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Extracellular ligand binding to G protein-coupled receptors (GPCRs) modulates G-protein and β-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific 19F-NMR labels in the β2-adrenergic receptor (β2AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium towards the G protein specific active state of helix VI. In contrast, β-arrestin-biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of β2AR in partial and biased agonist signaling.
Signaling through G protein-coupled receptors (GPCRs) is a fundamental component of eukaryotic cellular communication. As a result, GPCRs comprise a large fraction of the “druggable proteome”, with more than 30% of available pharmaceuticals targeting GPCRs (1). In performing their functions, GPCRs such as the β2-adrenergic receptor (β2AR) recognize a diverse array of ligands and transmit signals through the cellular membrane to cytoplasmic G-proteins. In parallel to G-protein signaling, activated β2AR can be phosphorylated and binds to β-arrestin, initiating desensitization, endocytosis of β2AR and β-arrestin-dependent signaling (2). A number of β2AR ligands, including the FDA-approved β-blocker carvedilol (3) and the agonist isoetharine (4), have been shown to impart differing degrees of signaling in G-protein and arrestin pathways, a phenomenon called “functional selectivity” or “biased signaling” (5). Understanding the mechanism of biased signaling can provide leads for designing more specific and efficient drugs.
Crystal structures have been determined for β2AR with bound inverse agonists, antagonists, and agonists (6–10), including active-state β2AR complexes with a G-protein mimetic nanobody (8) and with a heterotrimeric G-protein (11). The β2AR–G-protein structure, which has also been analyzed by EM (12), reveals the location of the G-protein binding site, showing that the G-protein interacts with helices V and VI, and the intracellular loops ICL2 and ICL3, but makes no substantial contacts with helices VII and VIII (Fig. 1A,B). A comparison of inactive and active-state crystal structures of β2AR, rhodopsin (13) and the A2A adenosine receptor (14, 15) suggest a common pattern of structural differences between inactive and active states in GPCRs (Fig.1C), where a coupled motion of helices V and VI, and changes in helices III and VII are accompanied by distinct side chain rotamer switches on the cytoplasmic side of the protein (16, 17). Activation of β2AR has been investigated previously via fluorescence-based biophysical experiments (18–25) and NMR (26), and conformational changes during receptor activation were observed in helix VI and the cytoplasmic surface.
In order to probe for subtle conformational changes involved in receptor–ligand binding (27), 19F-NMR spectroscopy was employed. This highly sensitive method was used to provide information based on the observation of line shapes and chemical shifts of strategically located 19F-labels in the cytoplasmic region of the receptor (Fig. 1A, B) (28). Similar approaches have previously been successfully applied to studies of a number of membrane proteins (29–31). Three native cysteine residues (Cys2656.27, Cys3277.54, Cys341) (32) of β2AR (33) in the cytoplasmic region of the receptor are accessible for covalent labeling with 2,2,2 trifluoroethanethiol (TET) (31), and their NMR signals were unambiguously assigned through the use of site-specific mutagenesis of these residues and all other native cysteine residues that might be affected by TET (Fig. 2A) (34). To probe for structural changes, we primarily used singly-labeled receptors, which have reduced complexity of the 19F-NMR spectra when compared with wild type TETβ2AR (Fig. 2A). Cys2656.27 and Cys3277.54 are located at the cytoplasmic ends of helices VI and VII, respectively, both of which are known to undergo large conformational changes during receptor activation (Fig. 1C). In contrast, Cys341 is located in the non-transmembrane helix VIII at the C-terminus and β2AR(C265A, C327S, TETC341) was observed to be unresponsive to binding of different ligands (Fig. 2B). This C-terminal probe therefore served as an internal negative control relative to Cys2656.27 and Cys3277.54 in studies of the effects from the binding of different pharmacological ligands.
