Biology of the Cell 95 (2003) 489–502
www.elsevier.com/locate/bicell
Review
Evolution and cell biology of dopamine receptors in vertebrates
Sophie Callier, Marina Snapyan, Stéphane Le Crom, Delphine Prou,
Jean-Didier Vincent, Philippe Vernier *
Développement, évolution, plasticité du système nerveux, UPR 2197, Institut de Neurobiologie Alfred Fessard, CNRS, Gif-sur-Yvette, France
Received 30 June 2003; accepted 2 July 2003
Abstract
Dopamine, one of main modulatory neurotransmitters of the nervous system acts on target cells through two classes of G protein-coupled
receptors, D1 and D2. The two dopamine receptor classes display different structures, interact with different regulatory partners (including
heterotrimeric G proteins) and, accordingly, have independent evolutionary origins. In vertebrates, each of these receptor classes comprises
several subtypes, generated by two steps of gene duplications, early in vertebrate evolution. In the D1 receptor class, the D1A, D1B, D1C and
D1D subtypes, and in the D2 class, the D2, D3 et D4 receptor subtypes have been conserved in most vertebrate groups. This conservation has
been driven by the acquisition, by each receptor subtype, of a small number of specific properties, which were selected for adaptive purpose
in vertebrates. Among these properties, affinity for dopamine, the natural ligand, intrinsic receptor activity, and agonist-induced desensitization clearly distinguish the receptor subtypes. In addition, each dopamine receptor subtype is addressed to a specific location within neuronal
networks, although detailed information is lacking for several receptor subtypes. Receptors localization at diverse subcellular places in
neurons may also differ from one subtype to another, resulting in different ways of regulating cell signalisation. One challenge for future
research on dopamine and its receptors would be to identify the nature of the protein partners and the molecular mechanisms involved in
localizing receptors to the neuronal plasma membrane. In this respect, the evolutionary approach we have undertaken suggests that, due to gene
duplications, a reasonable degree of freedom exists in the tight organisation of dopamine receptors in neurons. This “evolvability” of dopamine
systems has been instrumental to adapt the vertebrate species to nearly all the possible environments.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Receptor trafficking; Phylogeny; Desensitization; Cell signalling; Oligomerization
In Vertebrates, dopamine is a neurotransmitter acting as a
modulator of neuronal activity, regulating thereby many different functions in the central nervous system. This modulatory action applies to sensory perception in the retina and
olfactory bulb, regulation of prolactine release in the pituitary gland, control of body temperature, food intake and
sexual behavior in the hypothalamus, tuning of sensorimotor cues in the basal ganglia. In addition, dopamine systems are central to the maintenance and expression of the
qualitative values of novelty in life experiences and, thus,
motivation or aversion.
The various effects of dopamine on physiological systems
or organs are mediated by multiple metabotropic membrane
receptors coupled to heterotrimeric G proteins (GPCR). As
* Corresponding author: DEPSN, UPR 2197, Institut de Neurobiologie
A. Fessard, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette cédex,
France. Tel: 33–169 82 41 88; Fax: 33–169 82.
E-mail address: [email protected] (P. Vernier).
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
doi:10.1016/S0248-4900(03)00089-3
much as seven receptor proteins encoded by different genes
have been isolated so far in vertebrates. The interest for
dopamine receptors arose from the discovery of alteration in
dopamine transmission in several human pathologies, essentially Parkinson’s disease, addiction to drugs of abuse, disorders of mood and affect (schizophrenia), prolactinomas of
the anterior pituitary gland, hypertension or cardiovascular
failure. Accordingly, dopamine receptors are the targets of
major drug classes (neuroleptics, antiparkinsonian drugs,
etc. (Strange, 1993; Seeman and Van Tol, 1994; Sokoloff and
Schwartz, 1995).
The fact that dopamine is able to act on target cells via
several different receptor molecules is common to most neurotransmitters, but the necessity for such a molecular diversity, as well as its physiological consequences are far from
obvious. This is a frequently asked question in the field of
molecular biology and genetics. Dopamine receptor subtypes share similar sequences, structures and functions. Ac-
490
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
cordingly, they are encoded by a gene family, the members of
which have been generated by events of gene duplications,
followed by selection of the duplicated genes (Vernier et al.,
1995). Therefore, understanding how the genetic multiplicity
of G protein coupled receptors has been created, and which is
its functional counterpart, can only be achieved through an
evolutionary approach (Kirschner and Gerhart, 1998). Indeed, diversification of dopamine receptors is primarily an
historical phenomenon, and a better knowledge of this history is a prerequisite to any experimental analysis of the
structures and functions of dopamine receptors present in
different animal species. On the one hand, resemblances in
gene structures highlight the conservation of fundamental
functions of dopamine receptors in organisms. On the other
hand, differences rely on the occurrence of discrete or large
mutational events that led to modify tissue-specific gene
expression and function of corresponding proteins. These
differences have been selected for adaptive purpose all along
evolution. Comparative analysis that brings together molecular data and adaptive physiological features is more and more
necessary to build an integrated view of dopamine receptors,
from molecular neurobiology to animal behaviour.
1. A brief history of the evolution of dopamine receptor
genes in vertebrates
In vertebrates, dopamine receptors belong to two classes
of G protein-coupled receptors. The classes of dopamine
receptor were initially defined by their ability to modulate
adenylyl cyclase activity and cAMP accumulation in cells
(Kebabian and Calne, 1979). D1 receptors are able to activate, whereas dopamine D2 receptors decrease adenylyl cyclase activity. However, it is now established that the D2
receptors are mainly responsible for modulating the activity
of voltage-sensitive Ca++ and K+ channels. Lately, many
pharmacological compounds were used to discriminate these
two receptor classes, establishing the concept of multiple
receptors for dopamine (reviewed in Sokoloff and Schwartz,
1995; Missale et al., 1998).
As for most other neurotransmitter receptors, molecular
cloning confirmed that D1 and D2 receptors constituted two
different classes of dopamine receptors. In mammals, two
receptor subtypes have been assigned to the D1 receptor class
(D1A/D1 and D1B/D5), but other subtypes exist in nonmammalian vertebrates (D1C and D1D; reviewed in Kapsimali et
al., 2003). Similarly, three subtypes of D2 receptors have
been isolated in jawed vertebrates (D2, D3, D4). The overall
protein structure and topology deduced from receptor sequences reveals major differences between the two classes of
dopamine receptors. The D2 receptors have a long third
cytoplasmic loop and a short cytoplasmic C–terminal end,
whereas D1 receptors exhibit a shorter third cytoplasmic loop
but a very long C terminal tail. These protein segments are
involved in coupling with heterotrimeric G protein, and accordingly, D1 and D2 receptors interact with different classes
of heterotrimeric G proteins. The D1 receptors are coupled to
the Gs/Golf class of Ga proteins, whereas the D2 receptors
coupled essentially to Gi/Go proteins, therby modulating
different intracellular signalling pathways. These facts lead
to regard the two classes of dopamine receptors as very
distinct, except they are both able to bind dopamine as neurotransmitter. In addition, the existence of multiple receptor
subtypes, as found in vertebrates, cannot be understood without knowing more of their evolutionary origin and physiological roles.
The question of the evolutionary origin of the dopamine
receptor subtypes in vertebrates can been addressed by analyzing the molecular phylogeny of the corresponding genes.
When dopamine receptors are compared to other monoamine
receptors (adrenergic a1, a2, b, or trace amines, or serotonergic 5HT1, 5HT2, 5HT4, 5HT5, 5HT6, 5HT7), molecular phylogenies clearly indicate that D1 and D2 receptor classes are
unrelated, or at least not more closely related to each other
than they are to other classes of monoamine receptors
(Fig. 1). This is consistent with the distinct structural characteristics and functional properties of the D1 and D2 dopamine
receptors. It implicates that they have acquired independently and convergently the ability to bind dopamine, as in
the case of the other classes of monoamine receptors.
However, if molecular phylogeny highlights the structural
relationships of dopamine receptors, it cannot provide, alone,
robust hypotheses on the duplications and evolution of
dopamine receptors in the vertebrate phylum. With this perspective in mind, the whole complement of dopamine receptors has been isolated in species belonging to groups of
vertebrates diverging at different times of vertebrate evolution. The D1 receptors having no intron, all the members of
the gene family can be isolated rather easily from genomic
DNA. A similar approach is more difficult for the D2 receptors, the genes of which bear several large introns inside their
sequence.
In jawless vertebrates (agnathans), only one D1–like sequence has been found in lampreys and in hagfish. It is thus
very likely that only one type of D1 receptor exist in these
species that descend from the earliest diverging vertebrates.
Interestingly, these receptor sequences are not related to any
of the known D1 receptor sequences from jawed vertebrates.
Thus, they probably diverged from the ancestor of the vertebrate D1 receptor before the occurrence of gene duplications
generating the dopamine receptor subtypes found in jawed
vertebrates (gnathostomes; Fig. 1). Accordingly, from cartilaginous fish (the electric ray Torpedo marmorata) to amphibians (the toad Xenopus lævis), three different D1 receptor
sequences have been found (Sugamori et al., 1994). In these
species, the phylogenetic tree easily depicts three subtypes,
namely, D1A, D1B and D1C. In teleost fish (European eel,
puffer fish, medaka, zebrafish) extra D1 receptor sequences
are found (Cardinaud et al., 1997), probably corresponding
to gene duplication events that occurred specifically in teleost fish (Aparicio, 2000).
