These authors contributed equally to this work.
Identification and characterization of a novel amphioxus dopamine D1-like receptor
Article first published online: 23 JUL 2009
© 2009 The Authors. Journal Compilation © 2009 International Society for Neurochemistry
Journal of Neurochemistry
Volume 111, Issue 1, pages 26–36, October 2009
How to Cite
Burman, C., Reale, V., Srivastava, D. P. and Evans, P. D. (2009), Identification and characterization of a novel amphioxus dopamine D1-like receptor. Journal of Neurochemistry, 111: 26–36. doi: 10.1111/j.1471-4159.2009.06295.x
- Issue published online: 14 SEP 2009
- Article first published online: 23 JUL 2009
- Received April 7, 2009; revised manuscript received July 8, 2009; accepted July 9, 2009.
- cyclic AMP;
- dopamine D1-like receptor;
- expression in Chinese hamster ovary cells;
- expression in Xenopus oocytes;
- G protein-coupled receptor
Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus (Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. One of these receptors, AmphiD1/β, was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD1/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD1/β has a similar pharmacology to vertebrate D1 receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits ‘agonist-specific coupling’ depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD1/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.
Chinese hamster ovary
extracellular signal-related kinase
green fluorescent protein
G protein-coupled receptor
human embryonic kidney cells
mitogen-activated protein kinase
open reading frame
The biogenic amine dopamine plays an important role as a neurotransmitter in both vertebrates and invertebrates. In mammals, dopaminergic neurones regulate motor control, emotion, hormone release from the pituitary gland, nausea and vomiting. Dysregulation of dopaminergic transmission can result in pathological conditions such as Parkinson’s disease, Tourette’s syndrome and Schizophrenia (Rang et al. 1999). In invertebrates, dopaminergic neurones are known to be involved in learning and memory, motor control and fertility (Blenau and Baumann 2001; Clark et al. 2008). In both vertebrates and invertebrates, dopamine receptors can be divided into two groups based on sequence similarity, ligand affinity and effector coupling (Neve et al. 2004). Classically, the D1 subfamily of receptors couples to the Gαs G protein leading to adenylyl cyclase activation and a subsequent increase in cAMP levels. In contrast, the D2 subfamily couples to the Gαi G protein which causes an inhibition of adenylyl cyclase and a decrease in cAMP levels. However, D1 receptors also couple to both calcium mobilization (Surmeier et al. 1995) and to the activation of the mitogen-activated protein kinase (MAPK) cascade (Chen et al. 2004). Moreover, members of the D2 receptor subfamily also couple to the cell type-dependent modulation of phosphatidylinositol hydrolysis (Tang et al. 1994), to the inhibition of inward calcium currents (Seabrook et al. 1994), to an increase in outward potassium currents (Williams et al. 1989) and to the activation of both the phosphatidyl inositol-3 kinase pathway (Zhen et al. 2001) and the MAPK pathway (Wang et al. 2005).
In mammals, the D1 receptor subfamily is further subdivided into the D1 and D5 receptors. Pharmacologically, these two receptors can be distinguished by dopamine exhibiting a slightly higher affinity for the D5 receptor, by butaclamol having a slightly higher affinity for the D1 receptor and by the D5 receptor possessing a higher level of constitutive activity (Missale et al. 1998). There are at least three subtypes of D1 receptors in all jawed vertebrates, except mammals (Cardinaud et al. 1997, 1998; Le Crom et al. 2004). These receptors have been named the D1A, D1B, D1C and D1D receptors. Analysis of four D1 receptors from European eel (D1A1, D1A2, D1B and D1c) (Cardinaud et al. 1997) and three D1 receptors from Xenopus (D1A, D1B and D1C) (Le Crom et al. 2004) revealed that the D1A and D1B receptors had a similar pharmacology to the mammalian D1 and D5 receptors. Specifically, the D1B receptor had a higher affinity for dopamine and a higher level of agonist-independent activity than the D1A receptor. The D1C receptor was found to display an intermediate pharmacology, and it could be distinguished from the other receptors by its resistance to agonist-induced desensitization (Le Crom et al. 2004). A feature common to all vertebrate D1 receptors is their ability to be activated by the benzodiazepine agonist, SKF38393, and to be inhibited by the benzodiazepine antagonist, SCH23390.
Dopamine D1 receptors have also been identified and characterized from invertebrates including Drosophila (Gotzes et al. 1994; Feng et al. 1996; Reale et al. 1997), honeybee (Blenau et al. 1998) and Caenorhabditis elegans (Suo et al. 2002). The dopamine D1 receptors from invertebrates can be subdivided into two groups comprised of those that share high levels of sequence similarity with the mammalian D1 receptors and those that share higher levels of similarity with the invertebrate octopamine/tyramine receptors. Both subtypes have been found to couple to adenylyl cyclase activation and a subsequent increase in intracellular cAMP levels after stimulation with dopamine (Blenau and Baumann 2001; Mustard et al. 2005). Both subtypes of invertebrate D1 receptors are known to be much less responsive to the benzodiazepine ligands SKF38393 and SCH23390 than the mammalian D1 receptors (Blenau and Baumann 2001; Mustard et al. 2005).
