our MD simulations (Fig. 7A), we could visualize not only the intermolecular interactions calculated from the CGMD simulations (Fig. 4B), but also intramolecular salt bridges. In particular, the 355ERR357 cluster of charged residues lies distal from the dimeric interface, yet still forms several salt bridges (Fig. 7A, inset). This observation is supported by our experimental results showing that substituting this charged cluster with alanines reduces the total A2AR oligomer levels (Fig. 3C). However, it is unclear how such salt bridges involving this 355ERR357 cluster are enhanced by depletion interactions (Fig. 5), as electrostatic interactions are usually screened out at high ionic strengths. In our MD simulations, we also observed networks of salt bridges along the dimeric interface, for example between K315 of one monomer and D382 and E384 of the other monomer (Fig. 7A, inset). The innate flexibility of the C-terminus could facilitate the formation of such salt bridges, which then acts as a potential scaffold to stabilize A2AR dimers.
We also found that depletion interactions can enhance the diversity of interactions that stabilize A2AR oligomer formation (Fig. 5 and 6). Depletion interactions could be the key factor to the cooperative mechanism by which A2AR oligomerization occurs. As revealed by our MD simulations, an increasing number of contacts are formed along segment 291–334 when the rest of C-terminus is present (Fig. 4B and 4C). As more of the C-terminus is preserved, the greater extent of depletion interactions limits the available dimer arrangements, forcing segment 291–334 into an orientation that optimizes intermolecular interactions.
Our finding that A2AR forms homo-oligomers via multiple interfaces (Fig. 4A) agrees with the increasing number of studies reporting multiple and interconverting oligomeric interfaces in A2AR and other GPCRs(24–36). When translated to in vivo situations, GPCR oligomers can also transiently associate and dissociate(93–96). Such fast conformational changes require that the oligomeric interfaces be formed by interactions that can easily be modulated. This is consistent with our study, which demonstrates that depletion interactions via the intrinsically disordered, malleable C-terminus drive A2AR oligomerization. Because depletion interactions can be readily tuned by environmental factors, such as ionic strength, molecular crowding, and temperature, the formation of GPCR oligomeric complexes could be dynamically modulated in response to environmental cues to regulate receptor function.
Not only did we find multiple A2AR oligomeric interfaces, we also found that these interfaces can be either symmetric or asymmetric. This finding is supported by a growing body of evidence that there exists both symmetric and asymmetric oligomeric interfaces for A2AR(24) and many other GPCRs. Studies using various biochemical and biophysical techniques have shown that heterotetrameric GPCR complexes can be formed by dimers of dimers, including μOR-δOR(97), CXC4R-CC2R(98), CB1R/D2R(99) as well as those involving A2AR, such as A1R-A2AR(61, 100) and A2AR-D2R(101). The quaternary structures identified in these studies required specific orientations of each protomer, with the most viable model involving a stagger of homodimers with symmetric interfaces(102). On the other hand, since symmetric interfaces limit the degree of receptor association to dimers, the HMW oligomer of A2AR observed in this(24) and other studies(63, 103) can only be formed via asymmetric interfaces. It is indeed tempting to suggest that the formation of the HMW oligomer of A2AR may even arise from combinations of different interfaces. In any case, the wide variation of GPCR oligomerization requires the existence of both symmetric and asymmetric oligomeric interfaces.
In the case of A2AR, displacement of the transmembrane domains have been demonstrated to be the hallmark of receptor activation(104–107). However, no studies have linked receptor oligomerization with the arrangement of the TM bundles in A2AR. Our MD simulations revealed that C-terminus truncation resulted in structural changes in the heptahelical bundles of A2AR dimers. Specifically, as more of the C-terminus was preserved, we observed a progressive increase in the helical tilt of TM7 (Fig. 7B). This change in helical tilt occurred for the entire heptahelical bundle, with an increase in tilt for TM1, TM2, TM3, TM5, and TM7, and a decrease in tilt for TM4 and TM6 (Fig. S3). The longer C-terminus in the full-length A2AR permits greater rearrangements in the transmembrane regions, leading to the observed change in helical tilt. This result hints at potential conformational changes of A2AR upon oligomerization, necessitating future investigation on functional consequences.