In amniotes, the situation is even more complicated
(Fig. 1). A fourth D1 receptor-related sequence has been
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
491
Fig. 1. A phylogenetic tree of the monoamine receptor classes and hypothesis on the evolution of dopaminre receptors in vertebrates.
On the left part of the figure, the sequence relationships of dopamine receptor classes (D1 and D2) has been analyzed, together with those of the main other
monoamine receptor classes. After sequence alignment, a distance algorithm has been used to compute identities and differences, and the corresponding tree is
shown here. The tree has been rooted on the muscarinic receptor group, and branch length is proportional to the overall similarities between receptor sequences.
Only one example of receptor subtypes (human in general) has been used for the serotonin 5HT1, 5HT2, 5HT4 receptor classes (green boxes), the class of trace
amine receptors (TA; blue box) as well as for the adrenergic a1, a2 and b receptors (yellow boxes). The tree clearly shows that each class of monoamine receptor
is very distantly related to each other (including the D1 and D2 receptor class), suggesting a very ancient origin, long before the emergence of vertebrates. For
the dopamine receptors, the subtypes found in the various vertebrate phylum have been added, highlighting the presence of four subtypes in the D1 class and
three subtypes in the D2 class, overall. Hypothesis on the evolution of dopamine receptors drawn from the phylogenetic analysis is shown on the right part of the
figure. Two steps of gene duplications (1 and 2 in the figure), which occurred between the divergence of jawless vertebrates (agnathans) and the emergence of
jawed vertebrates (gnathostomes), generated the four D1 receptor paralogues (see text for details). Although it is very likely that a similar situation happened for
the D2 receptors, no strong eveidence is available yet. After gene duplications, the dopamine receptor subtypes acquired new functional characteristics
(dopamine affinity, square; intrinsic activity, circle; desensitization time course, triangle) that contributes to the conservation of the corresponding genes in the
numerous species expressing them. In contrast, coupling to either Gs/olf or to Gi/o proteins are ancient properties inherited from the common ancestor of D1
receptors, characterizing the D1 or the D2 receptor class. Accordingly, the agnathans D1 receptor (Agn. D1) also share this property with the other vertebrate D1
receptors. Finally, the ability to bind dopamine is a convergent character of D1 and D2 receptor classes.
492
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
isolated from chicken and crocodiles (archosaurs) and named
D1D (Demchyshyn et al., 1995). This subtype is also present
in lepidosaurs (lizzards and snakes). However, the third
group of amniotes, turtles, expresses D1A and D1B receptor
sequences, but they possess only one more sequence distantly related to the D1C subtype. In mammals, only D1A and
D1B receptor-related sequences have been isolated in eutherian (placental) mammals, as well as in metatherians (marsupials). Interestingly, in the two main species of prototherian mammals, platypus and echidnea, two D1 receptor
sequences have also been found, but they are related to the
D1A and D1D subtypes. The most parsimonious interpretation
of these data is that four different subtypes of D1–related
receptors existed in stem amniotes. Two of them have disappeared in mammals, D1B and D1C in prototherian mammals
and D1C and D1D in the other groups of mammals. In other
words, mammals are an exception among vertebrates since
the D1C receptor subtype is present in all the other groups of
jawed vertebrates, and the D1D subtype is present in all the
amniotes, with the exception of placental and marsupial
mammals.
A detailed analysis of the molecular phylogeny of the D2
receptor class is not possible yet. However, based on current
data, the phylogenetic picture and the evolutionary history of
the D2–like receptors is very similar to that of the D1 receptor
class. Again, three subtypes are easily identified, (D2, D3 and
D4 subtypes; Fig. 1), and they are found in most groups of
jawed vertebrate. Whether one or several D2–like receptors
exist in agnathans is not known at present, and we cannot
write a comprehensive history of the D2 receptor class yet.
Overall, the existence of one subtype of dopamine receptor in agnathans and of two, three or four paralogous receptors in gnathostomes, supports the hypothesis of a double
whole-genome duplication in the evolution of Craniates.
However, it is impossible to know precisely when these
duplications took place, except they should have occurred
between the emergence of the earliest vertebrates and that of
gnathostomes (Fig. 1). After these two gene duplications that
gave birth to four dopamine receptor paralogues, several
groups of species have lost one or two of these genes. For
example, bony fish and amphibians have lost one D1 receptor
sequence (D1D), and the mammals have lost two of these
genes (D1C and either D1B or D1D).
The D1A receptor exhibits the most conserved sequence
and it is the only one found in all the jawed vertebrate
species. It may thus be functionally the most important of the
dopamine receptors, a statement clearly confirmed by the
results of specific knock-out of the D1–related genes in
mouse (Sibley, 1999). In contrast, other dopamine receptor
sequences are relatively more divergent (apparent “evolutionary speed”), as shown by a “long branch” in the phylogenetic tree. This is for example the case for the D4 receptor
subtype in the D2 class, and for the D1C and D1D subtype in
the D1 class. This “long-branch” effect conceals the “true”
evolutionary relationships of the D1 receptors in vertebrates
(see Fig. 1) since a fast evolving sequence will branch deep in
the tree, although they have emerged late during evolution.
Such a biased relationship can only be detected when the all
the paralogous and orthologous genes have been isolated for
a large sample of species, as it is the case for the dopamine D1
receptors.
The molecular analysis of sequence relatedness (molecular phylogenies) cannot tell why some receptor sequences
have been conserved and why others have been lost. Acquisition of a novel functional character or a novel expression
territory, are means by which duplicated genes gain nonredundant characters and were conserved in ancestral species. The following paragraphs give an overview of our
current knowledge of the functional properties of dopamine
receptors.
2. Tissue distribution and subcellular localization
of dopamine receptors
A fundamental aspect of dopamine function in whole
organism is the localization of dopamine receptors in the
various areas of the nervous system or at the periphery of the
body. From a genetic point of view, as soon as two receptors
are differentially expressed in cells, they are no longer redundant, provided the cells remain submitted to the action of the
transmitter. Similarly, the precise subcellular localization of
receptor within the target cells are major features of receptor
function, especially in highly polarized cells such as neurons.
Obviously, a receptor located at a post-synaptic position on
the terminal dendrites will not play the same role as a presynaptic receptor located on nerve terminals. Differential expression or localization of receptors often occurs after gene
duplication, either by acquisition of “new” expression territories or by splitting the expression areas of the ancestral
gene (Aparicio, 2000).
2.1. General features of dopamine receptor distribution in
the vertebrate nervous system
A comparative analysis of dopamine receptors has been
carried out in several important groups of vertebrates including teleost fish (Kapsimali et al., 2000), xenopus (unpublished results), birds (Schnell et al., 1999) and mammals
(Tiberi et al., 1991; Meador-Woodruff et al., 1996; reviewed
in Kapsimali et al., 2003). The D1 and D2 receptors are
present in all of the known target areas of dopamine in the
central nervous system of vertebrates and their expression
territories exhibit considerable overlap. However the question about whether the different receptor subtypes are present
or not in the same cells has not been definitely resolved
(Gerfen, 2000). Mostly, however, neurons are not expressing
simultaneously D1 and D2 receptors, or only at very different
levels, revealing that the transcriptional regulation of the two
dopamine receptor classes is essentially non redundant.
The overall distribution of dopamine receptors in the central nervous system is probably conserved in many vertebrate
species, since, as far as we know, the distribution pattern of
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
493
Table 1
Distribution of dopamine receptors in vertebrate brains:
The table reports in a semi-quantitative manner the relative tissue distribution of the various dopamine receptor subtypes. This relative distribution is well
conserved for all the species of gnathostomes that have been analyzed so far
Brain region
Cortex
Hippocampus
Basal ganglia
Striatum ventral
Striatum dorsal
Nucleus accumbens
Olfactory tubercle
Globus pallidus
Ventral tegmental area
Substantia nigra
Midbrain and Brainstem
Nucleus tractus solitarius
Optic tract
Vegetative areas of spinal cord?
Thalamus
Hypothalamus
Dorsal hypothalamus
Ventral hypothalamus
Hypophyse
Amydaloid complex
Retina
Dopamine receptors
D1like
D1A/D1
D1B/D5
+
+
–
++
D2 like
D2
+
+
++
++
++
+
+
+
+
+
+
++
–
–
+
++
++
+
++
+
+
++
++
+
–
+
+
++
++
+
+
+
+
–
++
+
+
–
–
+
+
+
++
+
+
D3
+
+
D4
+
+
++
+
++
+
+
+
+
+
+
+
+
–
+
+
+
++
++ high expression, + low expression, – not detected
the D1 and D2 receptor subtypes is generally similar in fish,
amphibians birds and mammals. A summary of this tissue
distribution is given in Table 1. The distribution of the three,
well-studied paralogous D1 and D2 receptor subtypes displays a picture of large overlap, differential abundance, with
a few specific localizations. This overview indicates that,
after gene duplications, the new paralogous genes have conserved, to a large extent, a similar regulation of tissuespecific expression. However, the few –but significant– differences that exist between subtypes, certainly contributed to
the conservation of the duplicated genes at the origin of
jawed vertebrates and were crucial in setting up many of the
expression characteristics of dopamine receptors found in
modern vertebrates.
