Structural features for functional selectivity at serotonin receptors

Abstract

Drugs active at G protein-coupled receptors (GPCRs) can differentially modulate either canonical or noncanonical signaling pathways via a phenomenon known as functional selectivity or biased signaling. We report biochemical studies showing that the hallucinogen lysergic acid diethylamide, its precursor ergotamine (ERG), and related ergolines display strong functional selectivity for β-arrestin signaling at the 5-HT2B 5-hydroxytryptamine (5-HT) receptor, whereas they are relatively unbiased at the 5-HT1B receptor. To investigate the structural basis for biased signaling, we determined the crystal structure of the human 5-HT2B receptor bound to ERG and compared it with the 5-HT1B/ERG structure. Given the relatively poor understanding of GPCR structure and function to date, insight into different GPCR signaling pathways is important to better understand both adverse and favorable therapeutic activities.

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.

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.

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.

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*.

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.