C-terminal truncations prior to crystallization and structural studies may be the main reason for the scarcity of GPCR structures featuring oligomers. In that context, this study offers valuable insights and approaches to tune the oligomerization of A2AR and potentially of other GPCRs using its intrinsically disordered C-terminus. The presence of A2AR oligomeric populations with partial C-terminal truncations means that one can now study its oligomerization with less perturbation from the C-terminus. We also present evidence that the multiple C-terminal interactions that drive A2AR oligomerization can be easily modulated by ionic strength and specific salts (Fig. 5 and 6). Given that ~75% and ~15% of all class-A GPCRs possess a C-terminus of > 50 and > 100 amino acid residues(108), respectively, it will be worthwhile to explore the prospect of tuning GPCR oligomerization not only by shortening the C-terminus but also with simpler approaches such as modulating ionic strength and the surrounding salt environment.
CONCLUSION
This study emphasizes for the first time the definite impact of the C-terminus on A2AR oligomerization, which can be extended to include the oligomers formed by other GPCRs with a protracted C-terminus. We have shown that the oligomerization of A2AR is strongly driven by depletion interactions along the C-terminus, further modulating and enhancing the multiple interfaces formed via a combination of hydrogen, electrostatic, hydrophobic, and covalent disulfide interactions. The task remains to link A2AR oligomerization to functional roles of the receptor(109). From a structural biology standpoint, visualizing the multiple oligomeric interfaces of A2AR in the presence of the full-length C-terminus is key to investigating whether these interfaces give rise to different oligomer functions.
MATERIALS AND METHODS
Cloning, Gene Expression, and Protein Purification
The multi-integrating pITy plasmid(110), previously used for overexpression of A2AR in Saccharomyces cerevisiae(111), was employed in this study. pITy contains a Gal1–10 promoter for galactose-induced expression, a synthetic pre-pro leader sequence which directs protein trafficking(112, 113), and the yeast alpha terminator. The genes encoding A2AR variants with 10-His C-terminal tag were cloned into pITy downstream of the pre-pro leader sequence, using either splice overlapping extension(114) or USER cloning using X7 polymerase(115, 116), with primers provided in Table S3. The plasmids were then transformed into S. cerevisiae strain BJ5464 (MATα ura3-52 trp1 leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) (provided by the lab of Anne Robinson at Carnegie Mellon University) using the lithium-acetate/PEG method(117). Transformants were selected on YPD G-418 plates (1% yeast extract, 2% peptone, 2% dextrose, 2.0 mg/mL G-418).
Receptor was expressed and purified following the previously described protocol(118). In brief, from freshly streaked YPD plates (1% yeast extract, 2% peptone, 2% dextrose), single colonies were grown in 5-mL YPD cultures over night at 30°C. From these 5-mL cultures, 50-mL cultures were grown with a starting OD of 0.5 over night at 30°C. To induce expression, yeast cells from these 50-mL cultures were centrifuged at 3,000 x g to remove YPD before resuspended in YPG medium (1% yeast, 2% peptone, 2% D-galactose) at a starting OD of 0.5. The receptor was expressed for 24 hours over night at 30°C with 250 r.p.m shaking. Cells were pelleted by centrifugation at 3,000 x g, washed in sterile PBS buffer, and pelleted again before storage at – 80°C until purification.
Mechanical bead lysis of cells was done, per 250 mL of cell culture, by performing 12 pulses of 60 s intense vortexing (with at least 60 s of rest in between pulses) in 10 mL 0.5-mm zirconia silica beads (BioSpec, Bartlesville, OK, USA; #11079105z), 25 mL of lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10% (v/v) glycerol, pH = 8.0, 2% (w/v) n-Dodecyl-β-D-maltopyranoside (DDM; Anatrace, Maumee, OH, USA; #D310), 1% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Anatrace; #C216), and 0.2% (w/v) cholesteryl hemisuccinate (CHS; Anatrace; #CH210) and an appropriate amount of 100x Pierce Halt EDTA-free protease inhibitor (Pierce, Rockford, IL, USA #78439)). Beads were separated using a Kontex column. Unlysed cells were removed by centrifugation at 3,220 x g for 10 min. Receptor was let solubilized on rotary mixer for 3 hours before cell debris was removed by centrifugation at 10,000 x g for 30 min. Solubilized protein was incubated with Ni-NTA resin (Pierce; #88221) over night. Protein-resin mixture was then washed extensively in purification buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10% (v/v) glycerol, 0.1% (w/v) DDM, 0.1% (w/v) CHAPS and 0.2% (w/v) CHS, pH = 8.0) containing low imidazole concentrations (20– 50 mM). A2AR was eluted into purification buffer containing 500 mM imidazole. Prior to further chromatographic purification, imidazole was removed using a PD-10 desalting column (GE Healthcare, Pittsburgh, PA, USA; # 17085101).
