Structural Features for Functional Selectivity at Serotonin Receptors

Abstract

Drugs active at G protein-coupled receptors (GPCRs) can differentially modulate either canonical or non-canonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies that show that the hallucinogen lysergic acid diethylamide (LSD), its precursor ergotamine (ERG) and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-hydroxytryptamine (5-HT) receptor 5-HT_2B, while being relatively unbiased at the 5-HT_1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT_2B receptor bound to ERG, and compared it with the 5-HT_1B/ERG structure. Given the relatively poor understanding of GPCR structure-function to date, insight into different GPCR signaling pathways are important to better understand both adverse and favorable therapeutic activities.

Apart from canonical G protein mediated signaling, G protein-coupled receptors (GPCRs) also activate non-canonical G protein-independent pathways, frequently mediated by β-arrestins (1, 2). So-called ‘biased’ GPCR agonists differentially activate both signaling pathways with distinct efficacies and potencies as compared to unbiased agonists that activate both pathways equally (3). This preferential activation of one pathway over the other has been termed “functional selectivity” or “signaling bias” (2–5). Depending on the receptor, biased signaling patterns are key for mediating inflammation (6), apoptosis (7), and many other processes (2). Biased ligands have been proposed to stabilize receptor conformations that are distinct from those induced by unbiased ligands, and selectively change the propensity of GPCR coupling to either G proteins or β-arrestin (2).

Agonist-induced changes in “trigger motifs” of GPCRs (8) near the binding pocket facilitate large-scale helical movements that are accompanied by rearrangements in highly conserved residues called “micro-switches” (9) that prime GPCRs for subsequent G protein binding and activation (10). The structural features of a signaling biased receptor state remain elusive, and although complexes of two β-arrestin-biased ligands with the β₁ adrenergic receptor (β₁AR) have been recently solved (11), they did not reveal activation-related changes in the receptor.

To elucidate molecular and structural details of biased signaling, we characterized G protein- and β-arrestin-mediated signaling at G protein-coupled serotonin (5-HT; 5-hydroxytryptamine) receptors with several representative ergolines like LSD and ERG, and solved the crystal structure of the 5-HT_2B receptor in complex with ERG, which was identified as a highly biased agonist for the 5-HT_2B receptor (12).

To investigate potential differences of ergoline signaling at 5-HT receptors, we examined three prototypical serotonin receptors that interact with distinct G proteins. The 5-HT_1B receptor inhibits cyclic adenosine monophosphate (cAMP) production through G_i, the 5-HT_2B receptor mediates phospholipase C activation through G_q, and the 5-HT_7A receptor stimulates cAMP production through G_s (13). We compared G protein- and β-arrestin-mediated signaling at cloned human 5-HT_1B and 5-HT_2B receptors, and G protein-mediated signaling at 5-HT_7A receptors stimulated by selective and non-selective ligands in HEK293 cells (Fig. 1, table S1) (14).

Fig. 1.

Distinct signaling properties of lysergic acid diethylamide (LSD) and ergotamine (ERG) at 5-HT_1B, 5-HT_7A and 5-HT_2B receptors. We used luminescence-based assays to measure 5-HT_1B receptor mediated G_i activation and cAMP production; fluorescence-based calcium mobilization assays to measure 5-HT_2B receptor mediated G_q activation and β-arrestin translocation-dependent luciferase reporter assays to measure 5-HT_1B and 5-HT_2B receptors mediated β-arrestin recruitment, all in HEK293 derived cells. (A) Normalized concentration-response studies for LSD and ERG at human cloned 5-HT_1B receptor-mediated activation of G_i and non-canonical (Arrestin) signaling. (B) Normalized concentration-response studies for LSD and ERG at human cloned 5-HT_7A receptor-mediated activation of G_s signaling in the presence and absence of 5-Hydroxytryptamine (5-HT). (C) Normalized concentration-response studies for LSD and ERG at human cloned 5-HT_2B receptor-mediated activation of G_q and non-canonical (Arrestin) signaling. The solid circles for LSD-G_s signals are superimposed by the solid squares for ERG-G_s signals and thus, not visible. (D) Mean β-arrestin bias factors were calculated for serotonergic agonists (dihydroergotamine (DHE), methylergonovine (MTE), pergolide (PER) and cabergoline (CAB)), at 5-HT_2B and 5-HT_1B receptors. Concentration-responses curves were fit to the Black and Leff operational model to obtain transduction coefficients [Log(τ/K_A)] for each ligand at each corresponding pathway. The ΔLog(τ/K_A) was then calculated with 5-HT as a reference agonist for each pathway, and the ΔΔLog(τ/K_A) was calculated between two pathways for each ligand. The bias factor is unit-less and defined as 10^{ΔΔLog(τ/KA)} (28). Compounds with values close to one represent unbiased agonists while compounds with large numerical values, typically >100, represent extremely biased agonists. *p<0.0001 via two-way ANOVA comparing 5-HT_2B vs 5-HT_1B bias factors; N=3–6 separate experiments. ERG, DHE and, to a lesser extent, LSD, MTE and PER show strong β-arrestin bias at 5-HT_2B receptor, but not 5-HT_1B receptor.