The pharmacology and functionality of dopamine D1 receptors in the deuterostomian invertebrates so far remains uncharacterized. One such organism, the cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates (Delsuc et al. 2006; Putnam et al. 2008). A recent bioinformatic study revealed that amphioxus has at least three dopamine D1-like receptor sequences that show similarity to both vertebrate and invertebrate types (Burman et al. 2007). One of these receptors, AmphiD1/β, was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors (Vincent et al. 1998; Candiani et al. 2005; Burman et al. 2007). Thus, it is of interest to elucidate the pharmacological characteristics of AmphiD1/β, to discover if amphioxus does indeed have a vertebrate-type D1 receptor or possibly a (vertebrate-specific) β-adrenergic-like receptor. To this end, the present study describes the cloning of Amphi D1/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD1/β has a similar pharmacology to the vertebrate D1 receptors. Moreover, no pharmacological characteristics were observed in the present study to suggest that AmphiD1/β may be an amphioxus orthologue of the vertebrate β-adrenergic receptors.
Materials and methods
AmphiD1/β was amplified from an adult amphioxus (Branchiostoma floridae) head cDNA library (kindly supplied by Dr M. Matz, Whitney Laboratory University of Florida, St Augustine, FL, USA) using the Advantage 2 PCR system (BD Biosciences, Oxford, UK). PCR primers were manufactured by Sigma-Genosys (Cambridge, UK). The forward primer (5′-CACCATGTCGGCGAACACTAC-3′) was based on the 5′-end of the open reading frame (ORF) of AmphiD1/β, containing the start codon. The antisense reverse primer (5′-GTGAACTTGACGTTCCTTCACCG-3′) was located exactly at the end of the 3′-end of the ORF of AmphiD1/β, but excluding the stop codon. The reverse primer also contained an extra guanosine residue at its 5′-end, so that a cytosine residue was added at the 3′-end of the receptor to facilitate the in-frame cloning with the 3′-green fluorescent protein (GFP) tag of the pcDNA3.1/CT-GFP vector. The thermal profile consisted of an initial denaturation temperature of 95°C for 1 min, followed by 30 cycles of 95°C for 30 s, 62°C for 30 s and 68°C for 3 min. This was followed by a final extension of 68°C for 10 min. AmphiD1/β was cloned into the expression vector pcDNA3.1/CT-GFP via a TOPO cloning reaction (Invitrogen Life Sciences, Paisley, UK).
Expression in mammalian cell lines
Chinese hamster ovary (CHO)-K1 cells were maintained in Ham’s F-12 nutrient media (Invitrogen Life Sciences) supplemented with 10% charcoal-stripped foetal bovine serum (Hyclone, Cramlington, UK), 50 U/μg penicillin/streptomycin and 4 mM l-glutamine (Invitrogen Life Sciences) in 5% CO2 at 37°C. Stably transfected cell lines were generated by the transfection of CHO-K1 cells using Lipofectamine (Invitrogen Life Sciences) following the manufacturer’s instructions, followed by selection with 400/1000 μg/mL geneticin (G418) (Invitrogen Life Sciences). To derive a clonal cell line, single clumps of cells were picked and expanded in media containing 400 μg/mL G418. Human embryonic kidney cells (HEK293) were maintained in Ham’s F-12 nutrient media (Invitrogen Life Sciences) supplemented with foetal bovine serum (Hyclone), and 50 U/μg penicillin/streptomycin in 5% CO2 at 37°C. Transiently transfected HEK293 cells were generated using Fugene 6 (Roche, Lewes, UK) following the manufacturers instructions. The cells were transfected 48 h prior to stimulation and a ratio of 6 : 1 Fugene 6 : DNA was used.
cAMP levels in stably transfected CHO-K1 cells were determined as described previously (Maqueira et al. 2005) (see Appendix S1). cAMP levels were represented as a percentage of basal samples unless otherwise stated. anova was used to test for significant agonist-mediated effects in individual experiments. Unless otherwise stated, all data are shown as mean ± SEM. Forskolin was used both to increase basal cAMP levels to make it easier to detect increases and decreases in cAMP levels in the same experiments and also to potentiate responses to agonists to more accurately determine their threshold effects (see Insel and Ostrom 2003). A non-saturating 10 μM concentration of forskolin was used. Basal levels of CHO cell cAMP were 5.1 ± 1.7 pmoles/mg protein (n = 3) and these were raised to 1053.7 ± 4.8 pmoles/mg protein after exposure to 10 μM forskolin. Protein levels were determined using a Bradford assay (Bradford, 1976).