Among the D1 receptors, the D1A receptor subtype is the
most abundant, as well as the D2 receptor subtype in the D2
class. This holds true in all the vertebrate species examined
so far. In contrast, the D1C receptor, which is not found in
mammals, is only weakly expressed in the fish and amphibian brain and only in limited areas of the hypothalamus. The
D1B receptor subtype is also less abundant than the D1A
receptor in mammals, birds, amphibians and fish, but it is
specifically present in important functional areas such as the
hippocampus. This characteristic fits well with the higher
conservation of D1A sequences as compared to D1B or D1C
sequences in vertebrates. Similarly, in the D2 class, the D3
and D4 receptors are by far much less abundant in the brain,
as compared to D2 receptors. Moreover, the distribution of
the D4 receptor transcript strikingly overlaps with that of the
D2 receptor, in the dorsal striatum and cortical areas for
example, suggesting a large degree of redundancy between
the two receptor subtypes. Accordingly, the sequence D4
subtype is rapidely diverging, and null mutations are known
in human without phenotypic consequences (Nothen et al.,
1994). The D3 receptor is, on average, expressed at a lower
level than the D2 receptor subtype, but it is found in areas
where no or very little D2 receptors are present, such as in the
islands of Calleja and in the nucleus accumbens . In this latter
case, it is clearly co-express with the D1 receptor, suggesting
specific interactions between the two receptors (Sokoloff et
al., 2003).
These observations have important implications to understand how the various subtypes of the D1 or D2 receptors have
evolved after the duplication of the ancestral genes in vertebrates. The similarities of the expression territories of the D1
receptor genes implicates that the main cis–regulatory elements controlling tissue-specific transcription of genes have
been conserved after the gene duplication process. However,
a few -but significant– differences are observed among regional distributions of the D1 subtypes, probably sustaining
enough functional relevance in order to be conserved.
2.2. Subcellular localization of dopamine receptors
Recent investigations have pointed out that dopamine receptor subtypes exhibit discrete subcellular localizations
494
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
within neuronal networks. A detailed analysis has been made
for the D1A/D1, the D1B/D5 and the D2 receptor subtypes
only, due to the availability of good antibodies. Both D1A/D1
and D2 receptors are located at a post-synaptic location in
dopamine-target neurons in the mammalian brain. For instance, electron microscopy analysis has shown that, within
the pyramidal cells of the primate cortex, the D1 receptor is
expressed predominantly on dendritic spines, whereas the
D1B/D5 receptor is found primarily on dendritic shafts (Smiley et al., 1994; Bergson et al., 1995). In the rat striatum, D2
receptors are more concentrated on spiny dendrites and spine
heads of the medium spiny neurons than on the plasma
membrane of the somata (Levey et al., 1993). Similarly, in
the olfactory bulb, the D2 receptor is found mainly on the
distal part of dendrites and in the spines (Levey et al., 1993;
Yung et al., 1995).
In addition to this typical post-synaptic location, both the
D1A/D1 and the D2 receptor subtypes have been found at
presynaptic sites within axon terminals, although typically
not within the same projection pathway (Huang et al., 1992;
Levey et al., 1993; Smiley et al., 1994). In the dopaminergic
neurons of the nigrostriatal and tegmental ventral area, D2
receptors are highly concentrated in the distal region of
dendrites and proximal part of dendrites, but also on the
nerve terminals located far away from the cell bodies in the
striatum. Interestingly, the mammalian D2 receptor exists in
two isoforms generated by the alternative splicing of a small
region corresponding to the third cytoplasmic loop of the
receptor structure. Semi-quantitative PCR and immunohistochemical studies showed that the short D2S receptor predominates in the cell bodies and projection axons of dopaminergic neurons of the mesencephalon and hypothalamus
(Guivarch et al., 1995). In contrast, the long D2L receptor
isoform is more highly expressed in the neurons of striatum
and nucleus accumbens (Khan et al., 1998), suggesting that
the differential splicing of the D2 receptor may result in
preferential subcellular localization within neurons. These
studies also revealed an unusual localization of D2 receptors
in perinuclear and other intracellular membrane compartments, probably the endoplasmic reticulum (ER).
This observation agrees with a detailed analysis of the
subcellular localization of the mammalian dopamine D2 receptor carried out in various cell types (Prou et al. 2001).
Indeed, after cell transfection, most of the newly synthesized
receptors accumulate in large intracellular compartments,
whereas the plasma membrane was only weakly labelled.
Double-labelling experiments showed that the D2 receptor
was mostly retained in the ER, the long isoform more
strongly than the short one. This retention is accompanied by
a striking vacuolization of the ER, roughly proportional to
the expression levels of the two receptor isoforms. This
phenomenon seems to be a consequence of the high intrinsic
activity of the D2 receptor, promoting activation of heterotrimeric G protein inside the secretory pathway and blocking
further transport in the ER. Such a mechanism may contrib-
ute to the regulation of the intracellular trafficking of the D2
receptors isoforms (Prou et al., 2001).
However, the mechanisms involved in transport, sorting
and targeting of dopamine receptors subtypes to discrete
subcellular locations remain almost entirely unknown. Recently, the proximal C–terminus of the protein sequence was
shown to be crucial for the targeting of D1–like receptors to the
cell surface. A four amino acid spacing of hydrophobic
residues within the proximal C terminus of D1A/D1 and of the
D1B/D5, the FxxxFxxxF motif, is highly conserved among
GPCR and has been suggested to interact with caveolin
(Bermak et al., 2001). All three F residues within this motif
are required for normal D1 targeting to the plasma membrane. In fact, the single substitution of any of the F to A
resulted in improper processing and trapping of the mutated
receptor in the ER. The membrane-bound protein DRiP78,
which is specifically localized in the ER and physically
interacts with the FxxxFxxxF motif, is able to regulate transport of the D1 receptor from the endoplasmic reticulum to the
cell surface (Bermak et al., 2001). Whether similar mechanism is used for the D1B/D5 receptor is not known. However,
it was found, that N–linked glycosylation, which takes place
in the ER, is required for the correct membrane targeting of
D1B/D5 receptor, but not for the D1A/D1 subtype (Karpa et al.,
1999). Interaction between the c subunit of COP-I coatomer
and the C–terminus of D1 receptor has also been established,
suggesting that this interactions may have a role in receptor
transport (Bermak et al., 2002). Finally, the D1 receptors
have been shown to interact with neurofilament-M, these
latter being able to reduce the number of receptors in the
plasma membrane and to decrease agonist-induced desensitization of the receptor (Kim et al., 2002).
A few laboratories have undertaken two-hybrid screens in
yeast to identify dopamine receptor-interacting proteins that
may regulate intracellular trafficking of the dopamine receptors subtypes. The D2 receptors have now been shown to
interact with several proteins, such as spinophilin (Smith et
al., 1999), filamin-A (Lin et al., 2001), actin binding protein
ABP-280 (Li et al., 2000) and protein 4.1 N (Binda et al.,
2002, Kabbani et al., 2002). These proteins all are
cytoskeleton-associated proteins and they are specifically
enriched in neurones. However, the role they have in targeting or stabilizing dopamine receptors at specific subcellular
domains is not well understood yet. It is also possible that
these cytoskeletal proteins may participate as scaffolding
proteins in organizing the dopamine receptor-signalling
complex. Almost all the experiments performed to analyze
interactions between these proteins and dopamine receptors
were carried out in cells lines and we do not know if these
interactions also take place in neurons. The existence of
highly specific structures such as neurites, postsynaptic densities or synaptic boutons, together with the absence of many
protein partners present in neurons provide a level of complexity not easily mimicked in cells lines. Clearly, further
studies are needed to establish these cytoskeletal proteins as
functionally relevant in neuronal cells.
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
3. Functional properties of dopamine receptors
and modulation of signalling pathways
As already stated, dopamine receptors have been divided
into two groups, based on ligand affinity and specificity and
effector coupling. Differences in ligand binding affinity,
basal activity, efficacy and potency for G protein activation,
or desensitization mechanisms may be expected to be derived
characters that drove conservation of the dopamine receptor
subtypes during vertebrate evolution. These derived characters would be in fact the discriminating properties of receptor
subtypes found in modern vertebrate species. Here we show
that it is indeed the case. A detailed comparison of the
functional characteristics of dopamine receptors among species is only available for the D1 receptor class, but some
inferences can also be proposed for the D2 receptors.
3.1. D1 receptor signalling
Receptors of the D1 class, D1A/D1 and D1B/D5, activate
adenylyl cyclase through the G protein Gas in most tissues,
or Gaolf in the striatum and the olfactory bulb (Monsma et al.,
1990, Tiberi et al., 1991; Herve et al., 1993), resulting in
enhanced levels of cAMP in cells. As a consequence, cAMPdependent protein kinase (PKA) is turned on, and induces
phosphorylation of cellular protein, such as DARPPP-32,
ARPP-21, ARPP-16 and STEP. There is also evidence that
D1B/D5 receptor couples to Gaz and that D1A/D1 receptor
couples to Gao, thereby modulating the activity of Ca2+, K+,
and Na+ channels (Sidhu, 1998; Sidhu and Niznik, 2000).