Ligand affinity resin was prepared as previously described for purification of active A2AR.(119) (120) In brief, 8 mL of isopropanol-washed Affigel 10 resin (BioRad; # 1536099) was mixed gently in an Erlenmeyer flask for 20 h at room temperature with 48 mL of DMSO containing 24 mg of xanthine amine congener (XAC, high-affinity A2AR antagonist, KD = 32 nM; Sigma, St. Louis, MO, USA; #X103). The absorbance at 310 nm of the XAC-DMSO solution before and after the coupling reaction was measured in 10 mM HCl and compared to a standard curve. The amount of resin bound to ligand was estimated to be 5.6 μM. The coupling reaction was quenched by washing the resin with DMSO, then with Tris-HCl 50 mM (pH = 7.4), then with 20% (v/v) ethanol. The resin was packed into a Tricorn 10/50 column (GE Healthcare) under pressure via a BioRad Duoflow FPLC (BioRad).
For purification of active A2AR, the column was equilibrated with 4 CV of purification buffer. The IMAC-purified A2AR was desalted and diluted to 5.5 mL before applied to a 5-mL sample loop on the BioRad Duoflow FPLC, from which the sample was loaded onto the column at a rate of 0.1 mL/min. Inactive A2AR was washed from the column by flowing 10 mL of purification buffer at 0.2 mL/min, followed by 16 mL at 0.4 mL/min. Active A2AR was eluted from the column by flowing purification buffer containing 20 mM theophylline (low-affinity A2AR antagonist, KD = 1.6 μM; Sigma, St. Louis, MO, USA; #T1633). Western blot analysis was performed to determine 4-mL fractions with active A2AR collected with a BioFrac fraction collector (BioRad; Hercules, CA, USA), which were then concentrated through a 30-kDa MWCO centrifugal filter (Millipore, Billerica, MA, USA; # UFC803096) and desalted to remove excess theophylline. For the experiments where the salt concentrations were varied, the buffer exchange was done also by this last desalting step.
Size-Exclusion Chromatography
To separate oligomeric species of active A2AR, a prepacked Tricorn Superdex 200 10/300 GL column (GE Healthcare) connected to a BioRad Duoflow FPLC was equilibrated with 60 mL of running buffer (150 mM sodium chloride, 50 mM sodium phosphate, 10% (v/v) glycerol, 0.1% (w/v) DDM, 0.1% (w/v) CHAPS, 0.02% (w/v) CHS, pH = 8.0) at a flow rate of 0.2 mL/min. 0.5-mL fractions were collected with a BioFrac fraction collector in 30 mL of running buffer at the same flow rate. Analysis of SDS/PAGE and western blot was done to determine oligomeric states of the eluted A2AR.
SEC Peak Analysis
SEC chromatograms were analyzed using OriginLab using the nonlinear curve fit (Gaussian) function. The area under the curve and the peak width were manually defined in cases where the SNR of the SEC trace were too low. The R2 values reached > 0.96 for most cases. The population of each oligomeric species was expressed as the integral of each Gaussian this curve fit of the SEC signal. The HMW oligomer peak in some cases could not be fitted with one curve and thus was fitted with two curves instead. The reported standard errors were calculated from the variance of the fit and did not correspond to experimental errors. The results are detailed in Fig. S2 and Table S1.