Intracellular calcium measurements
Phosphorylated extracellular signal-related kinase (ERK) levels were determined essentially as described previously (Balmanno and Cook 1999; Swatton et al. 2004; Srivastava et al. 2005) (Minor modifications are described in Appendix S1 online where a full description of the methods used is given.). Values for the kinase expression and activity levels were defined as 100% in the control samples.
Expression in Xenopus oocytes
The Xenopus laevis oocyte experiments were carried out as described in Rogers et al. (2003). In short, capped sense cRNA was prepared using mMESSAGEmMACHINE T3 Kit (Ambion, (Europe) Ltd., Huntingdon UK) from linearized plasmid DNA containing a full-length cDNA encoding AmphiD1/β in pBS-MXT. Stages V and VI oocytes were prepared as described previously (Feng et al. 1996) and were injected with 50 ng of receptor sense cRNA. Injected oocytes were incubated at 19°C for 2–5 days before recording; uninjected oocytes and water injected oocytes were processed in parallel as controls. Recordings were made using a two-microelectrode voltage-clamp technique at a holding potential of −60 mV to measure oocyte currents (Van Renterghem et al. 1987). Oocytes were continuously superfused with ND96 medium (Feng et al. 1996) and test compounds were added to the superfusate.
The sources of the drugs used in these experiments are given in Appendix S1.
Cloning of AmphiD1/β
We initially identified AmphiD1/β from the NCBI non-redundant protein database as a potential Branchiostoma floridae orthologue of the Drosophila receptor, DmDopEcR (22% identity and 41% similarity), which could bind to and be activated by both dopamine and the ecdysteroids (Srivastava et al. 2005). AmphiD1/β was previously cloned from Branchiostoma lanceolatum and a short abstract (without any data) published, outlining some of its pharmacological characteristics (Vincent et al. 1998). In addition, Candiani et al. (2005) reported the cloning of AmphiD1/β from Branchistoma floridae together with a comparison of its cellular expression pattern with that for tyrosine hydroxylase. The sequence obtained for AmphiD1/β from Branchistoma floridae in the present study (see Fig. S1) is essentially identical to that reported by Candiani et al. (2005) except for the presence of a serine residue instead of an asparagine in its C-terminus (S380N), a valine instead of an isoleucine at the start of its Transmembrane helix I (V41I) and an alanine instead of a glycine (A109G) in its first extracellular loop. We have previously reported on a comprehensive bioinformatical analysis of the amino acid sequence of AmphiD1/β (Burman et al. 2007). It was found to show greater sequence similarity to both vertebrate dopamine D1 receptors (e.g. 44% identity and 62% similarity to Carp D1 sequence) and to β-adrenergic receptors (e.g. 48% identity and 67% similarity to Xenopusβ1 sequence) than to DmDopEcR (see Fig. S1). AmphiD1/β also showed some homology with vertebrate 5-hydroxytryptamine (5-HT) receptors (e.g. 40% identity and 55% similarity to Zebrafish 5-HT1A sequence). In addition, it was found phylogenetically to group with the β-adrenergic receptors (Burman et al. 2007). Further, the species variant of AmphiD1/β from B. lanceolatum showed 93% sequence identity and 98% similarity. In the present study, AmphiD1/β was amplified using PCR from adult B. floridae head and muscle cDNA libraries, using primers located at the start and end of the predicted ORF of AmphiD1/β (see Materials and methods). A PCR product of approximately 1.2 kb was amplified from both cDNA libraries, which was in agreement with the predicted size of the receptor (data not shown). The PCR product amplified from the head cDNA library was cloned into the mammalian expression vector pcDNA3.1/CT-GFP and into the multiple cloning site of the Xenopus oocyte expression vector, pBS-MXT, which was flanked by the Xenopusβ-globin 5′- and 3′-untranslated regions to promote stable mRNA expression in oocytes.
Biogenic amine specificity of AmphiD1/β
To explore how the structural parallels of the AmphiD1/β receptor, with vertebrate D1-like dopamine and β-adrenergic receptors, relate to the pharmacological properties of the receptor, we stably expressed AmphiD1/β in CHO cells. We then initially screened the effectiveness of a range of common biogenic amine ligands at a concentration of 1 μM for their abilities to alter forskolin-stimulated intracellular cAMP levels in the CHO cell line expressing AmphiD1/β. Table 1A shows that dopamine is the most effective ligand tested and that both adrenaline and noradrenaline also produced significant increases in cAMP levels. Tyramine, octopamine and histamine did not produce any significant increases in cAMP levels when tested at concentrations up to 1 μM. None of the biogenic amines tested were found to have an effect on cAMP levels in wild-type CHO cells (data not shown).