LSD, and especially ERG displayed bias for β-arrestin signaling at 5-HT_2B (Bias factors 101 and 228 respectively; Fig. 1D), minimal bias at 5-HT_1B (Bias factors 5 and 25 respectively; Fig. 1D), and G protein antagonism at 5-HT_7A receptors (Fig. 1B, table S1). We also found significant β-arrestin signaling bias for other ergolines, such as dihydroergotamine (DHE), methylergonovine (MTE), pergolide (PER), and cabergoline (CAB) at the 5-HT_2B receptor, whereas all other evaluated compounds showed no significant bias (Fig. 1D). ERG and DHE, both of which contain a large tripeptide moiety substitution at the amide scaffold, displayed more extreme signaling bias at the 5-HT_2B receptor compared to LSD.

To investigate the molecular details responsible for biased signaling, we crystallized an engineered 5-HT_2B receptor construct in complex with ERG, solved its structure at 2.7 Å (figure S1, S2, table S3), and compared it to the structure of 5-HT_1B/ERG reported in the companion manuscript (15), as well as to other known unbiased active-state GPCR structures.

Residues P^5.50, I^3.40 and F^6.44 (16, 17), the “P-I-F” motif, form an interface between helix V, helix III and helix VI near the base of the ligand binding pocket in the β₂ adrenergic receptor (β₂AR) and many other aminergic receptors, including all 5-HT GPCRs. In the active-state structures of β₂AR (8, 18), a chain of conformational rearrangements occur in the P-I-F residues, in which an inward shift of helix V residue P211^5.50 is coupled with: (i) a rotamer switch in I121^3.40, (ii) a large movement of the F282^6.44 side chain, and (iii) a corresponding rotation of helix VI on the cytoplasmic side (8). The 5-HT_1B and 5-HT_2B receptor structures display two different conformations of the P-I-F motif (Fig. 2). For the 5-HT_1B receptor, we observe that the P-I-F configuration is essentially identical to that of the active-state of β₂AR (β₂AR-R*, PDB ID: 3SN6, (18)) (Fig. 2B). Whereas the 5-HT_2B receptor adopts a similar conformation of P229^5.50 and I143^3.40, the side-chain conformation of F333^6.44 was similar to that observed in the inactive β₂AR (β₂AR-R, PDB ID: 2RH1, (19)) (Fig. 2C, figure S2B). The P-I-F motif, therefore, appears to be in an active-like conformation in the 5-HT_1B structure, but only in an intermediate active conformation in the 5-HT_2B receptor structure.

Fig. 2.

Trigger motif P-I-F displays active- and intermediate active-state for 5-HT_1B and 5-HT_2B receptors, respectively. Residues of the P-I-F motif are highlighted in a dashed red circle. (A) Overall architecture of the 5-HT_2B receptor (green) bound to ERG (magenta); residues of the P^5.50 I^3.40 F^6.44 motif are illustrated in space-filling representation. (B) Alignment between 5-HT_1B receptor (grey), β₂AR-R (magenta; PDB ID: 2RH1) and β₂AR-R*(yellow; PDB ID: 3SN6) indicates an activated P-I-F motif in the 5-HT_1B/ERG structure. Black arrows indicate likely rearrangements upon 5-HT_1B receptor activation according to analysis of β₂AR. (C) Alignment between 5-HT_2B receptor (green), β₂AR-R (magenta) and β₂AR-R* (yellow) suggests intermediate active-state of the P-I-F motif in the 5-HT_2B/ERG structure, with F^6.44 in an inactive conformation. Black arrows indicate likely rearrangements upon 5-HT_2B receptor activation according to analysis of β₂AR.