The 19F-NMR spectra ofβ2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) (Fig. 2C–E) show prominent changes upon addition of saturating concentrations of a collection of ligands (Table S1) which represent a full spectrum of ligand efficacy (35). In general, the signals at 280K of both β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC341A, C327) consist of two components, suggesting that the β2AR structure contains two independent equilibria between locally different conformations manifested at these two amino acid positions. While ligand-free and inverse agonist-bound TETβ2ARs have almost identical spectra, in which the populations of C327I and C265I are much more prominent than those of C327A and C265A, spectra of TETβ2AR bound to the full agonist isoproterenol exhibit approximately equal populations at both residues (Fig. 3A). Shifts of this equilibrium are observed with different agonists bound to TETβ2AR, suggesting that the peaks A and I are likely to represent the active and inactive state of both helices VI and VII of β2AR, respectively. The temperature dependence of the 19F-signals with regard to line width and peak intensity (Figs. 2C–E, S4) suggests that the active state of both helices VI and VII of β2AR exhibits a larger degree of conformational plasticity than the inactive state, i.e., the active state samples a wider range of conformers with slightly different chemical shifts, either in a static ensemble or in a manifold of conformers that also includes conformational exchange.
The 19F-NMR data were analyzed with a double-Lorentzian function to obtain quantitative information on the conformational equilibria (Fig. 3A). This revealed that positions and widths of the NMR peaks remain practically unchanged for all ligands studied, and confirmed that the major effects of agonist binding are population shifts from inactive state peaks (I) to those of active state (A). A plot of relative peak volumes for Cys265A versus those for Cys327A, which represent the populations of the active state in helix VI and helix VII, respectively, reveals varying degrees of agonist-mediated shifts towards the active state manifested by Cys2656.27 and Cys3277.54 (Fig. 3B). For most agonists the shifts along the two axes follow the same overall trend as their reported pharmacological efficacy on G-protein activation (36). For instance, the partial agonists tulobuterol, clenbuterol and norepinephrine have a less pronounced effect on Cys2656.27 than the full agonist isoproterenol, while the full agonist formoterol elicits a greater shift in conformational equilibrium on Cys2656.27. These results suggest that the degree to which agonists shift the equilibrium towards the active state of helix VI results in differing G-protein signaling capacity. The antagonist/weak partial agonist alprenolol can induce a small shift on Cys2656.27, but has a minimal effect on Cys3277.54, in agreement with previous findings that it is a weak partial agonist (25, 37). The inverse agonist carazolol, on the other hand, induces no apparent shifts as compared to the apo form. In contrast, the two β-arrestin biased ligands, isoetharine and carvedilol, cause large shifts of the equilibria in Cys3277.54 towards the active state. Interestingly, carvedilol, like other antagonists and inverse agonists, has little influence on the equilibrium of Cys2656.27, while isoetharine, like other agonists, also produces a prominent shift of Cys2656.27 towards the active state.
The large activation changes in Cys3277.54 caused by carvedilol and isoetharine suggest that helix VII primarily impacts β-arrestin signaling pathways. This is consistent with the result of a previous study that β2AR-phosphorylation by GPCR kinases, which is a prerequisite for β-arrestin binding, primarily targets a region directly adjacent to helix VII on helix VIII (38). Furthermore, recent crystallographic (11) and EM studies (12) of aβ2AR–Gαβγ heterotrimer complex show that the Gα subunit makes major contact with helices V and VI of β2AR, resulting in a 14 Å outward movement of the cytoplasmic ends of these helices, whereas there is no contact between Gαβγ and helix VII (Fig. 1A,B), suggesting that the conformational changes in helix VII are not directly involved in G-protein binding and signaling.
To further characterize the environment of Cys3277.54 in the active state, we evaluated the effect of the water-soluble paramagnetic relaxation agent gadopentetic dimeglumine (magnevist®) (39) on individual resonances of β2AR(C265A, TETC327, C341A) bound to isoproterenol. A comparison of the line widths of peaks A and I in the 1D 19F-NMR spectra at various magnevist concentrations revealed that peak I is more susceptible to the line broadening by magnevist than peak A (Fig. S1A), indicating that Cys3277.54 in the inactive state is more readily solvent-accessible than in the active state. This interpretation is consistent with crystal structure data, where Cys3277.54 was found to be solvent-exposed in the carazolol-bound structure (6), but buried in the trans-membrane helix bundle of the receptor in its complexes with a nanobody (8) or the G-protein (11) (Fig. S1B).