Nevertheless, all D1 receptor subtypes preferentially activate
Gas, the coupling to others Ga proteins depending on the
nature of the Ga proteins promiscuous to the receptors, on
the presence of associated proteins such as calcyon, and of
the activation level of the receptor (Kimura et al., 1991;
Kimura et al., 1995; Lezcano et al., 2000; Sidhu and Niznik,
2000).
One of the clearest distinctions between the three D1A,
D1B and D1C subtypes is made by the relative affinity of the
receptors for ligand and for the basal activity of the receptor.
The binding for ligands and coupling properties of the
D1B/D5 receptor differ from that of the D1A/D1 receptor by a
higher constitutive agonist-independent activity, an increased affinity and potency for dopamine and other agonists,
and a lower affinity for inverse agonist, these properties being
highly conserved among jawed vertebrates (Tiberi and Caron, 1994; Cardinaud et al., 1997; Sugamori et al., 1998). In
contrast, the D1C receptor subtype exhibits essentially the
same dopamine affinity and low intrinsic activity as the D1A
subtype. These characteristics could be related to the structural difference of cytoplasmic tails of each receptor since in
the three D1 receptor subtypes, it contains phosphorylation
motif, which are both subtype-specific and conserved in all
the species studied so far. Using chimeric constructions of
D1–like receptors, Jackon et al. (2000) have shown that the
cytoplasmic tail of human dopamine D1–like receptors regulate the binding affinity of dopamine and interacts with the
495
other intracellular loops of the receptor to modulate G
protein-coupling and second messenger activation. In addition, studies conducted in Xenopus with chimeric D1A, D1B
and D1C receptors showed that the cytoplasmic tail also
played a role in the desensitization mechanisms of these
D1–like receptors (Sugamori et al., 1998; see below).
3.2. D2 receptor signalling
No systematic analysis of the functional characteristics of
the three D2–like receptor subtypes has been undertaken, in
contrast to the D1–like receptors. However, a few general
features can be highlighted for the members of this dopamine
receptor class. Beside synthetic compounds, some of them
being fairly specific of either the D2, the D3 or the D4
receptor, the natural agonist dopamine has a significantly
higher affinity (ten-to-thirty fold) for the D3 and the D4 than
for the D2 receptor subtype (Sokoloff and Schwartz, 1995.
Although the inhibition of adenylyl cyclase activity is one
of the most conserved characteristics of the D2–like receptors,
the D3 receptor are not able to promote inhibition of cAMP
production in fibroblastic cell lines. It does so only in
neuronal-like cells, revealing that specific –but unknown–
intracellular components may be necessary for receptor activity (Cussac et al., 1999). All three D2–like receptors are also
able to modulate the activity of voltage-gated ionic channels.
The D2 receptor subtype inhibits several types of Ca2+ ionic
channels through Gao and activates K+ currents through Gai
in pituitary cells (Lledo et al., 1992). Similarly, the D3 and D4
receptor subtypes are able to inhibit L and N–types of Ca2+
currents in transfected pituitary and neuronal cell lines
(Seabrook et al., 1994).
Among the other effects of the D2–like receptors that have
been observed (mobilisation of intracellular calcium stores,
PKC activation, release of arachidonic acid, Na+/H+ exchange, inhibition of the Na+/K+ ATPase{ etc), no evidence
of a direct modulation of these effectors has been demonstrated (reviewed in Missale et al., 1998). However, a direct
activation of PLC (Vallar et al., 1990) or the modulation of
some of the MAP-kinase pathways (Lajiness et al., 1993) are
probably important but underestimated effects of the D2
receptors.
In amniotes, the D2 receptor subtype exists in two isofoms
of 444 (D2L) and 415 (D2S) amino acids generated by the
alternative splicing of the pre-mRNA (reviewed in Missale et
al., 1998). These two proteins are identical except for the
third intracytoplasmic loop, where there is an insertion of
29 amino acids in D2L, relative to D2S. The question of the
physiological counterpart of these two has long been debated, and a definitive answer has not been obtained yet. We
have discussed above the possibility of differential localization of the D2 receptor isoforms in neurons. At first sight, an
obvious idea was that the 29 amino acid insertion might elicit
differential coupling of the receptor to G proteins. As a
matter of fact, in some cell lines, D2L but not D2S, couple to
Gai2 to inhibit forskolin-stimulated adenylyl cyclase (Montmayeur et al., 1993; Liu et al., 1994; Guiramand et al., 1995).
496
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
This is not found in other cells where no discriminating
properties of the two D2 receptor isoforms could be seen
(Senogles, 1994; Gharemani et al., 1999). However, the ability of the D2 receptor isoforms to block Ca++ channels seems
to vary between D2S and D2L (Liu et al. 1994). In addition,
the D2L receptor activates K+ channels through PTXsensitive Ga subunits, whereas D2S receptor effect could
depend on Gas inactivation (Liu et al., 1996). In conclusion,
some clues exist for a differential activity of the two splicing
isoforms of the dopamine D2 receptor. However, it may
reflect a differential localization in microdomains of the
plasma membrane, as proposed above. It is the formation of a
local network of interacting proteins, regulating both transduction phenomenon as well as the dynamic of the receptors
between the subcellular compartments, which ultimately determines the cellular response to dopamine binding.
3.3. Dopamine receptors oligomerization and functional
protein-protein interactions
Among the molecular interactions that may be crucial for
dopaminergic transmission, the possibility that dopamine
receptors can form homo or heteromers shed a new light on
the mechanisms of transduction and receptor regulation.
Many single-transmembrane receptors including growthfactor receptors and cytokine receptors depend on dimerization for proper function. In addition, metabotropic glutamate
receptors and GABAB receptors obligatorily dimerize to be
transported to the plasma membrane and to transduce extracellular signals through G proteins. Whether monoamine G
protein-coupled receptors also undergo dimerization is still a
matter of debate. In addition to signal transduction, receptor
oligomerization may be a prerequisite to desensitization,
endocytosis and recycling of receptors (Hébert et al., 1996).
Agonist or antagonist exposure may change oligomer:monomer ratio suggesting that interconversion between monomers
and oligomers may be important for biological activity
(Hébert and Bouvier, 1998).
Dopamine receptors have also been shown to forms
dimers and higher order oligomers. Evidence has been provided for dopamine D2 dimerization in the human and rat
brain by showing discrepancy in binding parameters for
different ligands. Indeed binding measurements of labelled
benzamide and butyrophenone derivatives led to the hypothesis that [3H]–spiperone binds to the D2 receptor monomers
whereas [3H]–raclopride interacts with receptor dimers,
since [3H]–raclopride labelled about half the number of D2
receptor sites labelled by [3H]–spiperone (Zawarynski et al.,
1998). This finding was supported by the demonstration of
dimers and oligomers formation of D1 and D2 receptors in
transfected Sf9 cells (Ng et al., 1994a; Ng et al., 1996).
Indirect evidence of D2 receptor oligomerization has been
obtained by the inhibition of wild-type D2 receptor function
by truncated amino and carboxy-terminal D2 receptor portions (Lee et al., 2000). In addition, agonist-independent and
dependent oligomerization was suggested by using a biophysical method based on energy transfer from fluorescent
protein-tagged D2 receptors in close spacial proximity in
living cells (Wurch et al., 2001). However, no conclusive
proof has been provided yet to demonstrate that oligomerization is mandatory for dopamine receptor activity.
In addition to homodimerization, it has been suggested
that dopamine receptors not only form homodimers but also
heterodimers with other GPCRs or even ligand-gated ion
channels. Rocheville et al. (2000) have shown by a FRETbased assay that D2 receptor interacts with somatostatin
SSTR5 receptors, resulting in enhanced functional activity.
Evidence for heteromerization of other dopamine receptor
subtypes has been also provided. Oligomeric D2:D3 receptor
protein associations have been detected by coimmunoprecipitation in primate and rodent brain homogenates, as well as in rat GH3 cells stably expressing a D3
receptor (Nimchinsky et al., 1997). Scarselli et al. (2001)
used a truncated receptor approach to show that the N–terminal part of a D2 receptor could functionally associate with
C–terminal portion of a D3 receptor. In addition, evidences
exist for a direct interaction between D1 and D3 receptors in
ventral striatum and islands of Calleja (reviewed in Sokoloff
et al., 2003).
The D1 receptors certainly make hetero-oligomers with
adenosine A1 receptors (Gines et al. 2000). The A1 and D1A
receptors are highly co-localized and directly interact in
stable lines of mouse fibroblast Ltk– cells and in primary
cultures of cortical neurons. This selective A1/D1A receptor
heteromerization seems to depend upon the simultaneous
activation of the two receptors. Indeed, pre-treatment of the
cells by A1 receptor agonists prevents D1A receptor activation
and desensitization (Gines et al. 2000; Le Crom et al., 2002).
Another evidence of adenosine-dopamine receptor antagonism was obtained by Hillion et al, (2002), who showed that
adenosine A2A and D2 receptors are co-localized in membranes of SH-SY5Y human neuroblastoma cells stably transfected with human D2 receptors and in cultured striatal cells.
Moreover, long-term exposure to dopamine or adenosine
receptor agonists resulted in aggregation, internalization and
desensitization of both A2A and D2 receptors (Hillion et al.,
2002).
