Evolutionarily conserved phosphorylation and palmitoylation-dependent regulation of dopamine D 1-like receptors in vertebrates

In the vertebrate central nervous system, dopamine is a neurotransmitter, acting as a modulator of variety of neuronal functions, including cognition, emotion, locomotor activity, hunger and satiety, and endocrine system regulation. Dopamine receptors are composed of D1, D2, D3, D4 and D5 receptors, which are classified into two major subtypes, D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopamine receptors. D1-like receptors stimulate adenylyl cyclase (AC) activity, whereas D2-like receptors inhibit AC activity and these receptors modulate many signaling pathways in dopaminergic synapses. Previous studies showed that post-translational protein phosphorylation and palmitoylation of mammalian D1 receptors regulate functional properties, stability, cellular localization and membrane trafficking of the receptors. Here, I further focus on conservation of phosphorylation and palmitoylation sites found in vertebrate and invertebrate D1-like receptor homologs. Analysis of databases provides evidence to suggest that PKAand GRKs-catalyzed phosphorylation sites of D1-like receptors have been completely conserved in the vertebrate lineage, in spite of the divergence of D1-like receptors full-length amino acid sequences during molecular evolution. These intracellular phosphorylated serine and threonine residues of D1-like receptors are evolutionarily conserved against mutation pressure throughout vertebrate species with a couple of exceptions. Furthermore, a PKC-mediated phosphorylation site of D1-like receptors is conserved in gnathostomes (jawed vertebrates) and not exist in agnathans (jawless vertebrates). Another phosphorylation site for PKC has been additionally acquired only in mammalian D1 receptor orthologs. These findings suggest that the multiple kinases-dependent regulation of dopaminergic synapses by phosphorylation is so critical for complex vertebrate higher brain function. Namely, dynamic regulation of dopaminergic synapses made possible by reversible phosphorylation of D1-like receptors may ensure the specific refined functions of vertebrate nervous systems. In contrast, broadly conserved palmitoylation sites in both vertebrate and invertebrate D1-like receptor homologs indicate that palmitoylated cysteines play crucial roles for sustaining the structural and functional regulations of D1-like receptors.


Introduction
In the mammalian central nervous system, dopamine (DA) is a neurotransmitter, acting as a modulator of variety of neuronal functions, including emotion, cognition, locomotor activity, hunger and satiety, and endocrine system regulation.Dysfunction of dopaminergic systems induces several neurological and psychological disorders, such as schizophrenia, movement disorders, addiction to drugs of abuse and Parkinson's disease [1,2] .Dopaminergic signaling is mediated by five DA receptors (DARs), named D 1 , D 2 , D 3 , D 4 and D 5 receptors, all of which belong to the family of seven transmembrane domain G protein-coupled receptors (GPCRs).These DAR subtypes are classified into two major classes, D 1 -like and D 2 -like receptors, based on homology in their protein structure, pharmacological and physiological properties [3] .The five members of the DAR family are encoded by different genes, localized at independent chromosomal loci.Previous studies of DARs structure and function revealed that the D 1 -like receptor subclass is composed of the D 1 (also known as D 1A ) and D 5 (also known as D 1B ) receptors that activate Gα s/olf , resulting in activation of adenylyl cyclase (AC) to increase cyclic AMP (cAMP) production upon agonist stimulation.The D 2 -like receptor subclass consists of the D 2 , D 3 , and D 4 receptors that couple to Gα i/o , inducing inhibition of AC.In contrast to the postsynaptic D 1 -like receptors, D 2 and D 3 receptors are expressed both presynaptically and postsynaptically on dopaminergic synapses [4][5][6] .
One key modification of mammalian DARs is the post-translational protein modification, such as phosphorylation and palmitoylation.D 1 -like receptors are robustly phosphorylated by multiple protein kinases.Reversible phosphorylation at intracellular serine (Ser)/threonine (Thr) residues of the D 1 receptor by protein kinase A (PKA), G protein-coupled receptor kinases (GRKs) or protein kinase C (PKC) regulate desensitization and β-arrestin-mediated endocytosis of D 1 receptors [2] .Like phosphorylation, post-translational protein palmitoylation is reversible addition of the lipid palmitate to intracellular cysteine (Cys) residues.This process acts as a sticky 'tag' that can direct receptors to specific regions of the plasma membrane, or to specific intracellular membranes or vesicles [7-9]   .Genetic evidence strongly links impaired synaptic palmitoylation to abnormal mammalian brain development and/or function, including human neuropsychiatric disorders [10-15]   .Palmitoylation of DARs controls membrane trafficking and stability of DARs [16][17][18] .These post-translational protein modifications allow additional control of synaptic localization, protein-protein interactions, dynamic trafficking and desensitization of the receptors.
Many neurotransmitter receptors and ion channels, including DARs, appear to be evolutionarily conserved; orthologs with identical domains and transmembrane topology are found in organisms from worms to man [19][20][21][22] .Here, I further focused on the post-translational protein phosphorylation and palmitoylation sites of D 1 -like receptors found in the vertebrate and invertebrate lineages and comparative sequence analysis of databases provides evidence to suggest that only vertebrate lineage established the D 1 -like DARs phosphorylation during evolution.