SDS-PAGE and Western Blotting
10% SDS-PAGE gels were hand-casted in BioRad Criterion empty cassettes (BioRad; #3459902, 3459903). Lysate controls were prepared by lysis of 5 OD cell pellets with 35 μL of YPER (Fisher Scientific, Waltham, MA, USA # 8990) at RT for 20 min, incubation with 2x Laemmli buffer (4% (w/v) SDS, 16% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 167 M Tris, pH 6.8) at 37°C for 1 h, and centrifugation at 3,000 x g for 1 min to pellet cell debris. Protein samples were prepared by incubation with 2x Laemmli buffer at 37°C for 30 min. For all samples, 14 μL (for 26-well gel) or 20 μL (for 18-well gel) was loaded per lane, except for 7 μL of Magic Mark XP Western protein ladder (Thermo Scientific, Waltham, MA, USA; # LC5602) as a standard. Electrophoresis was carried out at 120 V for 100 min. Proteins were transferred to 0.2-μm nitrocellulose membranes (BioRad; # 170-4159) via electroblotting using a BioRad Transblot Turbo, mixed MW protocol. Membranes were blocked in Tris-buffered saline with Tween (TBST; 150 mM sodium chloride, 15.2 mM Tris-HCl, 4.6 mM Tris base, pH = 7.4, 0.1% (v/v) Tween 20 (BioRad; # 1706531)) containing 5% (w/v) dry milk, then probed with anti-A2AR antibody, clone 7F6-G5-A2 (Millipore, Burlington, MA, USA; # 05-717) at 1:500 in TBST with 0.5% (w/v) dry milk. Probing with secondary antibody was done with a fluorescent DyLight 550 antibody (Abcam, Cambridge, MA, USA; ab96880) at 1:600 in TBST containing 0.5% (w/v) milk.
Western blot was analyzed with Fiji. The Gels analysis plugin was used to define each sample lane, and to generate an intensity profile. Peaks were manually selected and integrated with the measure tool to determine the amount of protein present.
Coarse-Grained MD Simulations
Initial configuration of A2AR was based on the crystal structure of the receptor in the active state (PDB 5G53). All non-receptor components were removed, and missing residues added using MODELLER 9.23(121). Default protonation states of ionizable residues were used. The resulting structure was converted to MARTINI coarse-grained topology using the martinize.py script(122). The ELNeDyn elastic network(123) was used to constrain protein secondary and tertiary structures with a force constant of 500 kJ/mol/nm2 and a cutoff of 1.5 nm. To optimize loop refinement of the model, a single copy was embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer using the insane.py script, solvated with MARTINI polarizable water, neutralized with 0.15 M NaCl, and a short MD (1.5 μs) run to equilibrate the loop regions. Subsequently, two monomers of the equilibrated A2AR were randomly rotated and placed at the center of a 13 nm × 13 nm × 11 nm (xyz) box, 3.5 nm apart, with their principal transmembrane axis aligned parallel to the z axis. The proteins were then embedded in a POPC bilayer using the insane.py script. Sodium and chloride ions were added to neutralize the system and obtain a concentration of 0.15 M NaCl. Total system size was typically in the range of 34,000 CG particles, with a 280:1 lipid:protein ratio. Ten independent copies were generated for each A2AR truncated variant.
v2.2 of the MARTINI coarse-grained force field(124) was used for the protein and water, and v2.0 was used for POPC. All coarse-grained simulations were carried out in GROMACS 2016(125) in the NPT ensemble (P = 1 atm, T = 310 K). The Bussi velocity rescaling thermostat was used for temperature control with a coupling constant of τt = 1.0 ps(126), while the Parrinello-Rahman barostat(127) was used to control the pressure semi-isotropically with a coupling constant of τt = 12.0 ps and compressibility of 3 × 10–4 bar–1. Reaction field electrostatics was used with Coulomb cut-off of 1.1 nm. Non-bonded Lennard-Jones interactions were treated with a cut-off of 1.1 nm. All simulations were run with a 15 fs timestep, updating neighbor lists every 10 steps. Cubic periodic boundary conditions along the x, y and z axes were used. Each simulation was run for 8 μs.
