Biased Signaling Pathways in β2-Adrenergic Receptor Characterized by 19F-NMR

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Science  02 Mar 2012:
Vol. 335, Issue 6072, pp. 1106-1110
DOI: 10.1126/science.1215802


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 (fluorine-19 nuclear magnetic resonance) 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 toward 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 make up 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 U.S. Food and Drug Administration–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 (610), 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 electron microscopy (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. 1, A and B). A comparison of inactive and active-state crystal structures of β2AR, rhodopsin (13), and the A2A adenosine receptor (14, 15) suggests 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 with fluorescence-based biophysical experiments (1825) and nuclear magnetic resonance (NMR) (26), and conformational changes during receptor activation were observed in helix VI and the cytoplasmic surface.

Fig. 1

Locations of 19F-NMR labels in β2AR and activation-related changes in GPCR crystal structures. (A) Side view of β2AR in the active G protein–bound form [Protein DataBank (PDB) ID 3SN6], shown in green. The transmembrane helices I to VII and the C-terminal helix VIII are identified. The full agonist BI-167107 in the ligand-binding site is shown as a stick diagram. Green and yellow spheres highlight the three cysteine residues used for TET labeling, i.e., Cys2656.27 and Cys3277.54 at the cytoplasmic ends of helices VI and VII, respectively, and Cys341 at the C terminus. The bound G protein heterotrimer is shown as red ribbons and surfaces. (B) Cytoplasmic view of the structure in (A), with the G protein contact sites outlined by a broken red line. (C) Plot of distance root mean square deviations (RMSDs) of individual residues between crystal structures of inactive and active states of three GPCRs. Crystal structures used are as follows (from top to bottom): β2AR (PDB IDs 2RH1 versus 3SN6), rhodopsin (PDB IDs 1GZM versus 3DQB), and A2AAR (PDB IDs 3EML versus 3QAK). The horizontal axes represent the amino acid sequences (β2AR residues 34 to 341, bovine rhodopsin residues 38 to 320, and A2AAR residues 6 to 302). The vertical axis shows all-heavy-atom RMSDs per residue, and the color code defined in the upper right corner of the panel indicates corresponding Cα deviations. For each protein, selected residues are identified. The locations of the helices I to VIII are indicated at the top. Periplasmic loop regions are highlighted in red, and cytoplasmic loops and helix VIII are highlighted in blue. The cytoplasmic ends of helices VI and VII, which contain Cys2656.27 and Cys3277.54, are “hot spots” with large conformational rearrangements between the crystal structures of inactive and active states; other transmembrane helices and intracellular helix VIII, which includes Cys341, show only small displacements. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

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. 1, A and B) (28). Similar approaches have previously been successfully applied to studies of a number of membrane proteins (2931). Three native cysteine residues (Cys2656.27, Cys3277.54, and 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 nontransmembrane 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.

Fig. 2

One-dimensional 19F-NMR spectra of different TET-labeled β2AR constructs under variable solution conditions. (A) 19F-NMR resonance assignments for carazolol-bound TETβ2AR at 298 K. Spectra of the following constructs were recorded: wild-type (wt) TETβ2AR; β2AR(TETC265, C327S, C341A); β2AR(C265A, TETC327, C341A); and β2AR(C265A, C327S, TETC341). The vertical lines connect peaks in the 19F-NMR spectrum of TETβ2AR with the corresponding peaks in the spectra of the single-residue TET-labeled mutants. At the top, the peak assignments are indicated by the one-letter amino acid code and the residue number. (B) β2AR(C265A, C327S, TETC341) in complex with an inverse agonist (carazolol), a biased agonist (isoetharine), and a full agonist (isoproterenol) at 280, 298, and 310 K. (C, D, and E) β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) free and bound to nine different ligands at 280, 298, and 310 K. In (B) to (E), the temperature and the ligands are indicated at the top and on the right, respectively. For all experiments, the following parameter settings were used to collect and process the spectra: data size 1024 complex points, acquisition time 51 ms, and 24576 scans per increment. The data were multiplied with an exponential function with a line-broadening factor of 30 Hz and zero-filled to 2048 points before Fourier transformation.

The 19F-NMR spectra of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) (Fig. 2, C to E) show prominent changes upon addition of saturating concentrations of a collection of ligands (table S1) that represent a full spectrum of ligand efficacy (35). In general, the signals at 280 K of both β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) consist of two components, suggesting that the β2AR structure contains two independent equilibria between locally different conformations manifested at these two amino acid positions. Whereas 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 (Fig. 2, C to E, and fig. 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; that is, 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.