Conserved palmitoylation sites in vertebrate and invertebrate D 1 and D 5 receptor orthologs
Examination of the evolutionary conservation of Cys residues in mammalian D 1 receptor's palmitoylation sites (Cys347 and Cys351) showed that these palmitoylation sites are present in every animal species except for a few arthropods (Supplements-Table 1).Cys residues exist at the corresponding sites of vertebrate and invertebrate D 5 receptor orthologs examined (Supplements-Table 2).The broad conservation of the D 1 and D 5 receptors' palmitoylation sites in animal species, including those with only simple nervous systems, supports an idea that palmitoylation at the juxtamembrane region is evolutionarily ancient and regulates essential D 1 -like receptor functions.Hydrophobic palmitate attaches to the membrane, which enables to make an additional loop structure in the intracellular Carboxyl (C)-terminal region of D 1 -like receptors (Fig. 1A).Most species have two or three Cys residues around the C-terminal membrane proximal region, but there is no strict consensus rule in amino acid sequence around known palmitoylated cysteines [7][8][9] .One Cys residue may be adequate to make a C-terminal intracellular loop when it is palmitoylated.

Differential phosphorylation of D 1 and D 5 receptors occurred in vertebrates and invertebrates
Preferred consensus sequences for PKA consist of Arg-Arg/Lys-Xaa-Ser/Thr (R-R/K-X-S/T), R-X-X-S/T or R-X-S/T [23] .Previous studies revealed that Thr268 (K-R-E-T-K) in the third cytoplasmic loop of mammalian D 1 receptors is the phosphorylation site by PKA (Fig. 1A), which mediates DA agonist-induced receptor desensitization (Fig. 1B) [23,24] .As shown in Supplements-Table 1, Thr268 residue within a consensus recognition sequence for PKA living vertebrates, not in invertebrates, indicating that PKA-mediated D 1 receptor desensitization mechanism was specifically acquired in the common ancestor of all vertebrates just after the divergence from other chordates.The corresponding site is Thr292 (K/R/Q-K-E-T-K) in mammalian D 5 receptor, which is also conserved throughout vertebrates (K/R/Q/S-K/R-E-T-K), not in invertebrates (Supplements-Table 2).The PKA phosphorylation site of D 1 -like receptor orthologs is not detected in animals belonging to cephalochordates (e.g., lancelet and amphioxus), urochordates (also called tunicates, e.g., sea squirt) and the other phyla.
The human genome encodes seven different GRK isoforms (GRK1-7).The majority of the agonist-induced phosphorylation of the D 1 receptor is catalyzed by GRK2, GRK3, and GRK5 [25,26] .GRKs phosphorylate mammalian D 1 receptors at specific sites on their third intracellular loops (Ser256, Ser258, Ser259) and C-termini (Thr360) (Fig. 1A).It has been shown that the D 1 receptor associates with β-arrestin in GRK phosphorylation-dependent manner, leading the receptor for endocytosis (Fig. 1B).Ser258 and Ser259 are basically conserved from agnathans (jawless vertebrates) to gnathostomes (jawed vertebrates).Exceptionally, Ser259 is substituted with Cys in Australian echidna or with asparagine (Asn) in western European hedgehog, all reported birds and several reptiles (Supplements-Table 1).In tetrapods, Ser256 exists only in D 1 receptor orthologs of rodents (degu, Upper Galilee mountains blind mole rat, naked mole rat, thirteen-lined ground squirrel, Guinea pig, prairie vole, house mouse, Chinese hamster, Norway rat, prairie deer mouse, golden hamster, lesser Egyptian jerboa, Damara mole-rat, long-tailed chinchilla) and diprotodonts (common wombat, red kangaroo), whereas the site is replaced with proline (Pro) in other mammals, birds, reptiles and amphibians.This "triple Ser motif (S-E-S-S)" may be the original in primitive vertebrates, because hagfish and all examined fishes have three Ser or Thr there (Supplements-Table 1).Similarly, at least one Ser or Thr residue exists in the "triple Ser motif (Ser280, Ser282, Ser283)" of whole vertebrate D 5 receptor orthologs (Supplements-Table 2).
Thr360 in conjunction with Glu359 may comprise a motif necessary for GRK2-mediated D 1 receptor phosphorylation and desensitization [27] .The Glu359-Thr360 motif is conserved only in vertebrates with some exceptions in ray-finned fishes.In other phyla, hemichordates exceptionally have the phosphorylation motif besides vertebrates (Supplements-Table 1).Vertebrates D 5 receptor orthologs also have the motif (Glu385-Thr386) at the corresponding site with some exceptions in mammals, ray-finned fishes and hagfish (Supplements-Table 2).
PKC isoforms prefer basic residues (Arg and Lys) around the target Ser/Thr as their substrates [28] .It was reported that Ser397, Ser398, Ser417, Ser421, and Ser259 are phosphorylated upon PKC-activating phorbol ester treatment [5]   .PKC phosphorylation sites of mammalian D 1 receptor C-terminal region (Ser417 and Ser421) are highly conserved in gnathostomes, whereas agnathans (lampreys) has no PKC phosphorylation site (Supplements-Table 1).Almost all gnathostome D 5 receptor orthologs in upper taxa than bony fishes (actinopterygians; ray-finned fishes and sarcopterygians; flesh-finned fishes) have only one Ser or Thr residue at Ser444, which is lost in shark (Supplements-Table 2).Interestingly, an acidic residue (Asp or Glu) exists at 448 of most D 5 receptor orthologs, which may mimic "Ser417 and phospho-Ser421" motif in D 1 receptor C-terminal region (Supplements-Table 2).Another mammalian D 1 receptor C-terminal PKC sites (Ser397 and Ser398) exist only in mammals, which are not detected in other vertebrate classes, phyla and D 5 receptor orthologs.Some rodent D 5 receptors exceptionally have Ser420 at the corresponding site (Supplements-Tables 1 and 2).