|Biogenic amine||% Forskolin-stimulated cAMP levels||n|
|Dopamine||350.8 ± 26.1**||≥ 3|
|Noradrenaline||122.4 ± 8.4*||≥ 3|
|Adrenaline||120.8 ± 4.3**||≥ 3|
|Histamine||105.7 ± 4.2||≥ 3|
|Octopamine||102.5 ± 0.6||≥ 3|
|Tyramine||101.1 ± 7.8||≥ 3|
|Biogenic amine||% Forskolin-stimulated cAMP levels||n|
|Dopamine||319.20 ± 64.15**||3|
|5-HT||165.10 ± 14.28**||3|
|Biogenic amine||% of Response to 1 μM dopamine||n|
|5-HT||77.8 ± 13.9**||5|
|Noradrenaline||24.7 ± 7.4**||4|
|Tyramine||18.0 ± 2.1**||7|
|Adrenaline||8.9 ± 4.7*||4|
Full dose–response curves were obtained for the ability of dopamine, adrenaline and noradrenaline to increase cAMP levels in the CHO cells stably expressing AmphiD1/β (Fig. 1a). Dopamine showed a threshold response between 0.1 and 1 nM whilst noradrenaline and adrenaline showed much higher thresholds between 0.1 and 1 μM. Dopamine produced a much higher Emax than the other two biogenic amines and was at least two orders of magnitude more potent than both adrenaline and noradrenaline. (EC50: dopamine, 0.057 μM; adrenaline, 4.4 μM; noradrenaline, 12.0 μM). At higher concentrations, between 10 and 100 μM, the effectiveness of dopamine decreased, probably because of desensitization of the AmphiD1/β receptor.
The AmphiD1/β receptor also showed some structural parallels with vertebrate 5-HT receptors (see section, Results, Cloning of AmphiD1/β). However, we were unable to test the effectiveness of this biogenic amine on our stable transfected CHO cell line because wild-type CHO cells expressed endogenous 5-HT1B receptors which could decrease cAMP levels (Giles et al. 1996). Therefore, we transiently transfected AmphiD1/β into HEK293 cells because they did not express endogenous 5-HT receptors (Alberts et al. 2001). A comparison of the effectiveness of the ability of 5-HT and dopamine, at a concentration of 1 μM, to increase intracellular cAMP levels in such cells showed that dopamine was over three times more effective than 5-HT (Table 1B).
G protein-coupled receptors (GPCR) can be coupled to multiple second messenger pathways and such coupling may be agonist specific (Evans et al. 1995; Kenakin 1995) depending on the specific conformations of the GPCR induced by agonist binding. In addition, such second messenger interactions may also be cell specific depending on the complement of G proteins expressed in a particular cell type. To explore these possibilities we have also transiently expressed the AmphiD1/β receptor in Xenopus oocytes. Application of 2 min pulses of 1 μM dopamine generated inward currents of 87.3 ± 7.8 nA (n = 68) because of the activation of the endogenous inward calcium-dependent chloride current in oocytes expressing AmphiD1/β. Uninjected and water injected control oocytes showed no responses (data not shown). A comparison of the effectiveness of a range of biogenic amines to activate such inward currents in oocytes expressing AmphiD1/β is shown in Table 1C. It can be seen that dopamine is again the most effective agonist tested with 5-HT being less effective. The effects of both dopamine and 5-HT were again dose-responsive (Fig. 2) with dopamine showing a threshold effect between 1 and 10 nM and 5-HT a threshold between 10 and 100 nM. Dopamine had a higher Emax than 5-HT but both amines had similar potencies (EC50: dopamine, 0.67 μM; 5-HT 0.54 μM), even though 5-HT was only 77.8% as effective as dopamine at 1 μM. However, there were some differences between the effectiveness of the biogenic amines on AmphiD1/β depending on whether it was expressed stably in CHO cells or transiently in Xenopus oocytes. Thus, whilst noradrenaline was an effective agonist in both cell types, adrenaline was relatively more potent than tyramine on AmphiD1/β receptors expressed in CHO cells and tyramine was relatively more potent than adrenaline on AmphiD1/β receptors expressed in Xenopus oocytes (see Table 1C).
The activation of the inward calcium-dependent chloride currents by AmphiD1/β in Xenopus oocytes was presumably because of the receptor mediated activation of phospholipase C which increased phosphatidylinositol hydrolysis and stimulated the release of calcium from intracellular stores in oocytes [see Masu et al. (1987)]. Thus, we also examined the ability of a range of biogenic amines to alter intracellular calcium levels in CHO cells stably expressing AmphiD1/β using the fluorescent dye fura-2 [see Srivastava et al. (2005)]. Neither dopamine, adrenaline nor noradrenaline altered intracellular calcium levels in such cells when tested at concentrations up to 10 μM (data not shown). In contrast, control pulses of 10 μM ATP were effective at raising intracellular calcium levels (data not shown) when applied at the end of each experiment as reported previously (Iredale and Hill 1993). This suggests that the ability of agonist activated AmphiD1/β to couple to changes in intracellular calcium levels is cell type specific.