Atomistic MD Simulations
Three snapshots of symmetric dimers of A2AR for each respective truncated variant were randomly selected from the CG simulations as starting structures for backmapping. Coarse-grained systems were converted to atomistic resolution using the backward.py script(128). All simulations were run in Gromacs2019 in the NPT ensemble (P = 1 bar, T = 310 K) with all bonds restrained using the LINCS method(129). The Parrinello-Rahman barostat was used to control the pressure semi-isotropically with a coupling constant of τt = 1.0 ps and a compressibility of 4.5 × 10–5 bar–1, while the Bussi velocity rescaling thermostat was used for temperature control with a coupling constant of τt = 0.1 ps. Proteins, lipids, and solvents were separately coupled to the thermostat. The CHARMM36 and TIP3P force fields(130, 131) were used to model all molecular interactions. Periodic boundary conditions were set in the x, y, and z directions. Particle mesh Ewald (PME) electrostatics was used with a cut-off of 1.0 nm. A 2-fs time step was used for all atomistic runs, and each simulation was run for 50 ns.
Analysis of Computational Results
All trajectories were post-processed using gromacs tools and in-house scripts. We ran a clustering analysis of all dimer frames from the CG simulations using Daura et. al.’s clustering algorithm(132) implemented in GROMACS, with an RMSD cutoff of 1.5 Å. (An interface was considered dimeric if the minimum center of mass distance between the protomers was less than 5 Å.) This method uses an RMSD cutoff to group all conformations with the largest number of neighbors into a cluster and eliminates these from the pool, then repeats the process until the pool is empty. We focused our analysis on the most populated cluster from each truncated variant. Electrostatic interactions in the dimer were calculated from CG systems with LOOS(133) using a distance cutoff of 5.0 Å.
Transmembrane helical tilt angles were also calculated in LOOS from CG simulations. Hydrogen bonds were calculated from AA simulations using the hydrogen bonds plugin in VMD(134), with a distance cutoff of 3.5 Å and an angle cutoff of 20°. Only C-terminal residues were included in hydrogen bond analysis. PyMOL(135) was used for molecular visualizations.
Assessing A2AR Oligomerization with Increasing Ionic Strength
Na2HPO4 and NaH2PO4 in the buffer make up an ionic strength of 0.15 M, to which NaCl was added to increase the ionic strength to 0.45 M and furthermore to 0.95 M. The A2AR variants were purified at 0.45 M ionic strength and then exchanged into buffers of different ionic strengths using a PD-10 desalting column prior to subjecting the samples to SEC. The buffer composition is detailed below.
Isolated C-Terminus Purification
Escherichia coli BL21 (DE3) cells were transfected with pET28a DNA plasmids containing the desired A2AR sequence with a 6x His tag attached for purification. Cells from glycerol stock were grown in 10 mL luria broth (LB, Sigma Aldrich, L3022) overnight at 37°C and then used to inoculate 1 L of fresh LB and 10 μg/mL kanamycin (Fisher Scientific, BP906). Growth of cells were performed at 37°C, 200 rpm until optical density at λ = 600 nm reached 0.6–0.8. Expression was induced by incubation with 1 mM isopropyl-β-D-thiogalactoside (Fisher Bioreagents, BP175510) for 3 hrs.
Cells were harvested with centrifugation at 5000 rpm for 30 min. Harvested cells were resuspended in 25 mL Tris-HCl, pH = 7.4, 100 mM NaCl, 0.5 mM DTT, 0.1 mM EDTA with 1 Pierce protease inhibitor tablet (Thermo Scientific, A32965), 1 mM PMSF, 2 mg/mL lysozyme, 20 μg/mL DNase (Sigma, DN25) and 10 mM MgCl2, and incubated on ice for 30 min. Samples were then incubated at 30°C for 20 minutes, then flash frozen and thawed 3 times in LN2. Samples were then centrifuged at 10,000 rpm for 10 min to remove cell debris. 1 mM PMSF was added again and the resulting supernatant was incubated while rotating for at least 4 hrs with Ni-NTA resin. The resin was loaded to a column and washed with 25 mL 20 mM sodium phosphate, pH = 7.0, 1 M NaCl, 20 mM imidazole, 0.5 mM DTT, 100 μM EDTA. Purified protein was eluted with 15 mL of 20 mM sodium phosphate, pH = 7.0, 0.5 mM DTT, 100 mM NaCl, 300 mM imidazole. The protein was concentrated to a volume of 2.5mL and was buffer exchanged into 20 mM ammonium acetate buffer, pH = 7.4, 100 mM NaCl using a GE PD-10 desalting column. Purity of sample was confirmed with SDS-PAGE and western blot.