Palmitoylation and phosphorylation sites in D 1C /D 1D /D 1X receptor homologs
Besides D 1 /D 1A and D 5 /D 1B receptors, additional D 1 -like receptor genes, D 1C , D 1D and D 1X receptor, have been identified in non-mammalian vertebrates, yet biochemically and pharmacologically uncharacterized.Previous researches proposed a hypothesis that the first gene duplication of the D 1 -like receptor ancestral gene should have occurred between the emergence of the earliest vertebrates and that of gnathostomes, and then the two paralogs were further duplicated before the divergence of gathostome lineage [20][21][22] .One or two of them had been secondarily lost in some class lineages.Corresponding sites with D 1 /D 1A receptor palmitoylation sites (Cys347 and Cys351), D 1 /D 1A receptor PKA phosphorylation site (Thr268), D 1 /D 1A receptor GRKs phosphorylation sites (Ser256, Ser258, Ser259) and D 1 /D 1A receptor PKC phosphorylation sites (Ser417, Ser421) in these D 1 -like receptor homologs are conserved throughout tetrapods (Supplements-Table 3).D 1 /D 1A receptor GRK2 phosphorylation site (Thr360) exists in reptile D 1C /D 1D /D 1X receptors, but not in birds and amphibians.Another D 1 /D 1A receptor PKC phosphorylation sites (Ser397, Ser398) are absent in these non-mammalian D 1 -like receptor homologs.