Action of synthetic agonists on AmphiD1/β
The AmphiD1/β receptor showed the highest structural parallels to vertebrate β-adrenergic receptors, but surprisingly, functionally displayed a preference for dopamine over the other catecholamines. Thus, we proceeded to determine its pharmacological properties by testing a wide range of synthetic biogenic amine agonists for their abilities to alter intracellular cAMP levels in the CHO cell line stably expressing AmphiD1/β. Table 2 shows that at a concentration of 1 μM, the classical dopamine D1 receptor partial agonist, SKF38393, and the classical dopamine D1 receptor agonist, 6-Chloro-APB [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], were both able to cause a significant increase in cAMP levels in stably transfected CHO cells, as could the α2-adrenoreceptor agonist, UK14304. Full dose–response curves were formulated for the dopamine D1 receptor agonists, SKF38393 and 6-chloro-APB (Fig. 1b), and both agonists increased intracellular cAMP levels in a dose-dependent manner. They had similar potencies (EC50: SKF38393 0.258 μM; 6-Chloro-APB 0.32 μM) but 6-Chloro-APB was found to produce a much higher Emax value and was three times as effective as SKF38393 at 1 μM. Therefore, D1-like synthetic agonists (tested at a concentration of 1 μM) were the most effective at activating AmphiD1/β and the receptor had the following rank order of potency when stably expressed in CHO cells: dopamine > 6-chloro-APB > SKF38393 = adrenaline = noradrenaline.
|Synthetic agonist||Specificity||% Forskolin-stimulated cAMP levels||n|
|(±)-6-Chloro-APB||D1||178.1 ± 10.5**||≥ 3|
|R(+)-SKF38393||D1||125.9 ± 11.1**||≥ 3|
|UK14304||α2||124.0 ± 3.3**||≥ 3|
|(±)-Isoprenaline||β||105.0 ± 10.2||≥ 3|
|Phenylephrine||α1||101.1 ± 1.4||≥ 3|
|Quinpirole||D2||94.6 ± 3.2||≥ 3|
|Synthetic agonist||Specificity||% of Response to 1 μM dopamine||n|
|(±)-6-Chloro-PB||D1||37.8 ± 9.0**||6|
|R(+)-SKF38393||D1||22.9 ± 2.9**||6|
|(±)-6-Chloro-APB||D1||14.9 ± 1.7**||6|
|Quinpirole||D2||14.3 ± 4.4**||3|
|R(+)-6-Bromo-APB||D1||10.9 ± 2.4**||4|
|Quinelorane||D2||5.9 ± 2.1**||6|
|(±)-PPHT||D2||5.0 ± 1.4**||4|
|PD-128907||D3||2.2 ± 0.9||3|
|(±)-Isoprenaline||β||1.3 ± 0.6||6|
To determine if the AmphiD1/β receptor showed agonist-specific coupling when expressed in different cell types, where it is coupled to different effector systems in the same way as a Drosophila D1-like dopamine receptor (Reale et al. 1997), we also compared its synthetic agonist rank order of potency when it was expressed in Xenopus oocytes. Table 2B shows that when expressed in Xenopus oocytes dopamine D1 receptor agonists, such as 6-Chloro-PB [(+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] and SKF38393, were again more effective than the D2 agonists, such as Quinpirole and Quinelorane, the β-adrenergic receptor agonist, Isoprenaline and the α-adrenergic agonists, UK14304 and phenylephrine. However, SKF38393 was more effective on the receptor than 6-Chloro-APB when it was expressed in oocytes, whilst the converse was true when the receptor was expressed in CHO cells. In addition, UK14304 was much more effective on the receptor when it was expressed in CHO cells than when it was expressed in oocytes. Further, Quinpirole was a more effective agonist when the receptor was expressed in oocytes than when it was expressed in CHO cells. Thus, AmphiD1/β appeared to show agonist-specific coupling to different second messenger pathways for a range of synthetic agonists.
Action of synthetic antagonists on AmphiD1/β
To further increase our knowledge of the pharmacological profile of AmphiD1/β we tested the effects of a wide range of pharmacological antagonists known to act at various mammalian biogenic amine receptor subtypes. We tested these compounds on AmphiD1/β expressed in both CHO cells and Xenopus oocytes because recent evidence suggested that in some cases synthetic antagonists might also display ligand selectivity, depending on which second messenger pathway the receptor was coupled (see Galandrin and Bouvier 2006).