Additional insight from analysis of the 19F-NMR results can be obtained by examining the chemical structures of the agonists and their interactions with the individual helices in the β2AR binding pocket (Fig. 4). The majority of known high-affinity adrenergic ligands have a common structural motif consisting of an aromatic “head group” and an ethanolamine “tail group”. The head group (e.g., catechol) of agonists is directly connected to the ethanolamine tail, while antagonists and inverse agonists, both of which act as competitive inhibitors to agonists and either have no impact or a negative impact on the receptor basal activity, have an additional two-atom linker between the head and tail groups. Crystal structures of the β2AR complexes with various ligands (6–11), as well as biochemical (40–42) and modeling studies (43, 44) have established that the aromatic head groups interact with helices V and VI, while the ethanolamine tails are anchored by ionic and polar interactions at helices III and VII (Fig. 4A and Fig. S2). For agonists, polar interactions of their head groups with helix V side chains thereby mediate an inward shift of Pro2115.50, and subsequent conformational changes involving Ile1213.40 and Phe2826.44 (8) result in the activation move of the cytoplasmic tip of helix VI.
The changes observed by 19F-NMR (Fig. 3) provide additional insight into correlations between the chemical structure of the ligands and their interactions with β2AR. Binding of full agonists (such as isoproterenol and formoterol) imparts a large shift in the conformational equilibrium of both helices VI (Cys2656.27) and VII (Cys3277.54). Binding of partial agonists, which either completely lack (as in tulobuterol) or have much weaker (as in clenbuterol) polar features on their aromatic heads, has a smaller influence on the conformational equilibrium of helix VI than full agonists, but has almost as much impact on helix VII. In addition, the β-arrestin-biased ligands carvedilol and isoetharine, which differ from their corresponding “unbiased” prototypes (inverse agonist carazolol and full agonist isoproterenol, respectively) only in the tail moieties, strongly impact the helix VII equilibrium. A key observation is that the 1D 19F-NMR spectrum of β2AR(C265A, C327S, TETC341) bound to isoetharine, which is one of the most potent biased agonists, is similar to those of β2AR(C265A, C327S, TETC341) bound to carazolol and isoproterenol (Fig. 2B). This shows that the C-terminus of the non-transmembrane helix VIII, where Cys341 is located, is not directly involved in the β-arrestin signaling pathway. In the absence of structural data on a complex of β2AR with β-arrestin, it is tempting to further hypothesize from this data that the position of Cys341 on helix VIII is located outside of the interface with bound arrestin.
These observations on structure–activity relationships for β-arrestin biased β2AR ligands, and unbiased full and partial agonists, provide insights into β2AR signaling pathways that suggest the existence of parallel pathways for G-protein and β-arrestin signaling (Fig. S3). Although these pathways appear interconnected on several levels and may involve other activation-related rearrangements in the cytoplasmic and periplasmic loops (45), the present 19F-NMR results point to a significant level of decoupling of the two pathways. The implicated links between ligand structure and function may serve as a platform for future rational design of specific ligands.
Given the above observations on the correlation between ligand structure and receptor response, a connection can now be made between the chemical structures of pharmacological ligands and their signaling pathways acting either through G-proteins or β-arrestins. The general principle that links the chemical space of the ligands to the specific changes in conformational plasticity at the signaling cytoplasmic receptor surface may be preserved in other GPCRs. The use of 19F-NMR may thus prove to be a widely applicable tool for structure–activity relationship studies of GPCR ligands.
This work was supported in part by NIH Roadmap Initiative grant P50 GM073197 for technology development and purchase of the 19F-NMR probe, and by PSI:Biology grant U54 GM094618 for GPCR biology studies. The authors thank J. Velasquez for help on molecular biology; C. Cornillez-Ty, T. Trinh, and K. Allin for help on baculovirus expression; J. Gatchalian for ligand binding assays; P. Stanczak for assistance with the paramagnetic titration experiment; V. Cherezov for helpful discussions; I. Wilson for careful review and scientific feedback on the manuscript; K. Kadyshevskaya for assistance with figure preparation; and A. Walker for assistance with manuscript preparation. R.C.S. is a founder and member of the board of directors of Receptos, a GPCR structure-based drug discovery company.