Fig. 3

Relative populations of active (A, red) and inactive (I, blue) states of β2AR derived from the 1D 19F-NMR spectra at 280 K. (A) Peak volumes for the individual components in the 1D 19F-NMR signals of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) obtained by a nonlinear least-squares fit to a double-Lorentzian function. The experimental data and the double-Lorentzian are indicated by thin and thick black lines, respectively. The fit used the chemical shift positions of peaks A and I indicated by the red and blue vertical lines. (B) Plot of the relative peak volumes for C265A versus the relative peak volumes for C327A. The relative peak volumes are the ratios of the volume of peak A and the sum of the volumes of peaks A and I. Agonists [tulobuterol, clenbuterol, norepinephrine (NE), isoproterenol, and formoterol] are shown as black circles highlighted by a yellow background, biased ligands (carvedilol, isoetharine) as red triangles highlighted by a green background, a neutral antagonist (alprenolol) as a black square, an inverse agonist (carazolol) as a black diamond, and apo as an open square.

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 toward 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, whereas 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 toward 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 toward the active state. Interestingly, carvedilol, like other antagonists and inverse agonists, has little influence on the equilibrium of Cys2656.27, whereas isoetharine, like other agonists, also produces a prominent shift of Cys2656.27 toward the active state.

The large activation changes in Cys3277.54 caused by carvedilol and isoetharine suggest that helix VII primarily affects 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 a β2AR-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. 1, A and 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, Bayer HealthCare Pharmaceuticals, New Jersey, USA) (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 one-dimensional (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 transmembrane 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, whereas 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 (611), as well as biochemical (4042) and modeling studies (43, 44) have established that the aromatic head groups interact with helices V and VI, whereas 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.

Fig. 4

Features of ligand binding in the β2AR structure and a conceptual model of signaling pathways to G proteins and arrestins in β2AR activation. (A) Side view of the structure of active-state β2AR in the complex with the agonist BI-167107 (PDB ID 3SN6), with helices V/VI and III/VII color-coded orange and blue, respectively, to indicate that they interact with the correspondingly colored fragments of the ligands in (B). (B) Chemical structures of the ligands used in the current 19F-NMR studies. Orange highlights the head groups, green the common ethanolamine moieties, and blue the substituents to the amino group of the ethanolamine tail. Ligand names are shown on the right, with published pharmacological efficacy indicated in parentheses.

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 affect the helix VII equilibrium. Another important 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 nontransmembrane 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 high 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.

Supporting Online Material

Materials and Methods

Figs. S1 to S4

Tables S1 and S2

References (4654)

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Ballesteros-Weinstein numbering is shown as a superscript. The most conserved residue in each transmembrane helix is designated as x.50, where x is the number of the transmembrane helix, and the second number (50) indicates the relative position of the specific residue. The second number decreases toward the N terminus and vice versa. Only residues on transmembrane helices are noted.
  3. To produce β2AR samples that are stabilized enough for NMR measurement, the C terminus was truncated, the part of intracellular loop 3 that is not involved in G protein recruitment was removed, and a stabilizing E122W mutation was added. Constructs containing these modifications were also crystallized and characterized as functionally similar to the unmodified β2AR.
  4. The three 19F signals in labeled wild-type TETβ2AR/carazolol were identified by comparison with signals from β2AR constructs containing single-labeled residues, i.e., β2AR(TETC265, C327S, C341A); β2AR(C265A, TETC327, C341A); and β2AR(C265A, C327S,TETC341).
  5. Ligand-binding properties of the cysteine mutants were examined on the membranes from β2AR expressing Sf9 cells. Radioligand binding assay used [3H]Dihydroalprenolol as the reporter ligand, and competition assay used isoproterenol as the competing ligand. The binding affinity of antagonist to the wild-type receptor (1.4 nM) is identical to all cysteine mutants (Cys265 = 1.1 ± 0.1 nM; Cys327 = 1.4 ± 0.4 nM; and Cys341 = 1.1 ± 0.1 nM), and the affinity of full agonist to the wild-type receptor (323 nM) is similar to all cysteine mutants (Cys265 = 100nM; Cys327 = 118 nM; and Cys341 = 138 nM).
  6. Acknowledgments: 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.
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