Dopamine receptors heterodimerization can also modulate trafficking and activity of ligand-gated ion channels
through direct protein-protein interactions. The D1 receptor
was shown to interact with the N-methyl-D-aspartate
(NMDA) receptor, mediating changes in the NMDAdependent currents Lee et al., 2002). Two regions in the D1
receptor carboxyl tail directly and selectively couple the
subunits NR1a and NR2A of the NMDA receptor. One interaction inhibits NMDA receptor-gated currents, and the other
attenuates NMDA receptor-mediated excitotoxicity through
a PI-3 kinase-dependent pathway (Lee et al., 2002). Direct
interaction was also shown between D5 receptor of dopamine
and ligand-gated ion channel GABAA receptor (Liu et al.,
2000). GABAA–ligand–gated channels complex selectively
with D5 receptors through the direct binding of the D5
carboxy-terminal domain with the second intracellular loop
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
497
Fig. 2. Molecular mechanisms of desensitization, internalization and trafficking of dopamine receptors.
Activation of dopamine receptors by agonist leads to GRK-dependant phosphorylation of residues that favours the translocation and the binding of b-arrestin
proteins to the receptor. These b-arrestin proteins act as adaptor that binds with high affinity clathrin and facilitates internalization of the receptors into
clathrin-coated pits. The receptors are then addressed to acidic endosomal compartment. Here, either the ligand dissociates, the receptor dephosphorylated by
a phosphatase and then recycled to the plasma membrane, or targeted for degradation into lysosomes or proteasomes.
of the c2 subunit (short) of the GABAA receptor. This physical association makes possible reciprocal inhibitory interactions between these receptors.
These data highlight new mechanisms of signal transduction regulation that increase the dynamic of receptor regulation and functioning at synapses in the central nervous system, independently of classically defined second-messenger
systems. Thus, it is probable that, in a near future careful
examination of the dopamine receptor homo et heterooligomerization will lead to a better understanding of the
specificity and diversity of dopaminergic signalling. The
long-separated fields of receptor transduction and receptor
localization are clearly merging now, to provide a more
unitary view of receptor function.
4. Regulation of dopamine receptor activity
As other G-protein-coupled receptors, dopamine receptors undergo a variety of regulatory changes, among which
agonist-induced desensitization and internalization are the
most important. Desensitization and internalization are the
two faces of a similar process that tends to diminish cell
responsiveness to the presence of the neurotransmitter in the
cell vicinity. Although receptor internalization into endocytotic vesicles may be the first step of a catalytic process
leading ultimately to receptor degradation, it is also part of a
recycling process (Fig. 2) involved in receptor trafficking and
localization at appropriate sites in the cells. Previous studies
of dopamine receptors have revealed a great variability of
agonist-induced desensitization subtypes.
4.1. Regulation of D1 receptor activity
The D1–like receptors undergo multiple levels of regulation
after exposure to agonists. In the short term, agonists promote rapid functional uncoupling of the D1 receptor from G
protein, which rely on receptor phosphorylation and palmitoylation (Gardner et al., 2001). The phosphorylation of D1
receptor is strictly dependent upon agonist occupancy/
498
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
activation of the receptor (Gardner et al., 2001). The second
step consists in agonist-induced redistribution of surface
receptor from a diffuse pattern of distribution to aggregates.
Then, internalization of the ligand-receptor complex takes
place within small vesicles in the cytoplasm (Trogadis et al.,
1995). The molecular mechanism of G protein coupled –
receptor internalization has not been fully elucidated, but
several lines of evidence indicate that protein-kinase A, G
protein-coupled receptors kinases (GRKs), arrestin, as well
as clathrin and dynamin are involved (Fig.2). As mentioned
above, desensitization and internalization of D1–like receptors
may represent two distinct –although interconnected– biochemical processes. For example, in SF9 cells, pretreatment
with concavalin A or sucrose completely blocked agonistinduced internalization but not desensitization (Ng et al.,
1995).
The respective roles of the different protein kinases,
GRKs or second messenger-regulated kinases in the agonistinduced desensitization of D1 receptor is controversial.
While some authors have underlined the central implication
of GRKs (see Ng et al., 1994a; Tiberi et al., 1996), others
have clearly shown that PKA may also play a role (Zhou et
al., 1991). One of the phosphorylation sites located in the
C–terminal end of the third cytoplasmic loop of the D1–like
receptors is conserved in the sequences of all the subtypes of
every species known to date. This site is phosphorylated by
PKA in agonist-induced desensitization, and mutation of this
site attenuated the rate of agonist-induced desensitization of
D1 receptors (Jiang and Sibley, 1999; Lamey et al., 2002).
Interestingly, each of the D1 receptor subtypes bears also
putative phosphorylation sites that are both subtype-specific
and conserved in all the species analyzed so far. Whether they
are the substrates of different kinases has not yet been investigated. It is possible that they underlie the differences of
internalization rates displayed by the D1 receptor subtypes.
Desensitization of the D1A receptor is occurring very fast,
50% of receptor activity being lost during the first five minutes. As far as the internalization process is concerned, D1A
dopamine receptors seem to preferentially use a b-arrestin
and dynamin-dependent mechanism (Vickery and von Zastrow, 1999, Iwata et al., 1999; Ito et al., 1999). However, how
this process contributes to receptor recycling or degradation
is not known yet.
The human D1B/D5 receptor undergoes a similar process
of desensitization but with lower amplitude than the D1A/D1
receptor (Jarvie et al., 1993; Iwasiow et al., 1999). Again, this
characteristic is also found for the D1B receptors of the other
vertebrate species (Tiberi and Caron, 1994; Cardinaud et al.,
1997; reviewed in Kapsimali et al., 2003). We have proposed
that the differential kinetics of desensitization between D1A
and D1B receptors may be due to constitutive activity of the
D1B receptor, this latter favoring a “constitutive” desensitization of the receptor. The desensitization of the D1B/D5 receptor is functionally coupled to endocytosis, as for the D1A/D1
receptor (unpublished observations), but details of the process need to be investigated further.
In sharp contrast with the two other subtypes of dopamine
D1 receptors, D1C receptors from fish and amphibians are
neither able to desensitize, nor to be internalized. Thus,
although it still needs to be fully proven, it is very probable
that the lack of desensitization is an important discriminating
property of the D1C receptors. To conclude, desensitization
response to agonist exposure has been clearly diversified in
the different D1 receptor subtypes after gene duplications in
early vertebrates, and this property has probably contributed
to the conservation of these subtypes later in evolution.
4.2. Regulation of receptor activity in the D2 class
The regulation of the D2–like receptors is rather different
from that of the D1–like receptors and remains highly controversial. For the D2 receptor subtype, some authors have
described desensitization of D2 receptors as a process closely
correlated with agonist occupancy (Barton et al., 1991), and
somehow related to receptor sequestration (Itokawa et al.,
1996). Others have reported that D2 receptor desensitization
occurred, but slowly and only after prolonged agonist exposure, with some differences between the two D2L and D2S
receptor isoforms (Zhang et al., 1994). Finally and very
surprisingly, an up regulation of D2 receptors promoted by
agonist stimulation on cultured cells has also been shown
(Filtz et al., 1993; Starr et al., 1995; Ng et al., 1997). Reasons
for such discrepancies are not clear at all, except to say that
the regulation of the D2 receptor subtype may have some
peculiarity, perhaps the requirement of specific cell components, which is not easily recapitulated in heterologous transfected cells.
Among eccentricities of the D2 receptor subtype, a high
intrisic activity has been described (Gardner et al., 1996;
Prou et al, 2001) that may promote a constitutive endocytosis
and recycling of the receptor at the cell surface (Vickery and
von Zastrow, 1999; Prou et al., 2001). Nevertheless, early
steps of D2 receptor internalization seem to involve a rather
common dynamin-dependent mechanism of endocytosis in
early endosomes (Vickery and von Zastrow, 1999, Iwata et
al., 1999). Interstingly, in COS-7 and HEK-293 cells the D2
receptor sequestration induced by agonist treatment is observed only after coexpression with GRK2 or GRK5 (Ito et
al., 1999; Iwata et al., 1999), but not in Sf9 cells (Ng et al.,
1994b).
Very few studies have carefully compared the desensitization rate and mechanisms of the D2–like receptor subtypes.
However, it has recently been shown that D2 and D3 receptors
exhibit some differences in both desensitization and trafficking properties (Kim et al., 2001). Contrasting with the D2
receptor, the D3 subtype is not phosphorylated upon agonist
exposure, in the absence of GRK. Overexpression of GRK
caused only a subtle increase in the level of phosphorylation
and the D3 receptor seems to have a reduced ability to
translocate b-arrestin to the plasma membrane, as compared
to the D2 receptor. Thus, as for the D1–like receptors, the D2
receptor subtypes are likely to be distinguishable by their
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
499
properties of regulation in cells, the mechanisms of which are
also tightly linked to the intracellular trafficking and subcellular localization of the receptors.