Initially, synthetic antagonists were screened at a concentration of 1 μM for their ability to inhibit the increase in intracellular cAMP levels mediated by 100 nM dopamine in CHO cells stably expressing AmphiD1/β (Table 3A). The rank order of effectiveness for the antagonists is as follows: flupenthixol > R(+)SCH23390 > (+)-butaclamol = phentolamine > dl-propranolol. None of the antagonists tested were found to alter cAMP levels in stably transfected CHO cells when tested alone (data not shown). Therefore, butaclamol and flupenthixol, which acted as inverse agonists at vertebrate D1 receptors (Cai et al. 1999), did not act as inverse agonists at AmphiD1/β. Moreover, in both stably transfected CHO cells and transiently transfected HEK293 cells, the basal level of cAMP was not altered by the expression of AmphiD1/β (data not shown), thus indicating that AmphiD1/β was not constitutively active.
|Antagonist||Specificity||% Dopamine-stimulated cAMP levels||n|
|Flupenthixol||D1/D2||5.0 ± 7.3**||≥ 6|
|R(+)SCH23390||D1||23.5 ± 8.9**||≥ 6|
|(+)-Butaclamol||D1/D2||53.6 ± 14.6**||≥ 6|
|Phentolamine||α||71.7 ± 7.9**||≥ 6|
|dl-Propranolol||β||82.2 ± 12.6*||≥ 6|
|Spiperone||D2||110.7 ± 6.3||≥ 6|
|Antagonist||Specificity||% of Response to 1 μM dopamine||n|
|Flupenthixol||D1/D2||33.4 ± 6.3**||7|
|R(+)SCH23390||D1||68.7 ± 1.0**||4|
|Domperidone||Peripheral D2||72.9 ± 7.6**||5|
|S(−)-Eticlopride||D2||73.9 ± 11.8**||5|
|dl-Propranolol||β||74.2 ± 7.8**||9|
|Phentolamine||α||79.8 ± 3.9*||3|
|(+)-Butaclamol||D1/D2||84.1 ± 8.6||9|
|Spiperone||D2||87.8 ± 5.5||11|
|S(−)-Sulpiride||D2||89.6 ± 4.3||8|
A similar rank order of potency was observed for the effects of synthetic antagonists at inhibiting the actions of 1 μM dopamine on inward currents generated in Xenopus oocytes expressing AmphiD1/β (Table 3B). Again flupenthixol and R(+)SCH23390, agents with vertebrate dopamine D1-like actions were the most effective blocking agents tested, whilst a range of other antagonists with vertebrate dopamine D2 and α- and β-adrenergic-like actions were less effective. Thus, in both preparations AmphiD1/β displayed vertebrate D1-like pharmacological properties, independently of the second messenger pathway to which the receptor was coupled.
Action of AmphiD1/β on the mitogen-activated protein kinase cascade
Both vertebrate dopamine D1 receptors (Chen et al. 2004) and β-adrenergic receptors (Luttrell et al. 1999; Drube et al. 2006; Galandrin and Bouvier 2006) have also been shown to signal via the MAPK cascade. Therefore, dopamine was tested on CHO cells stably transfected with AmphiD1/β to see if it could alter the levels of phosphorylated ERK. Figure 3 shows that exposure of CHO cells stably transfected with AmphiD1/β to 1 μM dopamine for 2, 5, 10 and 15 min led to an increase in phosphorylated ERK levels. Control experiments with wild-type CHO cells showed that incubation with 1 μM dopamine for 5 min did not cause an increase in phosphorylated ERK levels (data not shown). The activation of ERK by dopamine was also found to be dose-dependent (Fig. 4). At low concentrations of dopamine (3 nM–100 nM) a decrease in ERK phosphorylation was observed when compared with basal (vehicle treated) levels. However, a significant increase in phosphorylated ERK levels was observed when stably transfected CHO cells were treated with 1 μM dopamine. Concentrations of dopamine above 1 μM were not tested because of the fact that 10 and 100 μM dopamine increased the levels of phosphorylated ERK in wild-type CHO cells (data not shown), possibly because of cross-reactivity with endogenous receptors in CHO cells, such as the 5-HT1B receptor (Giles et al.1996).