At the cytoplasmic side of the receptors, GPCR activation is generally characterized by the displacement of helices V, VI and VII (10). The magnitude of the helical motions depends on the activation-state; for example, the outward displacement of the intracellular tip of helix VI ranges between 3 and 14 Å, whereas helix VII shifts between 3 to 5 Å towards the receptor core (10, 18–22). These concerted rearrangements induce an opening of the helical bundle at the cytoplasmic side, which facilitates the binding and subsequent activation of G proteins. Analysis of the 5-HT_1B and 5-HT_2B receptor structures shows that the conformation in the intracellular half of the helical bundle is notably shifted towards that seen in active-state GPCR structures (Fig. 3, figures S3 and S4), and distinct from those of inactive-state GPCR structures (Fig. 3, figures S5 and S6). In the 5-HT_1B and 5-HT_2B receptor structures, the helix VI intracellular part is located at least 2 to 4 Å further away from the receptor core than in the inactive-state structures of other aminergic GPCRs (figure S5). This conformation of helix VI is close to the one observed in active-state structures of the A_2A adenosine receptor (A_2AAR) (figure S3), and rhodopsin (Rho) (figure S4), though the outward shift of the helix is smaller in magnitude compared to that of the G protein-bound β₂AR (Fig. 3A, 3C). The only difference that we observed in helix VI between 5-HT receptor subtypes was a small clockwise rotation in the 5-HT_1B receptor towards the active-state that is absent in the 5-HT_2B structure (Fig. 2B, 2C, 3A, 3C). Helix VII in both 5-HT receptor structures also displays intermediate active-states when compared to β₂AR (Fig. 3B, 3D), where it is shifted further towards the receptor core than in inactive-state structures of other aminergic GPCRs (figure S6). Whereas the 5-HT_2B/ERG receptor structure shows less pronounced active-like changes in helix VI, helix VII appears to be in a more active conformation than it is in the 5-HT_1B/ERG receptor structure (Fig. 3B, 3D).

Fig. 3.

Structural alignment with β₂AR-R and β₂AR-R* reveals distinct active-state 7TM conformations of the 5-HT_1B and 5-HT_2B receptor structures. All structures were analyzed based on the last membrane embedded residue to minimize the effect of G proteins and fusion partners on the relative helix positions (see supplementary section). Center panel shows overall 7TM configuration of β₂AR-R (magenta; PDB ID: 2RH1), β₂AR-R*(yellow; PDB ID: 3SN6), 5-HT_1B (grey) and 5-HT_2B (green) receptors aligned through helices I–IV. Membrane boundaries are indicated by grey dots according to the OPM database (29). (A, C) Intracellular view of helix V and helix VI in β₂AR-R, β₂AR-R* compared to (A) the 5-HT_1B, or (C) the 5-HT_2B receptor. Residues on the intracellular side of the membrane have been removed for better comparison of ligand-induced helical rearrangements (see main text). Helix VI of the 5-HT_1B and 5-HT_2B receptors is in an intermediate active-state compared to β₂AR. (B, D) Side view of helix VII and helix VIII in β₂AR-R, β₂AR-R* and (B) 5-HT_1B receptor, or (D) 5-HT_2B receptor. Helix VII of the 5-HT_1B and 5-HT_2B receptors is in an intermediate active-state compared to β₂AR, with more pronounced activation features for the 5-HT_2B receptor.

Another important aspect of GPCR activation are rearrangements of side chains in highly conserved motifs D(E)/RY (helix III) and NPxxY (helix VII), which are referred to as “micro-switches” (9). Thus, the D(E)/RY motif of all inactive-state and most active-state GPCR structures shows an intact salt bridge between the side chains of D(E)^3.49 and R^3.50. Importantly, this salt bridge is broken only in the active-state structures of β₂AR-Gαβγ (18) and Opsin-GαCT (21), where R^3.50 interacts instead with the G protein and Gα peptide, respectively. The salt bridge is preserved between the side chains of R153^3.50 and D152^3.49 in the 5-HT_2B receptor structure, but it is broken in the 5-HT_1B receptor structure (Fig. 4A, 4B, 4D, figure S2C). In the 5-HT_1B receptor structure, the D146^3.49 side chain forms a hydrogen bond to Y157 in ICL2, and the R147^3.50 side chain interacts with the main chain carbonyl in a loop of the fusion protein BRIL (residue L1048), which is partially inserted into the G protein binding crevice (Fig. 4A). Thus, the conformation of the D(E)RY motif mimics the active state of β₂AR in the 5-HT_1B structure, but resembles the inactive state in the 5-HT_2B structure (Fig. 4A, 4C and figure S7).

Fig. 4.

Activation state of the D(E)/RY and NPxxY motifs in 5-HT_1B and 5-HT_2B receptors compared to β₂AR-R and β₂AR-R*. Configuration of the D(E)/RY motif in (A) the 5-HT_1B receptor (grey), (B) the 5-HT_2B receptor (green), (C) β₂AR-R* (yellow; PDB ID: 3SN6) and (D) β₂AR-R (magenta; PDB ID: 2RH1). Residues of the G protein in β₂AR-R* (C) and G protein mimicking BRIL loop (A) are highlighted in orange. The conformation of the D(E)/RY motif in the 5-HT_1B receptor is similar to that observed in β₂AR-R*, whereas the configuration of the 5-HT_2B receptor compares to that of β₂AR-R. (E, F) Conformational states of Y^7.53 of the NPxxY motif and the proceeding residue 7.54 in β₂AR-R, β₂AR-R* and (E) the 5-HT_1B, or (F) 5-HT_2B receptor. When compared to β₂AR, the conformation of the NPxxY motif in the 5-HT_1B receptor is in an intermediate active-state, whereas the configuration of the 5-HT_2B receptor is similar to β₂AR-R*.