Aggregation Assay to Assess A2AR C-Terminus Assembly
Absorbance was measured at 450 nm using a Shimadzu UV-1601 spectrophotometer with 120 μL sample size. Prior to reading, samples were incubated at 40°C for 5 minutes. Samples were vigorously pipetted to homogenize any precipitate before absorbance was measured. Protein concentration was 50 μM in a 20 mM ammonium acetate buffer (pH = 7.4).
Differential Scanning Fluorimetry (DSF)
DSF was conducted with a Bio-rad CFX90 real-time PCR machine. A starting temperature 20°C was increased at a rate of 0.5°C per 30 seconds to a final temperature of 85°C. All samples contained 40 μL of 40 μM A2AR C-terminus, 9x SYPRO orange (ThermoFisher S6650), 200 mM NaCl, and 20 mM MES. Fluorescence was detected in real-time at 570 nm. All samples were conducted in triplicate.
Hydrophobicity and Charge Profile of C-Terminus
The hydrophobicity profile reported in Fig. S4 was determined with ProtScale using method described by Kyte & Doolittle(136), window size of 3.
FUNDING AND ACKNOWLEDGMENTS
This material is based upon work supported by (1) the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM136411, (2) the National Institute of Mental Health of the National Institutes of Health under Small Business Innovation Research Award Number 1R43MH119906-01, and (3) the National Science Foundation under Award Number MCB-1714888 (E.S. and B.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Many of the experiments were completed with the assistance from Rohan Katpally. The pITy expression vector and S. cerevisiae BJ5464 strain were generously provided by Prof. Anne Robinson’s lab at Carnegie Mellon University. The X7 polymerase was a gift from Dr. Morten Nørholm, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark. Computational time was provided through WVU Research Computing and XSEDE allocation no. TG-MCB130040.
Footnotes
Classifications: Biological Sciences – Biophysics and Computational Biology
SIGNIFICANCE G protein-coupled receptors (GPCRs) are important drug targets in medicine. While it is widely known that these receptors can form oligomers with unique functional consequences, the driving factor of receptor oligomerization remains unclear. The intrinsically disordered C-terminus of GPCRs is often thought to play no major role in receptor function and is thus usually removed to simplify biophysical studies. Using the human adenosine A2A receptor as a model GPCR, we find instead that its C-terminus drives oligomer formation via an intricate network of interactions. This finding suggests that the distinct properties associated with GPCR oligomerization may prevail only when the C-terminus is present.
REFERENCES
- 1.↵
- 2.↵
- 3.↵
- 4.↵
- 5.↵
- 6.
- 7.
- 8.
- 9.↵
- 10.↵
- 11.
- 12.
- 13.↵
- 14.↵
- 15.
- 16.↵
- 17.↵
- 18.↵
- 19.
- 20.
- 21.
- 22.
- 23.↵
- 24.↵
- 25.
- 26.
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.
- 39.↵
- 40.↵
- 41.
- 42.
- 43.
- 44.
- 45.
- 46.
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.
- 54.
- 55.
- 56.
- 57.
- 58.
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.
- 75.↵
- 76.↵
- 77.
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵
- 88.↵
- 89.
- 90.
- 91.↵
- 92.↵
- 93.↵
- 94.
- 95.
- 96.↵
- 97.↵
- 98.↵
- 99.↵
- 100.↵
- 101.↵
- 102.↵
- 103.↵
- 104.↵
- 105.
- 106.
- 107.↵
- 108.↵
- 109.↵
- 110.↵
- 111.↵
- 112.↵
- 113.↵
- 114.↵
- 115.↵
- 116.↵
- 117.↵
- 118.↵
- 119.↵
- 120.↵
- 121.↵
- 122.↵
- 123.↵
- 124.↵
- 125.↵
- 126.↵
- 127.↵
- 128.↵
- 129.↵
- 130.↵
- 131.↵
- 132.↵
- 133.↵
- 134.↵
- 135.↵
- 136.↵