(Received for publication, October 20, 1994)
From the
Three genomic clones encoding dopamine D1-like receptors were
isolated from the avian species Gallus domesticus. Two of
these genes encode proteins of 451 and 488 amino acids, which, based on
deduced amino acid sequence identity and homology of exhibited
pharmacological profiles, appear to be species homologs of mammalian
and vertebrate D1/D1A and D5/D1B receptors, respectively. The third
genomic clone, termed D1D, encodes a protein of 445 amino acids
displaying a deduced amino acid sequence identity within putative
trans-membrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B
receptors with overall sequence homologies of only 49% and 46%,
respectively. Membranes from COS-7 cells transfected with D1D DNA bound
[H]SCH-23390 in a saturable manner with high
affinity (
300 pM) and with a pharmacological profile
clearly indicative of a dopamine D1-like receptor. The D1D receptor
exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine
10-fold higher than D1/D1A receptors, characteristic of the D5/D1B
receptor subfamily. In contrast, the D1D receptor bound dopaminergic
agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with
affinities 10-fold higher than other cloned mammalian or vertebrate
D1A/D1B receptor subtypes, while both clozapine and haloperidol
displayed considerably lower affinity for the D1D receptor. Based on
the low overall amino acid sequence identity (54%) and unique
pharmacological profile, the avian dopamine D1D receptor does not
appear to be a species homolog of the recently cloned vertebrate D1C
receptor (Sugamori, K. S., Demchyshyn, L. L., Chung, M., and Niznik, H.
B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91,
10536-10540). As with all cloned mammalian and vertebrate D1-like
receptors, the D1D receptor stimulates adenylate cyclase activity in
the presence of dopamine or SKF-82526. Northern blot analysis reveals
the selective expression of both avian D1D and D1A receptor mRNAs only
in brain with the D1B receptor more widely distributed and localized in
tissues such as brain, kidney, and spleen. The isolation of four
distinct vertebrate dopamine D1 receptor subtypes suggests the
existence of additional mammalian D1 like receptor genes that may
account for the observed pharmacological and biochemical multiplicity
of dopamine D1-like receptor mediated events.
Dopamine receptors have been historically classified into two
major subtypes, termed D1 and D2(1) . Operationally, native D1
receptors are defined by their ability to promote adenylate cyclase
activity and bind agonists (e.g. fenoldopam, SKF-38393) ()and antagonists (SCH-23390) of the benzazepine and
benzonapthazine class of compounds with high
affinity(2, 3) . Dopamine D2 receptors inhibit
adenylate cyclase activity and bind selectively to agonists (e.g. quinpirole) and antagonists (haloperidol, spiperone, emonopride)
of numerous structural classes. The mammalian dopamine receptor family
is comprised of five distinct genes, two of which encode D1 like
receptors, termed D1/D1A and D5/D1B, and three genes encoding dopamine
D2 like receptors, termed D2, D3, and D4, which in addition yield
numerous functional splice and polymorphic forms of these
receptors(4, 5, 6, 7, 8) .
Based on pharmacological, biochemical, and behavioral observations,
it has been postulated that additional members of the dopamine D1
receptor gene subfamily exist(7, 9) . Thus, in native
mammalian systems, selective D1 receptor stimulation has been linked to
the activation of phospholipase C leading to phosphoinositide
hydrolysis in both the brain and periphery all independent of adenylate
cyclase activity (10, 11, 12, 13, 14, 15, 16) and
inhibition of Na/H
exchange (17) . Moreover, distinct behavioral effects mediated by
selective D1 receptor stimulation also suggest the existence of D1-like
subpopulations that either display unique pharmacological profiles for
some D1 selective compounds or are not classically defined as adenylate
cyclase-coupled D1
receptors(18, 19, 20, 21, 22) .
Cloned D1/D1A and D5 D1B receptors are found only to stimulate
adenylate cyclase activity in numerous cells
lines(23, 24, 25) , and the weak D1
receptor-mediated alteration in Ca
efflux observed in
some cells also appears to be
cAMP-dependent(26, 27, 28) . Thus, the
proposed dissociation of dopamine D1-like receptor-mediated events from
the stimulation of adenylate cyclase activity or in known responsivity
to various structural classes of agonist/antagonist ligands are
disparate with the observed pharmacological and signal transduction
profiles of the cloned D1A/D1B receptors. Although there has been some
molecular evidence for the existence of additional mammalian D1 like
receptors(12) , the gene encoding this protein has yet to be
identified.
Localization studies have thoroughly mapped the
neuroanatomical distribution pattern of dopamine-containing cells in
the avian brain, and dopamine D1-like receptors have been identified by
both autoradiographic and ligand binding
techniques.(29, 30, 31, 32, 33) .