5. Conclusions
Despite many uncertainties on how dopamine receptors
are targeted to the plasma membrane and regulated in situ,
the evolutionary approach to dopamine receptors has brought
to light several important information on the role played by
the receptors in vertebrate organisms (Fig. 3). Firstly, although no major changes in expression territories have been
evidenced between the dopamine receptor subtypes in vertebrates, there are sufficient differences to rationalize the conservation of both the D1A/D1 and the D1B/D5 subtypes in
most jawed vertebrates. Both in mammals and fish, the tissue
distribution of D1B/D5 receptors overlap with that of the
D1A/D1 receptors, but the D1B/D5 receptor subtype is the
only one present in the hippocampal areas, a fact significant
enough to justify the conservation of the D1B receptor subtype. Conversely, the D1C receptor mRNA that has a highly
restricted distribution in the fish brain being present mainly
in the dorsal and medial hypothalamic nuclei and ventral
habenula (Kapsimali et al., 2000). The precise role of these
neuronal nuclei is still poorly known, but they have, for most
of them no obvious homologues in mammals. This suggests
that the role of the D1C receptor is related to adaptive neuroendocrine functions in fish, amphibians and most amniotes, functions that have been lost in mammals, together
with the D1C receptors.
Other physiologically-relevant observations came out of
our studies. The subcellular localisation of D1A/D1 receptors
at the basis of the dendritic spines, in close proximity to the
dopamine release sites in the mammalian basal ganglia,
should be related to its “low” affinity and fast desensitization
rate. The D1A receptor is thus mainly a “synaptic” receptor
and it is the most highly expressed in the brain (Fig. 3). At the
cellular level, D1B/D5 receptors are found on dendritic shafts,
far from synapses. This localization may account for the high
affinity and high intrinsic activity that characterized the vertebrate D1B/D5 receptors, fitting with a “non-synaptic” neurohormonal type of receptor. In contrast, the D1C receptor
subtype is resistant to desensitization, a property compatible
with a regulatory role in cells permanently submitted to
dopamine influence, as might be the case in the periventricular hypothalamic areas where the receptors (Fig. 3) are
present. Similar suggestions can be made for the D2 receptor
subtypes with the D2 subtype also being closer to the dopamine release sites than the D3 receptor, this latter playing a
central role at the end of the development of the nervous
system and during adaptation of the limbic areas to environmental changes.
Thus, the molecular properties of each of the receptor
subtypes could account for the conservation of the duplicated
dopamine receptor subtypes. Each of the D1 receptor subtypes has indeed distinct functional properties that have obvi-
Fig. 3. The dopaminergic “synapse”.
Although terminals of dopamine synthesizing neurons do not make true
synapses, they are apposed close to the neck of dendritic spines in many
structures such as the mammalian striatum. The biosynthetic pathway of
dopamine is shown (TH: tyrosine hydroxylase, AAADC: Aromatic amino
acid decarboxylase) as well as the storage into vesicles mediated by vesicular monoamine transporter (VMAT). After being released in the extracellular
space, dopamine is rapidly taken up by the dopamine transporter (DAT).
Dopamine also acts on D1–like (coupled to Gs proteins) and D2–like receptors
(coupled to Go/i proteins). Subcellular localization determines the cellular
effects promoted by receptor activation. Dopamine D2 receptors are localized both in presynaptic terminals where they inhibit dopamine release by
decreasing Ca++ activity, and postsynaptically where they activate K+ channels. In contrast, D1–like receptors are basically postsynaptic where they
activate adenylyl cyclase. They may be located close to the synapse (mainly
D1A) where they are able to interact with true synaptic receptors such as
NMDA receptors. In contrast, D1B receptors are supposed to be located
further away from the sites of dopamine release (see text for details).
ous adaptive consequences for the modulation of neuronal
activity in regulatory networks. Taken together, these data
suggest that the duplication of dopamine receptor genes in
vertebrates has given some degree of freedom to species for
adapting the morphological and functional organisation of
their nervous system to various environments and behaviour.
In particular, it has allowed areas controlled by dopamine,
such as the basal ganglia and frontal cortex to undergo large
quantitative changes, especially in mammals, without dramatically modifying the general properties of the dopamine
systems. Thus, as very often during evolution, significant
physiological changes have been reached at low genetic
costs.
500
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
Acknowledgements
Work in our laboratory is supported by CNRS, Université
Paris XI and Institut Universitaire de France.
References
Aparicio, S., 2000. Vertebrate evolution: recent perspectives from fish.
Trends Genet 16, 54–56.
Barton, A.C., Black, L.E., Sibley, D.R., 1991. Agonist-induced desensitization of D2 dopamine receptors in humanY–79 retinoblastoma cells. Mol.
Pharmacol. 39, 650–658.
Bergson, C., Mrzljak, L., Smiley, J.F., Pappy, M., Levenson, R., GoldmanRakic, P.S., 1995. Regional, cellular, and subcellular variations in the
distribution of D1 and D5 dopamine receptors in primate brain. J Neurosci. 15, 7821–7836.
Bermak, J.C., Li, M., Bullock, C., Zhou, Q.Y., 2001. Regulation of transport
of the dopamine D1 receptor by a new membrane-associated ER protein.
Nat Cell Biol 3, 492–498.
Bermak, J.C., Li, M., Bullock, C., Weingarten, P., Zhou, Q.Y., 2002. Interaction of gamma-COP with a transport motif in the D1 receptor C–terminus. Eur J Cell Biol 81, 77–85.
Binda, A.V., Kabbani, N., Lin, R., Levenson, R., 2002. D2 and D3 dopamine
receptor cell surface localization mediated by interaction with protein
4.1N. Mol Pharmacol 62, 507–513.
Cardinaud, B., Sugamori, K.S., Coudouel, S., Vincent, J.D., Niznik, H.B.,
Vernier, P., 1997. Early emergence of three dopamine D1 receptor
subtypes in vertebrates. Molecular phylogenetic, pharmacological, and
functional criteria defining D1A, D1B, and D1C receptors in European
eel Anguilla anguilla. J Biol Chem 272, 2778–2787.
Cussac, D.A.N.-T., Pasteau, V., Millan, M.J., 1999. Human dopamine D(3)
receptors mediate mitogen-activated protein kinase activation via a phosphatidylinositol 3–kinase and an atypical protein kinase C–dependent
mechanism. Mol Pharmacol 56, 1025–1030.
Demchyshyn, L.L., Sugamori, K.S., Lee, F.J., Hamadanizadeh, S.A.,
Niznik, H.B., 1995. The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1–like receptors from
Gallus domesticus. J Biol Chem 270, 4005–4012.
Filtz, T.M., Artymyshyn, R.P., Guan, W., Molinoff, P.B., 1993. Paradoxical
regulation of dopamine receptors in transfected 293 cells. Mol Pharmacol 44, 371–379.
Gardner, B., Hall, D.A., Strange, P.G., 1996. Pharmacological analysis of
dopamine stimulation of (35S)–GTP gamma S binding via human
D2short and D2long dopamine receptors expressed in recombinant cells.
Br J Pharmacol. 118 (6), 1544–1550.
Gardner, B., Liu, Z.F., Jiang, D., Sibley, D.R., 2001. The role of
phosphorylation/dephosphorylation in agonist-induced desensitization
of D1 dopamine receptor function: evidence for a novel pathway for
receptor dephosphorylation. Mol Pharmacol 59, 310–321.
Gerfen, C.R., 2000. Molecular effects of dopamine on striatal-projection
pathways. Trends Neurosci. 23 (Suppl), S64–S70.
Gines, S., Hillion, J., Torvinen, M., Le Crom, S., Casado, V., Canela, E.I.,
Rondin, S., Lew, J.Y., Watson, S., Zoli, M., et al., 2000. Dopamine D1
and adenosine A1 receptors form functionally interacting heteromeric
complexes. Proc Natl Acad Sci U S A 97, 8606–8611.
Guiramand, J., Montmayeur, J.P., Ceraline, J., Bhatia, M., Borrelli, E., 1995.
Alternative splicing of the dopamine D2 receptor directs specificity of
coupling to G–proteins. J. Biol. Chem. 270, 7354–7358.
Guivarc’h, D., Vernier, P., Vincent, J.D., 1995. Sex steroid hormones change
the differential distribution of the isoforms of the D2 dopamine receptor
messenger RNA in the rat brain. Neuroscience 69, 159–166.
Hébert, T.E., Moffet, S., Morello, J.-P., Loisel, T.P., Bichet, D.G., Barret, C.,
Bouvier, M., 1996. A peptide derived from a ß2–adrenergic receptor
transmembrane domain inhibits both receptor dimerization and activation. Journal of Biological Chemistry 271, 16384–16392.
Hébert, T.E., Bouvier, M., 1998. Structural and functional aspects of G
protein-coupled receptor oligomerization. Biochem. Cell Biol. 76, 1–11.
Herve, D., Levi-Strauss, M., Marey-Semper, I., Verney, C., Tassin, J.P.,
Glowinski, J., Girault, J.A., 1993. G(olf) and Gs in rat basal ganglia:
possible involvement of G(olf) in the coupling of dopamine D1 receptor
with adenylyl cyclase. J Neurosci 13, 2237–2248.
Hillion, J., Canals, M., Torvinen, M., Casado, V., Scott, R., Terasmaa, A.,
Hansson, A., Watson, S., Olah, M.E., Mallol, J., et al., 2002. Coaggregation, cointernalization, and codesensitization of adenosine A2A receptors and dopamine D2 receptors. J Biol Chem 277, 18091–18097.
