Evolution-guided discovery and recoding of allosteric pathway specificity determinants in psychoactive bioamine receptors

Abstract

G protein-coupled receptors for dopamine and serotonin control signaling pathways targeted by many psychoactive drugs. A puzzle is how receptors with similar functions and nearly identical binding site structures, such as D2 dopamine receptors and 5-HT2A serotonin receptors, could evolve a mechanism that discriminates stringently in their cellular responses between endogenous neurotransmitters. We used the Difference Evolutionary Trace (Difference-ET) and residue-swapping to uncover two distinct sets of specificity-determining sequence positions. One at the ligand-binding pocket determines the relative affinities for these two ligands, and a distinct, surprising set of positions outside the binding site determines whether a bound ligand can trigger the conformational rearrangement leading to G protein activation. Thus one site specifies affinity while the other encodes a filter for efficacy. These findings demonstrate that allosteric pathways linking distant interactions via alternate conformational states enforce specificity independently of the ligand-binding site, such that either one may be rationally rekeyed to different ligands. The conversion of a dopamine receptor effectively into a serotonin receptor illustrates the plasticity of GPCR signaling during evolution, or in pathological states, and suggests new approaches to drug discovery, targeting both classes of sites.

Fig. 1.

Identification of candidates for specificity-determining residues. (A) The Cα atoms, in blue, mapped onto the β2-adrenergic structure (2RH1) of all positions in the top 20th percentile rank following ET analysis of the transmembrane domains of 402 bioamine receptor sequences. (B) Cα atoms, in cyan, of top 20th percentile positions after ET analysis of all 2512 class A GPCR sequences. (C) Bioamine-specific determinants suggested by subtracting set B (the Class A trace) from set A (the bioamine trace). The residues that cluster in C define a bioamine-specific core that was further extended to include more bioamine-specific neighbors (D) or more residues important in all class A receptors (E) at ranks up to the 40th percentile (see Fig. S1 and Table S1 for details). This set represents 48 candidates for functionally important positions in bioamine receptors (F), of which 15 have different residues in D2R and 5-HT2AR (G).

Fig. 2.

Mutation of ET positions in D2R alters dopamine response. (A) Typical Ca²⁺ transients elicited in response to 10 μM dopamine, as measured by fluorescence intensity (relative fluorescence units, RFU) changes in a microplate reader. HEK 293-derived cells stably expressing G_α16 were transiently transfected with plasmids expressing D2R WT or indicated mutants, loaded with dye, and treated with dopamine at t = 10 s. (B) Dopamine maximal responses normalized by receptor surface expression. D2R WT response was defined as 100% (Dashed Line). Eight mutations (Red) reduced receptor responsiveness to dopamine. Data represent mean ± S.E.M. (*, P < 0.05; **, P < 0.001). (C) Dopamine-dose-response curves for D2RWT (▴), I48T (•), N124H (▾) and S193G (♦). Curves are four parameter fits as described in SI Text. (D) Positions that reduced dopamine response (α C) mapped onto the β2-adrenergic receptor structure.

Fig. 3.

Enhanced serotonin responses in D2R with ET residue swaps. (A) Typical Ca²⁺ transients, recorded as in Fig. 2, in response to 10 μM 5-HT. (B) Dose dependence of responses of I48T and D2RWT to serotonin. (C) Maximal 5-HT responses normalized by surface expression (mean ± SEM) with WT D2R response defined as 100% (Dashed Line). Four mutations (Red) yielded significant enhancement (p < 0.01). (D) Mutations with enhanced 5-HT responses (Red) and inactivating position L379 (Blue) are mapped onto the β2-adrenergic structure. One mutation, S193G, lowered dopamine affinity (E) and enhanced serotonin affinity (F). Specifically bound [³H] binding was measured in the presence of the indicated concentrations of competing ligands (mean ± SEM). g–j. Receptor internalization was determined by loss of surface expression using immunofluorescence in HEK WT (G and H) or cells stably expressing G_α16 (I and J), that were cotransfected with receptor and β-arrestin-EGFP cDNAs. Cells were treated with either 10μM dopamine (G and I) or 10 μM serotonin (H and J).

Fig. 4.

Enhanced effects of combined mutations. Activation and binding data for dopamine (A and B) or 5-HT (C and D) for D2R WT, I48T, S193G, and the indicated double mutants are shown. Activation data are representative traces for 10 μM dopamine (A) or 5-HT (C). Specific binding (B and D) was determined and curves calculated as in Fig. 3.