Evolution of dopamine receptors: phylogenetic evidence suggests a later origin of the DRD2l and DRD4rs dopamine receptor gene lineages

Abstract

Dopamine receptors are integral membrane proteins whose endogenous ligand is dopamine. They play a fundamental role in the central nervous system and dysfunction of dopaminergic neurotransmission is responsible for the generation of a variety of neuropsychiatric disorders. From an evolutionary standpoint, phylogenetic relationships among the DRD₁ class of dopamine receptors are still a matter of debate as in the literature different tree topologies have been proposed. In contrast, phylogenetic relationships among the DRD ₂ group of receptors are well understood. Understanding the time of origin of the different dopamine receptors is also an issue that needs further study, especially for the genes that have restricted phyletic distributions (e.g., DRD_2l and DRD_4rs). Thus, the goal of this study was to investigate the evolution of dopamine receptors, with emphasis on shedding light on the phylogenetic relationships among the D₁ class of dopamine receptors and the time of origin of the DRD_2l and DRD_4rs gene lineages. Our results recovered the monophyly of the two groups of dopamine receptors. Within the DRD₁ group the monophyly of each paralog was recovered with strong support, and phylogenetic relationships among them were well resolved. Within the DRD₁ class of dopamine receptors we recovered the sister group relationship between the DRD_1C and DRD_1E, and this clade was recovered sister to a cyclostome sequence. The DRD₁ clade was recovered sister to the aforementioned clade, and the group containing DRD₅ receptors was sister to all other DRD₁ paralogs. In agreement with the literature, among the DRD₂ class of receptors, DRD₂ was recovered sister to DRD₃, whereas DRD₄ was sister to the DRD₂/DRD₃ clade. According to our phylogenetic tree, the DRD_2l and DRD_4rs gene lineages would have originated in the ancestor of gnathostomes between 615 and 473 mya. Conservation of sequences required for dopaminergic neurotransmission and small changes in regulatory regions suggest a functional refinement of the dopaminergic pathways along evolution.

Keywords: Dopamine receptors; Gene family evolution; Neuroscience; Whole genome duplications.

Figure 1. Maximum likelihood tree depicting evolutionary relationships among dopamine receptors in vertebrates.

Numbers on the nodes correspond to maximum likelihood ultrafast bootstrap support values. Human ADRA_1A, ADRA_1B, and ADRA_1D sequences were used as outgroups.

Figure 2. Patterns of conserved synteny in the chromosomal regions that harbor the DRD₁ class of dopamine receptors.

(A) Chromosomal region that harbors the DRD₁ gene; (B) Chromosomal region that harbors the DRD_1C gene; (C) Chromosomal region that harbors the DRD_1E gene; (D) Chromosomal region that harbors the DRD₅ gene. Asterisks denote that the orientation of the genomic piece is from 3′ to 5′, gray lines represent intervening genes that do not contribute to conserved synteny whereas dashed lines represent genes that are not present.

Figure 3. Patterns of conserved synteny in the chromosomal regions that harbor the DRD₂ class of dopamine receptors.

(A) Chromosomal region that harbors DRD₂ gene; (B) Chromosomal region that harbors DRD₂₁ gene; (C) Chromosomal region that harbors DRD₃ gene; (D) Chromosomal region that harbors DRD₄ gene; (E) Chromosomal region that harbors DRD_4rs gene. Asterisks denote that the orientation of the genomic piece is from 3′ to 5′, gray lines represent intervening genes that do not contribute to conserved synteny whereas dashed lines represent genes that are not present.

Figure 4. Maximum likelihood trees depicting evolutionary relationships among DRD₂ and DRD_2l dopamine receptors in vertebrates.

Numbers on the nodes correspond to maximum likelihood ultrafast bootstrap support values. This tree topology does not represent novel phylogenetic analyses; they are the DRD₂/DRD_2l clade that was recovered from Fig. 1.

Figure 5. Phyletic distribution of dopamine receptor genes in vertebrates.

The cyclostome check between the DRD_1C and DRD_E indicates that the duplication event that gave rise to these genes occurred after the divergence between cyclostomes and gnathostomes. Therefore, cyclostomes retained the ancestral condition of a single gene copy. A similar situation applies to DRD₂/DRD_2l and to DRD₄/DRD_4rs.