Discussion
Generally, structurally or functionally important amino acid residues are conserved during molecular evolution against mutation pressure.Despite divergences in amino acid sequences among D 1 -like receptors, this study revealed that some palmitoylation sites in D 1 , D 5 and D 1C /D 1D /D 1X receptors are broadly conserved throughout animal species, which means critical roles of D 1 -like receptors palmitoylation in the regulation of dopaminergic synapses.Alignment data also indicate stepwise acquisition of phosphorylation sites in D 1 -like receptors during vertebrate evolution (Fig. 2A).Multi protein kinases-dependent regulation of D 1 -like receptors has been acquired and established in the temporal order of PKA, GRK and finally PKC (Fig. 2B).While biochemical evidences have shown D 1 receptor phosphorylation by these protein kinases, it still remains unclear whether similar phosphorylation events are used for the functional regulation of D 5 and D 1C /D 1D /D 1X receptors.However, previous researches suggested that D 1 /D 1A , D 5 /D 1B and D 1C /D 1D /D 1X receptors were generated by duplication from a common ancestral gene in early vertebrate evolution [20][21][22] .Moreover, this study showed that the high degree of conservation of the corresponding  1 and 2. (B) A model of divergence and conservation of vertebrate D1-like receptors from the viewpoint of post-translational protein modifications.
phosphorylation sites among vertebrate D 1 -like receptor orthologs contrasts with the poor conservation of the phosphorylation sites in invertebrates.Combining these findings with previous phylogenetic analysis, post-translational protein modifications, which precisely control protein localization and activity, support higher brain functions.Especially, PKA-mediated phosphorylation site in D 1 -like receptors are completely conserved in the vertebrate lineage from the superclass Agnatha (jawless fishes, such as hagfish and lamprey) to the superclass Gnathostomata (jawed vertebrates).A site for PKA phosphorylation was readily acquired in the vertebrate D 1 -like receptors' common ancestral gene by simple gain-of-function mutations around 500 million years ago in the late Cambrian to the early Ordovician periods (Fig. 2B), which appears to act as a qualitative downregulation mechanism of D 1 -like receptors (Fig. 1B).It seems that the initially established PKA-dependent negative feedback regulation system was followed by GRK-dependent internalization of the receptor from plasma membrane in the subphylum Vertebrata (Fig. 2B), which quantitatively control dopaminergic synapses (Fig. 1B).Two PKC-dependent phosphorylation sites were then acquired only in the superclass Gnathostomata or in the class Mammalia, respectively (Fig. 2B).In contrast, invertebrate species have no Ser or Thr residue at the corresponding sites of D 1 -like receptor homologs, presumably because of random mutations in molecular evolution.In summary, these facts mean that the phosphorylation-dependent regulation of the D 1 -like receptors was established only in the common vertebrate ancestral species for refined regulation of receptor functions.The vertebrate specific phosphorylation mechanisms have been completely maintained up to the present time.Concerning other neurotransmitter-mediated synapses, we have already reported evolutionarily conserved palmitoylation-dependent regulation of glutamatergic synapses in vertebrates [29][30][31][32][33][34][35][36][37] .Future genome analyses will reveal detailed processes of acquisition and establishment of the post-translational protein modifications in complex vertebrate central nervous system.

Figure 1 .
Figure 1.Regulation of D1-like receptors by phosphorylation and palmitoylation.(A) Schematic of mammalian D1 receptors, showing location of phosphorylation and palmitoylation sites.(B) Summary of molecular mechanisms of phosphorylation-dependent desensitization, internalization and trafficking of D1 receptors.Binding of dopamine (DA) to D1 receptors leads Gs α subunit binds to and activation of adenylyl cyclase (AC), which, in turn, potently elevates cyclic AMP (cAMP) level.Then, downstream PKA is activated in cAMP-dependent manner, followed by phosphorylation and desensitization of D1 receptors by PKA in feedback inhibition system.GRK-dependent phosphorylation of D1 receptors induces the endocytosis of surface D1 receptors through the binding of β-arrestin proteins to the receptors.β-arrestin protein acts as an adaptor, which binds with clathrin and facilitates internalization of the receptors into clathrin-coated pits.

Figure 2 .
Figure 2. Acquisition of phosphorylation and palmitoylation sites of D1 and D5 receptors in animal evolution.(A) Summary of vertebrate D1 and D5 receptor evolution shown in Supplements-Tables1 and 2. (B) A model of divergence and conservation of vertebrate D1-like receptors from the viewpoint of post-translational protein modifications.
37. Thomas GM, Hayashi T. Smarter neuronal signaling complexes from existing components: how regulatory modifications were acquired during animal evolution: evolution of palmitoylation-dependent regulation of AMPA-type ionotropic glutamate receptors.Bioessays 2013; 35:929-939.Supplements Table 1.The BLAST alignments of phosphorylation and palmitoylation sites in D 1 receptor orthologs Table 2.The BLAST alignments of phosphorylation and palmitoylation sites in D 5 receptor orthologs Table 3.The BLAST alignments of phosphorylation and palmitoylation sites in D 1C , D 1D and D 1X receptor homologs in non-mammalian vertebratesAmino acid sequences around PKA phosphorylation site (Thr268), GRKs phosphorylation sites (Ser256, Ser258, Ser259), GRK2 phosphorylation site (Thr360), PKC phosphorylation sites (Ser417, Ser421), PKC phosphorylation sites (Ser397, Ser398) and palmitoylation sites (Cys347, Cys351) in D1-like receptor homologs of birds, reptiles, amphibians and human and mouse as representative of mammals are shown.