The present study suggests that AmphiD1/β functions as a dopamine D1-like receptor when expressed in either CHO cells or Xenopus oocytes. Dopamine was found to be the most potent agonist tested, and receptor activation by dopamine was coupled with an increase in cAMP when the receptor was expressed in CHO cells, a characteristic of other D1-like dopamine receptors (Neve et al. 2004). AmphiD1/β could be activated by the classical D1 receptor agonists, SKF38393 and 6-chloro-APB, with SKF38393 acting as a partial agonist, as it did on vertebrate D1 receptors (O’Boyle et al. 1989). Despite showing a high amount of structural similarity to the β-adrenergic receptors (Vincent et al. 1998; Candiani et al. 2005; Burman et al. 2007), noradrenaline and adrenaline, the cognate ligands for the β-adrenergic receptors, could only activate AmphiD1/β with much lower Emax values and higher EC50 values than dopamine. Moreover, isoprenaline, a β-adrenergic receptor agonist, was unable to activate AmphiD1/β to cause an increase in cAMP in CHO cells or to cause a significant inward current in Xenopus oocytes expressing AmphiD1/β. Thus, one might expect that the ligand binding pocket of AmphiD1/β might show more commonalities with that of vertebrate D1-like receptors than with that of β-adrenergic receptors. However, AmphiD1/β shared only 2 (3.36 and 7.43) out of the 7 classical specific vertebrate D1 receptor binding residues and only 1 (2.52) out of the 4 classical specific β-adrenergic receptor binding residues (see Xhaard et al. 2006).
The ability of dopamine D1-like receptors to stimulate inositol phosphate production and to induce intracellular calcium mobilization is controversial (see Reale et al. 1997) and may vary from one cell type to another. Thus, many cloned D1-like receptors do not couple to inositol phosphate production when expressed in CHO or baby hamster kidney cells (Pedersen et al. 1994) or in COS-7 cells (Demchyshyn et al. 1995). Similarly, AmphiD1/β (present study) did not generate changes in intracellular calcium levels when expressed in CHO cells. Conversely, dopamine D1-like receptors did affect calcium levels when expressed in Ltk cells (Bouvier et al. 1993) or in HEK 293 cells (Frail et al. 1993). In addition, several studies have demonstrated that D1-like receptors when expressed in Xenopus oocytes can generate inward currents caused by the activation of endogenous calcium-dependent chloride currents. Thus, dopamine D1-like receptors coupling to inositol phosphate production and calcium mobilization were demonstrated when rat striatal mRNA was expressed in Xenopus oocytes (Mahan et al. 1990). Further, mRNA encoding the Drosophila D1-like receptor, DopR99B (Feng et al. 1996; Reale et al. 1997), when expressed in Xenopus oocytes, generated intracellular calcium signals which were pertussis toxin insensitive, suggesting that they were likely to be mediated by G proteins of the Gq or G11 subclass. Similarly, in the present study, AmphiD1/β was able to generate inward currents by the activation of the endogenous calcium-dependent chloride currents when expressed in Xenopus oocytes. Thus, the strength of coupling of dopamine D1-like receptors to either the production of cAMP or to the generation of intracellular calcium signals, can vary from one cell type to another depending on their local G protein environments.
AmphiD1/β displayed ‘agonist-specific coupling’ (also called ‘agonist trafficking’, ‘biased agonism’ or ‘ligand-biased signalling’) to various second messenger pathways for different synthetic agonists and biogenic amines. Parallel findings have been made for a wide range of GPCRs including the dopamine D1-like receptor, DopR99B (Reale et al. 1997), and the Octopamine/Tyramine receptor (Robb et al. 1994) from Drosophila and for vertebrate β-adrenergic receptors (Galandrin and Bouvier 2006; Weitl and Seifert 2008). It is now well established that different agonists can differentially stabilize different conformations of GPCRs which can each exhibit different coupling efficiencies to different second messenger pathways [see Evans et al. (1995); Kenakin (1995, 2007); Galandrin et al. (2008); Wess et al. (2008)].
At the antagonist level, AmphiD1/β also showed a dopamine D1-like pharmacology in both expression systems. The most potent antagonists tested, flupenthixol and SCH23390, also blocked the vertebrate D1 receptors. Interestingly, AmphiD1/β was found to respond to 1 μM of both SKF38393 (agonist) and SCH23390 (antagonist). These benzodiazepine ligands that act at vertebrate dopamine D1 receptors, are generally thought to act only at much higher concentrations, or not at all, at the invertebrate dopamine D1-like receptors (Mustard et al. 2005). However, the Drosophila DopR99B dopamine D1-like receptor, although only responding poorly to SKF 38393, was significantly blocked by 1 μM SCH23390 (Reale et al. 1997). Hence, because the ability to respond to SKF38393 and SCH23390 is generally regarded as a property of only vertebrate D1 receptors, it suggests that amphioxus has a D1 receptor that displays some properties of vertebrate-specific D1 receptors. The β-adrenergic receptor antagonist, propranolol, only caused a small inhibition of the effects mediated by dopamine stimulation of AmphiD1/β. This suggests that AmphiD1/β does not share any pharmacological properties with the vertebrate β-adrenergic receptors. In the present study AmphiD1/β was found not to display any constitutive agonist-independent activity. Moreover, flupenthixol and butaclamol, which usually acts as inverse agonists at vertebrate D1 receptors (Cardinaud et al. 1997; Cai et al. 1999), were not found to act as inverse agonists at AmphiD1/β when it was expressed in mammalian cell lines. The lack of constitutive activity, and of inverse agonist activity by flupenthixol and butaclamol at AmphiD1/β, is a feature that distinguishes it from the vertebrate D1 receptors (Cardinaud et al. 1997; Cai et al. 1999).