The highly conserved NPxxY motif at the cytoplasmic end of helix VII is another key micro-switch of GPCR activation (9). Upon GPCR activation, (i) the intracellular end of helix VII moves towards the receptor core and (ii) a rotation of Y^7.53 around the helical axis moves the side chain further into the 7TM bundle (10). Both 5-HT receptor structures show active-state conformations of the NPxxY motif when compared to β₂AR, A_2AAR and Rho (Fig. 4D–E and figures S2D and S7), with more pronounced activation features in the 5-HT_2B receptor.

Our analysis indicates that the 5-HT_1B/ERG structure has most of the attributes of a classical agonist-induced active-like state, consistent with our biochemical findings that ERG is a comparatively unbiased agonist at the 5-HT_1B receptor. In contrast, the 5-HT_2B/ERG structure exhibits conformational characteristics of both the active- and inactive-state. The structure of β₂AR-Gαβγ complex, along with recent NMR and fluorescence studies of β₂AR, implicate helix VI predominantly in G protein signaling, whereas conformational changes in helix VII are associated with enhanced β-arrestin signaling (18, 23, 24). Thus, an active-like state in the helix VII conformation of the 5-HT_2B receptor, but only partial changes in helix VI, mirrors the strong β-arrestin bias of ERG at 5-HT_2B receptors observed in pharmacological assays.

A likely structural explanation for the distinct conformational features and biased pharmacology of ERG between 5-HT_1B and 5-HT_2B receptors can be found in the region of the extracellular loop 2 (ECL2) junction with helix V. In the 5-HT_2B receptor structure, E212-R213-F214 form an additional helical turn which is stabilized by a structured water molecule and forms a sharp angle to the extracellular tip of helix V (Fig. 5). As a result, the segment of ECL2 that connect helices III and V via the conserved disulfide bond is shortened in the 5-HT_2B receptor inducing an inward shift and creating a conformational constraint on the position of the extracellular tip of helix V. Because of these rearrangements, ERG forms additional hydrophobic contacts with M218^5.39, L347^6.58, V348^6.59, L362^7.35 and K211^ECL2 (figure S8A) and stabilizes a closer distance between helices V and VI, which form hydrophobic contacts between L219^5.40, V348^6.59 and L349^6.60, along with a hydrogen bond between S222^5.43 and N344^6.45 (figure S8B). These extensive ligand-mediated interactions between helices V and VI in the 5-HT_2B/ERG complex may be inferred to prevent rearrangements in helix VI and the corresponding rotation of F^6.44 (8), observed in the 5-HT_1B receptor and structures of other active-state GPCRs. The strengthened interactions of helix V and VI through ligand-mediated hydrogen bonds to both helices have also been linked to inhibition of G protein signaling at β₂AR by the most efficacious inverse agonist (25).

Fig. 5.

Structural differences in extracellular configuration of helix V between 5-HT_1B and 5-HT_2B receptors likely explain β-arrestin functional selectivity at the 5-HT_2B receptor. Side view (left) and top view (right) of 5-HT_1B (grey) and 5-HT_2B (green) receptors shows a kink in the extracellular end of helix V in the 5-HT_2B receptor. A water molecule (red sphere) was found to stabilize the kink through hydrogen bonds (grey dashed lines) with the E212^5.33 main-chain carbonyl oxygen and the main-chain nitrogens of D216^5.37 and G215^5.36. Main-chain atoms of both residues are shown as lines.

Ergolines predominantly signal through β-arrestin pathways at 5-HT_2B receptors, whereas signaling at 5-HT_1B receptors appears non-biased. The differential signaling patterns are mirrored in the crystal structures, which show features of an intermediate active-state for the 5-HT_1B receptor, and a β-arrestin-biased activation-state for the 5-HT_2B receptor. We propose a mechanism by which ERG stabilizes a conformation of the 5-HT_2B receptor that selectively interferes with G protein signaling (figure S9). The tripeptide moiety of ERG appears to function as a negative allosteric regulator of G protein signaling at the 5-HT_2B receptor, as ERG exhibits strongly increased β-arrestin signaling bias compared to LSD and MTE. Because both therapeutic (26) and adverse (27) drug effects have been associated with β-arrestin recruitment by GPCRs, identifying the features of biased GPCR states by known and yet to be discovered intracellular signaling proteins may facilitate the development of safer and more effective therapeutics with selective signaling profiles.