Figure 6. Alignment of the human dopamine receptor 2 (DRD₂) with zebrafish (Danio rerio), coelacanth (Latimeria chalumnae) and spotted gar (Lepisosteus oculatus) dopamine receptor 2l (DRD_2l).

Shaded regions denote transmembrane domains according to UniProt. Dopamine binding sites, agonist and antagonist binding sites were predicted with theoretical and computational techniques (Yashar et al., 2004) and experimental evidence (Shi & Javitch, 2002) . Amino acids in the third intracellular loop conferring G protein subunit Gαi specificity (Senogles et al., 2004) are indicated by orange asterisks.

Figure 7. Maximum likelihood trees depicting evolutionary relationships among DRD₄ and DRD_4rs dopamine receptors in vertebrates.

Numbers on the nodes correspond to maximum likelihood ultrafast bootstrap support values. This tree topology does not represent novel phylogenetic analyses; they are the DRD₄/DRD_4rs clade that was recovered from Fig. 1.

Figure 8. Alignment of the human dopamine receptor 4 (DRD₄) with zebrafish (Danio rerio), coelacanth (Latimeria chalumnae) and spotted gar (Lepisosteus oculatus) dopamine receptor 4rs (DRD_4rs).

Shaded regions denote transmembrane domains according to UniProt. Dopamine binding sites (red dots) were determined by site directed mutagenesis (Cummings et al., 2010) and homology to DRD₂. Antagonist binding sites and hydrophobic pocket-including selectivity region-were obtained from mutagenesis studies (Cummings et al., 2010) and from the crystal structure of the receptor coupled to the antagonist nemonapride (Wang et al., 2017). Non-conserved amino acids in the nemonapride binding pocket are labeled with green asterisks. Binding sites for the selective agonist UCSF924 are also shown (light blue dot).

Figure 9. Structural details of human DRD₄ binding site to the antipsychotic drug nemonapride (in blue) based on the molecular file PDB ID: 5WIV (Wang et al., 2017).

(B) Conserved amino acids within 4 Å of the drug molecule are shown with functional groups (in red). Non-conserved amino acids (in green) were changed (inset A) to the residue present in the fish species: F91Y and V193I. This mutagenesis was simulated choosing the rotamer (orientation of the side chain) with the highest probability (Y rotamer: 72.6%; I rotamer: 79% probability) see methods for additional details. (C) Partial alignment of the human dopamine receptor 4 (DRD₄) with zebrafish (Danio rerio), coelacanth (Latimeria chalumnae) and spotted gar (Lepisosteus oculatus) dopamine receptor 4rs (DRD_4rs) showing the numbers corresponding to the human DRD₄ sequence (NP_000788). Conserved and non-conserved aminoacids shown in (B) are indicated with red and green dots respectively. Non-conserved aminoacids within the region are also shown in green fonts.

Figure 10. An evolutionary hypothesis regarding the origin of dopamine receptor genes in vertebrates.

The vertebrate ancestor possessed two dopamine receptors, one of each class. However, after the two rounds of whole genome duplications (WGD) that occurred in the ancestor of the group each ancestral gene (DRD_1anc and DRD_2anc) gave rise to four genes. In the case of the DRD₁ class of receptors three out of the four genes were retained in the genome of the vertebrate ancestor. In the gnathostome ancestor, the DRD_1C∕E gene underwent a duplication event that gave rise to the actual DRD_1C and DRD_1E genes. Thus, the gnathostome ancestor had a repertoire of four DRD₁ genes: DRD₁, DRD₅, DRD_1C and DRD_1E. In the case of the DRD₂ group of receptors, the vertebrate WGDs originated four genes, three of which were maintained in the genome of extant species (DRD_2∕2l, DRD₃ and DRD_4∕4rs). In the ancestor of gnathostomes, the DRD_2∕2l gene underwent a duplication event that gave rise to an extra copy of the gene. Similarly, the DRD_4∕4rs gene also underwent a duplication event that gave rise to an extra copy of the gene. Thus, the ancestor of gnathostome vertebrates possessed a repertoire of five DRD₂ genes: DRD₂, DRD_2l, DRD₃, DRD₄ and DRD_4rs.