Many GPCRs have been found to couple to the modulation of the MAPK cascade, including both dopamine D1 receptors (Chen et al. 2004) and β-adrenergic receptors (Luttrell et al. 1999; Drube et al. 2006). In the present study AmphiD1/β was found to couple to a concentration-dependent modulation of the MAPK cascade, as measured by changes in phosphorylated ERK levels. At low concentrations of dopamine AmphiD1/β coupled to a decrease in phosphorylated ERK levels. A possible explanation is that the dopamine-mediated increase in cAMP levels is causing an inhibitory input to the MAPK pathway. For example, protein kinase A, which is activated upon increasing concentrations of intracellular cAMP, is known to inhibit Raf activation, thereby leading to attenuation of the MAPK cascade (Schmitt and Stork 2002). In addition, AmphiD1/β may not efficiently activate the receptor internalization machinery and thus have a low efficiency for ERK phosphorylation. Conversely, 1 μM dopamine was found to increase phosphorylated ERK levels and this concentration of dopamine was also found to maximally activate cAMP levels. It is likely that the cAMP response elicited by 1 μM dopamine acting at AmphiD1/β is limited by the number of available Gαs G proteins. AmphiD1/β may then be able to couple to other G proteins, such as Gαi, to couple to the activation of the MAPK cascade. A similar scenario has been observed with the α2C-adrenergic receptor (Eason et al. 1992), whereby low concentrations of UK14304 caused receptor activation, Gαi coupling and inhibition of adenylyl cyclase, yet high concentrations of UK14304 cause receptor activation, Gαs coupling, and adenylyl cyclase activation.
AmphiD1/β has been shown to have pharmacological properties similar to vertebrate-type dopamine D1-like receptors. A study analysing the expression pattern of AmphiD1/β during amphioxus development revealed that its expression pattern is homologous to that of vertebrate D1 receptors (Candiani et al. 2005). AmphiD1/β was found to be expressed in cells that corresponded to ganglion cells of the vertebrate retina and in cells in the infundibular organ, which was thought to be a homologue of the vertebrate subcommisural organ (Candiani et al. 2005). Correspondingly, the vertebrate retina (Fujieda et al. 2003) and subcommisural organ (Olsson et al. 1994) also express D1 receptors. Additionally, expression of AmphiD1/β did not overlap with that of the dopamine-synthesizing enzyme tyrosine hydroxylase (Candiani et al. 2005). This is also consistent with role of AmphiD1/β as a dopamine D1 receptor, because vertebrate D1 receptors are known not to act as autoreceptors which are expressed on the same pre-synaptic neurones that synthesize and release dopamine. On the other hand, both β-adrenergic receptors (Gothert 1985) and dopamine D2 receptors (Missale et al. 1998) are known to act as autoreceptors. Importantly, dopamine and tyrosine hydroxylase have been found to be expressed in both the anterior and posterior brain-like region of amphioxus (Moret et al. 2004). However, only very small amounts of noradrenaline were found to be present in the head, trunk and tail of amphioxus. In fact, the authors concluded that the noradrenaline present in amphioxus was a byproduct of the biosynthesis of dopamine and octopamine (Moret et al. 2004).
Based on the pharmacological characterization it seems that AmphiD1/β is likely to be an amphioxus orthologue of vertebrate-type dopamine D1 receptors. When expressed heterologously in mammalian cell lines AmphiD1/β couples to an increase in intracellular cAMP, a characteristic of dopamine D1 receptors. Additionally, it responds to both agonists and antagonists that specifically target vertebrate-type dopamine D1 receptors. It is interesting that amphioxus, a deuterostomian invertebrate thought to be the closest living relative of the most basal chordate, has a dopamine D1 receptor with many vertebrate characteristics. Future studies focussed on the pharmacological characterization of the two remaining uncharacterized amphioxus D1-like receptors, AmphiAmR1 and AmphiAmR2, are required to elucidate the specific complement of dopamine D1 receptors in amphioxus. It is hoped that these studies will be able to provide insights into how dopamine D1 receptors have evolved throughout chordate evolution into the well-characterized D1 receptor subtypes found in mammals.
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Materials and Methods
Fig. S1 Multiple sequence alignment of AmphiD1/β, dopamine D1 receptor (carp, D1R), β1-adrenergic receptor (Xenopus, B1AR), 5-HT 1A receptor (Zebrafish, 5-HT1A) and Drosophila, DmDopEcR.
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