Huang, Q., Zhou, D., Chase, K., Gusella, J.F., Aronin, N., DiFiglia, M.,
1992. Immunohistochemical localization of the D1 dopamine receptor in
rat brain reveals its axonal transport, pre– and postsynaptic localization,
and prevalence in the basal ganglia, limbic system, and thalamic reticular
nucleus. Proc Natl Acad Sci U S A 89, 11988–11992.
Ito, K., Haga, T., Lameh, J., Sadee, W., 1999. Sequestration of dopamine D2
receptors depends on coexpression of G-protein-coupled receptor
kinases 2 or 5. Eur J Biochem 260, 112–119.
Itokawa, M., Toru, M., Ito, K., Tsuga, H., Kameyama, K., Haga, T., Arinami, T., Hamaguchi, H., 1996. Sequestration of the short and long
isoforms of dopamine D2 receptors expressed in Chinese hamster ovary
cells. Mol Pharmacol 49, 560–566.
Iwasiow, R.M., Nantel, M.F., Tiberi, M., 1999. Delineation of the structural
basis for the activation properties of the dopamine D1 receptor subtypes.
J Biol Chem 274, 31882–31890.
Iwata, K., Ito, K., Fukuzaki, A., Inaki, K., Haga, T., 1999. Dynamin and rab5
regulate GRK2–dependent internalization of dopamine D2 receptors.
Eur J Biochem 263, 596–602.
Jackson, A., Iwasiow, R.M., Tiberi, M., 2000. Distinct function of the
cytoplasmic tail in human D1–like receptor ligand binding and coupling.
FEBS Lett 470, 183–188.
Jarvie, K.R., Tiberi, M., Silvia, C., Gingrich, J.A., Caron, M.G., 1993.
Molecular cloning, stable expression and desentization of the human
dopamine D1B/D5 receptor. J. Recept.Res. 13, 573–590.
Jiang, D., Sibley, D.R., 1999. Regulation of D(1) dopamine receptors with
mutations of protein kinase phosphorylation sites: attenuation of the rate
of agonist-induced desensitization. Mol Pharmacol 56, 675–683.
Kabbani, N., Negyessy, L., Lin, R., Goldman-Rakic, P., Levenson, R., 2002.
Interaction with neuronal calcium sensor NCS-1 mediates desensitization of the D2 dopamine receptor. J Neurosci 22, 8476–8486.
Kapsimali, M., Vidal, B., Gonzalez, A., Dufour, S., Vernier, P., 2000. Distribution of the mRNA encoding the four dopamine D(1) receptor subtypes
in the brain of the european eel (Anguilla anguilla): comparative
approach to the function of D(1) receptors in vertebrates. J Comp Neurol
419, 320–343.
Kapsimali, M., Le Crom, S., Vernier, P., 2003. Origin and evolution of
dopamine receptors and transporters in vertebrates. In: Sidhu, M.L.A.,
Vernier, P. (Eds.), Dopamine receptors and transporter. Marcel Dekker,
New York, pp. 1–43.
Karpa, K.D., Lidow, M.S., Pickering, M.T., Levenson, R., Bergson, C.,
1999. N–linked glycosylation is required for plasma membrane localization of D5, but not D1, dopamine receptors in transfected mammalian
cells. Mol Pharmacol 56, 1071–1078.
Kebabian, J.W., Calne, D.B., 1979. Multiple receptors for dopamine. Nature
277, 93–96.
Khan, Z.U., Gutierrez, A., Martin, R., Penafiel, A., Rivera, A., De La
Calle, A., 1998. Differential regional and cellular distribution of dopamine D2–like receptors: an immunocytochemical study of subtypespecific antibodies in rat and human brain. J Comp Neurol 402, 353–371.
Kim, K.M., Valenzano, K.J., Robinson, S.R., Yao, W.D., Barak, L.S.,
Caron, M.G., 2001. Differential regulation of the dopamine D2 and D3
receptors by G protein-coupled receptor kinases and beta-arrestins. J
Biol Chem 276, 37409–37414.
Kim, O.J., Ariano, M.A., Lazzarini, R.A., Levine, M.S., Sibley, D.R., 2002.
Neurofilament-M interacts with the D1 dopamine receptor to regulate
cell surface expression and desensitization. J Neurosci 22, 5920–5930.
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
Kimura, K., Sela, S., Bouvier, C., Grandy, D.K., Sidhu, A., 1995. Differential coupling of D1 and D5 dopamine receptors to guanine nucleotide
binding proteins in transfected GH4C1 rat somatomammotrophic cells. J
Neurochem 64, 2118–2124.
Kirschner, M., Gerhart, J., 1998. Evolvability. Proc Natl Acad Sci U S A 95,
8420–8427.
Lajiness, M.E., Chio, C.L., Huff, R.M., 1993. D2 dopamine receptor stimulation of mitogenesis in transfected Chinese hamster ovary cells: relationship to dopamine stimulation of tyrosine phosphorylations. J. Pharm.
Exp. Ther. 267, 1573–1581.
Lamey, M., Thompson, M., Varghese, G., Chi, H., Sawzdargo, M.,
George, S.R., O’Dowd, B.F., 2002. Distinct residues in the carboxyl tail
mediate agonist-induced desensitization and internalization of the
human dopamine D1 receptor. J Biol Chem 277, 9415–9421.
Le Crom, S., Prou, D., Vernier, P., 2002. Autocrine Activation of Adenosine
A1 Receptors Blocks D1A but not D1B Dopamine Receptor Desensitization. J. Neurochem. 82, 1549–1552.
Lee, F.J., Xue, S., Pei, L., Vukusic, B., Chery, N., Wang, Y., Wang, Y.T.,
Niznik, H.B., Yu, X.M., Liu, F., 2002. Dual regulation of NMDA receptor functions by direct protein-protein interactions with the dopamine D1
receptor. Cell 111, 219–230.
Lee, S.P., Xie, Z., Varghese, G., Nguyen, T., O’Dowd, B.F., George, S.R.,
2000. Oligomerization of dopamine and serotonin receptors. Neuropsychopharmacology 23, S32–S40.
Levey, A.I., Hersch, S.M., Rye, D.B., Sunahara, R.K., Niznik, H.B.,
KItt, C.A., Price, D.L., Maggio, R., Brann, M.R., Ciliax, B.J., 1993.
Localization of D1 and D2 dopamine receptors in brain with subtypespecific antibodies. Proc. Natl Acad. Sci. USA 90, 8861–8865.
Lezcano, N., Mrzljak, L., Eubanks, S., Levenson, R., Goldman-Rakic, P.,
Bergson, C., 2000. Dual signaling regulated by calcyon, a D1 dopamine
receptor interacting protein. Science 287, 1660–1664.
Li, M., Bermak, J.C., Wang, Z.W., Zhou, Q.Y., 2000. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280). Mol
Pharmacol 57, 446–452.
Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P., Levenson, R., 2001.
Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via
interaction with filamin A. Proc Natl Acad Sci U S A 98, 5258–5263.
Liu, F., Wan, Q., Pristupa, Z.B., Yu, X.M., Wang, Y.T., Niznik, H.B., 2000.
Direct protein-protein coupling enables cross-talk between dopamine
D5 and gamma-aminobutyric acid A receptors. Nature 403, 274–280.
Liu, L.X., Monsma Jr., F.J., Sibley, D.R., Chiodo, L.A., 1996. D2L, D2S,
and D3 dopamine receptors stably transfected into NG108–15 cells
couple to a voltage-dependent potassium current via distinct G protein
mechanisms. Synapse 24, 156–164.
Liu, Y.F., Jakobs, K.H., Rasenick, M.M., Albert, P.R., 1994. G protein
specificity in receptor-effector coupling. Analysis of the roles of G0 and
Gi2 in GH4C1 pituitary cells. J Biol Chem 269, 13880–13886.
Lledo, P.M., Homburger, V., Bockaert, J., Vincent, J.D., 1992. Differential G
protein-mediated coupling of D2 dopamine receptors to K+ and Ca2+
currents in rat anterior pituitary cells. Neuron 8, 455–463.
Meador-Woodruff, J.H., Damask, S.P., Wang, J., Haroutunian, V.,
Davis, K.L., Watson, S.J., 1996. Dopamine receptor mRNA expression
in human striatum and neocortex. Neuropsychopharmacology 15,
17–29.
Missale, C., Nash, S.R., Robinson, S.W., Jaber, M., Caron, M.G., 1998.
Dopamine receptors: from structure to function. Physiol Rev 78, 189–
225.
Monsma, F.J., Mahan, L.C., McVittie, L.D., Gerfen, C.R., Sibley, D.R.,
1990. Molecular cloning and expression of a D1 dopamine receptor
linked to adenylyl cyclase activation. Proc. Natl Acad. Sci. USA 87,
6723–6727.
Montmayeur, J.P., Guiramand, J., Borrelli, E., 1993. Preferential coupling
between dopamine D2 receptors and G–proteins. Mol Endocrinol. 7,
161–170.
Ng, G.Y., Mouillac, B., George, S.R., Caron, M., Dennis, M., Bouvier, M.,
O’Dowd, B.F., 1994. Desensitization, phosphorylation and palmitoylation of the human dopamine D1 receptor. Eur. J. Pharmacol. 267, 7–19.
501
Ng, G.Y., O’Dowd, B.F., Caron, M., Dennis, M., Brann, M.R., George, S.R.,
1994. Phosphorylation and palmitoylation of the human D2L dopamine
receptor in Sf9 cells. J Neurochem 63, 1589–1595.
Ng, G.Y., Trogadis, J., Stevens, J., Bouvier, M., O’Dowd, B., George, S.R.,
1995. Agonist-induced desensitization of dopamine D1 receptorstimulated adenylyl cyclase activity is temporally and biochemically
separated from D1 receptor internalization. Proceedings of the National
Academy of Sciences of the United States of America 92, 10157–10161.
Ng, G.Y.K., O’Dowd, B.F., Lee, S.P., Chung, H.T., Brann, M.R., Seeman, P.,
George, S.R., 1996. Dopamine D2 receptor dimers and receptorblocking peptides. Biochem. Biophys. Res. Com. 227, 200–204.
Ng, G.Y.K., Varghese, G., Chung, H.T., Trogadis, J., Seeman, P.,
O’Dowd, B.F., George, S.R., 1997. Resistance of the dopamine D2(L)
receptor to desensitization accompanies the up-regulation of receptors
on to the surface of Sf9 cells. Endocrinology 138, 4199–4206.
Nimchinsky, E.A., Hof, P.R., Janssen, W.G.M., Morrison, J.H.,
Schmauss, C., 1997. Expression of dopamine D–3 receptor dimers and
tetramers in brain and in transfected cells. J Biol Chem 272, 29229–
29237.
Nothen, M.M., Cichon, S., Hemmer, S., Hebebrand, J., Remschmidt, H.,
Lehmkuhl, G., Poustka, F., Schmidt, M., Catalano, M., Fimmers, R., et al., 1994. Human dopamine D4 receptor gene: frequent
occurrence of a null allele and observation of homozygosity. Hum Mol
Genet. 3, 2207–2212.
Prou, D., Gu, W.J., Le Crom, S., Vincent, J.D., Salamero, J., Vernier, P.,
2001. Intracellular retention of the two isoforms of the D(2) dopamine
receptor promotes endoplasmic reticulum disruption. J Cell Sci 114,
3517–3527.
Rocheville, M., Lange, D.C., Kumar, U., Patel, S.C., Patel, R.C., Patel, Y.C.,
2000. Receptors for dopamine and somatostatin: formation of heterooligomers with enhanced functional activity. Science 288, 154–157.
Scarselli, M., Novi, F., Schallmach, E., Lin, R., Baragli, A., Colzi, A.,
Griffon, N., Corsini, G.U., Sokoloff, P., Levenson, R., et al., 2001.
D2/D3 dopamine receptor heterodimers exhibit unique functional properties. J Biol Chem 276, 30308–30314.
Schnell, S.A., You, S.Y., El Halawani, M.E., 1999. D1 and D2 dopamine
receptor messenger ribonucleic acid in brain and pituitary during the
reproductive cycle of the turkey hen. Biol Reprod 60, 1378–1383.
Seabrook, G.R., Knowles, M., Brown, N., Myers, J., Sinclair, H., Patel, S.,
Freedman, S.B., McAllister, G., 1994. Pharmacology of high-threshold
calcium currents in GH4C1 pituitary cells and their regulation by activation of human D2 and D4 dopamine receptors. Br J Pharmacol 112,
728–734.
Seeman, P., Van Tol, H.H., 1994. Dopamine receptor pharmacology. Trends
Pharmacol Sci 15, 264–270.
Senogles, S.E., 1994. The D2 dopamine receptor isoforms signal through
distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with
site-directed mutant Gi alpha proteins. J Biol Chem 269, 23120–23127.
Sibley, D.R., 1999. New insights into dopaminergic receptor function using
antisense and genetically altered animals. Annu Rev Pharmacol Toxicol
39, 313–341.
Sidhu, A., 1998. Coupling of D1 and D5 dopamine receptors to multiple G
proteins: Implications for understanding the diversity in receptor-G
protein coupling. Mol Neurobiol 16, 125–134.
Sidhu, A., Niznik, H.B., 2000. Coupling of dopamine receptor subtypes to
multiple and diverse G proteins. Int J Dev Neurosci 18, 669–677.
Smiley, J.F., Levey, A.I., Ciliax, B.J., Goldman-Rakic, P.S., 1994. D1
dopamine receptor immunoreactivity in human and monkey cerebral
cortex: predominant and extrasynaptic localization in dendritic spines.
Proc. Natl Acad. Sci. USA 91, 5720–5724.
Smith, F.D., Oxford, G.S., Milgram, S.L., 1999. Association of the D2
dopamine receptor third cytoplasmic loop with spinophilin, a protein
phosphatase-1-interacting protein. J Biol Chem 274, 19894–19900.
Sokoloff, P., Schwartz, J.C., 1995. Novel dopamine receptors half a decade
later [see comments]. Trends Pharmacol Sci 16, 270–275.
502
S. Callier et al. / Biology of the Cell 95 (2003) 489–502
Sokoloff, P., Griffon, N., Guillin, O., Pilon, C., Diaz, J., Schwartz, J.C., P.,
2003. The dopamine D3 receptor in the pathophysiology and treatments
of neuropsychiatric disorders. In: Sidhu, M.L.A., Vernier, P. (Eds.),
Dopamine receptors and transporter. Marcel Dekker, NewYork, pp. 211–
253.
Starr, S., Kozell, L.B., Neve, K.A., 1995. Drug-induced up-regulation of
dopamine D2 receptor on cultured cells. J. Neurochem. 65, 569–577.
Strange, P.G., 1993. New insights into dopamine receptors in the central
nervous system. Neurochem Int 22, 223–236.
Sugamori, K.S., Demchyshyn, L.L., Chung, M., Niznik, H.B., 1994. D1A,
D1B, and D1C dopamine receptors from Xenopus laevis. Proc Natl Acad
Sci USA 91, 10536–10540.
Sugamori, K.S., Scheideler, M.A., Vernier, P., Niznik, H.B., 1998. Dopamine D1B receptor chimeras reveal modulation of partial agonist activity
by carboxyl-terminal tail sequences. J Neurochem 71, 2593–2599.
Tiberi, M., Jarvie, K.R., Silvia, C., Falardeau, P., Gingrich, J.A., Godinot, N., Bertrand, L., Yang-Feng, T.L., Fremeau Jr, R.T., Caron, M.G.,
1991. Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype:
differential expression pattern in rat brain compared with the D1A
receptor. Proc Natl Acad Sci U S A 88, 7491–7495.
Tiberi, M., Caron, M.G., 1994. High agonist-independent activity is a
distinguishing feature of the dopamine D1B receptor subtype. J Biol
Chem 269, 27925–27931.
Tiberi, M., Nash, S.R., Bertrand, L., Lefkovitz, R.J., Caron, M.G., 1996.
Differential regulation of dopamine D1A receptor responsiveness by
various G protein-couped receptor kinases. Journal of Biological Chemistry 271, 3771–3778.
Trogadis, J.E., Ng, G.Y., O’Dowd, B.F., George, S.R., Stevens, J.K., 1995.
Dopamine D1 receptor distribution in Sf9 cells imaged by confocal
microscopy: a quantitative evaluation. J Histochem Cytochem 43, 497–
506.
Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J.,
Civelli, O., 1990. Differential coupling of dopaminergic D2 receptors
expressed in different cell types. Stimulation of phosphatidylinositol
4,5– bisphosphate hydrolysis in LtK– fibroblasts, hyperpolarization, and
cytosolic-free Ca2+ concentration decrease in GH4C1 cells. J Biol Chem
265, 10320–10326.
Vernier, P., Cardinaud, B., Valdenaire, O., Philippe, H., Vincent, J.D., 1995.
An evolutionary view of drug-receptor interaction: the bioamine receptor family. Trends Pharmacol Sci 16, 375–381.
Vickery, R.G., von Zastrow, M., 1999. Distinct dynamin-dependent and
-independent mechanisms target structurally homologous dopamine
receptors to different endocytic membranes. J Cell Biol 144, 31–43.
Wurch, T., Matsumoto, A., Pauwels, P.J., 2001. Agonist-independent and
-dependent oligomerization of dopamine D(2) receptors by fusion to
fluorescent proteins. FEBS Lett 507, 109–113.
Yung, K.K., Bolam, J.P., Smith, A.D., Hersch, S.M., Ciliax, B.J., Levey, A.I.,
1995. Immunocytochemical localization of D1 and D2 dopamine receptors in the basal ganglia of the rat: light and electron microscopy.
Neuroscience 65, 709–730.
Zawarynski, P., Tallerico, T., Seeman, P., Lee, S.P., O’Dowd, B.F.,
George, S.R., 1998. Dopamine D2 receptor dimers in human and rat
brain. FEBS Lett 441, 383–386.
Zhang, L.J., Lachowicz, J.E., Sibley, D.R., 1994. The D2S and D2L dopamine receptor isoforms are differentially regulated in Chinese hamster
ovary cells. Molecular Pharmacology 45, 878–889.
Zhou, X., Sidhu, A., Fishman, P.H., 1991. Desensitization of the human
dopamine D1 receptor: Evidence for involvement of both cyclic AMPdependent and receptor-specific kinases. Mol. Cell. Neurosci. 2, 264–
272.