As with mammalian systems(34) , developmental discrepancies
have been observed to exist between the number of D1 like receptors, as
indexed by [H]SCH-23390 binding sites and the
magnitude of D1 receptor-mediated cAMP accumulation in the avian
retina(35, 36) . Similarly, selective D-1 receptor
stimulation of avian retinal cells mediates K
efflux,
which has been shown to act independently of adenylate cyclase
activity(37) .
The existence of genes encoding for additional D1-like receptor subtypes, at least in vertebrates, has recently received direct support. Thus, a gene encoding a unique D1 receptor subtype, termed D1C, has been isolated from Xenopus laevis, which is distinguishable from cloned Xenopus D1A and D1B receptors on the basis of its primary structure, receptor mRNA distribution, and expressed pharmacological profile(38) . Using a strategy based on low stringency homology screening, we report here on the isolation and characterization of an additional member of the vertebrate D1 receptor subfamily, termed D1D, from the avian species Gallus domesticus (chicken).
The recent isolation and characterization of a novel D1-like
receptor gene from Xenopus laevis, termed D1C(38) ,
has provided direct molecular evidence for the heterogeneity of the D1A
and D1B receptor subfamily. In preliminary experiments, the generality
of this observation was further strengthened by Southern blot analysis
of avian genomic DNA probed with P-labeled dopamine D1 and
D5 receptor fragments encoding trans-membrane domains 2-5, which
revealed the presence of more than two distinct hybridizing bands (data
not shown). Selective stringency screening of an avian genomic library
was therefore initiated, and positives were isolated according to their
distinctive hybridization patterns. Three types of signals were
observed: 1) positives that strongly hybridized to D1 but not
D5, 2) positives that strongly hybridized to D5 and weakly to
D1, and 3) positives that hybridized moderately to both D1 and
D5. Twenty-four selections from these differential hybridization
patterns resulted in the isolation of three genomic fragments (LDChi-1,
LDChi-2, and LDChi-6).
Sequence analysis indicated that all three clones shared strong deduced amino acid sequence homology to the mammalian dopamine D1-receptor family. Like their mammalian counterparts, all three genes were intronless within their putative coding sequence. Consensus nucleotide sequences for putative initiating methionines (40) were found in all three clones. The first two clones (LDChi-2 and LDChi-6) contained open reading frames of 1353 and 1464 nucleotides encoding 451- and 488-amino acid proteins with estimated molecular masses of 49,142 and 53,110 Da, respectively. The third genomic clone (LDChi-1) revealed an open reading frame of 1335 nucleotides encoding for a 445-amino acid protein with an estimated molecular mass of 49,246 Da.
Comparison of the deduced amino acid sequence of all three clones depicted in Fig. 1suggests that LDChi-2 may be a homolog of the mammalian D1/D1A receptor, while LDChi-6 may be the species equivalent of the D5/D1B receptor. LDChi-2 was found to be 94% identical, within putative transmembrane domains, and displays 80% overall amino acid sequence identity to the mammalian (human/rat) D1/D1A receptor. Comparisons to mammalian D5/D1B receptors revealed that LDChi2 displays 81% identity within transmembrane domains and only 53% overall amino acid sequence homology. The LDChi-6 gene, however, exhibited greatest sequence resemblance to D1B receptors with 90% identity within transmembrane domains to both the mammalian (human/rat) D5/D1B receptors and to the amphibian D1B receptor homolog. Overall amino acid sequence identity of the avian D1B receptor to mammalian or amphibian D5/D1B receptors was considerably lower at 66 and 67%, respectively. The lower overall similarities between these genes and the avian D1B receptor is primarily attributable to sequence divergence within the amino-terminal, second and third intracellular-extracellular loops and portions of the carboxyl tail and is consistent with the view that this receptor subfamily is subject to an accelerated rate of accumulated evolutionary drift (41, 42) compared with the D1/D1A receptor family. One particular area of significant sequence divergence is evident within the third extracellular loop of the avian D1B receptor when compared with its mammalian counterparts. Thus, while mammalian D5/D1B receptors contain relatively large loops between transmembrane 4 and 5, the avian D1B receptor is significantly larger containing an additional 32 amino acids to encompass a total loop size of 65 amino acids. Although the functional significance third extracelluar loop is currently unknown, RT-PCR of brain D1B receptor mRNA and sequence analysis of the amplified DNA suggests that, at least in this tissue, the D1B receptor is not subject to alternative splicing (data not shown). In any event, based on the strong overall amino acid sequence homologies to either mammalian D1/D1A and D5/D1B receptors aligned in Fig. 1, we propose to term LDChi-2 and LDChi-6 the avian D1A and D1B receptor, respectively.
Figure 1:
Deduced
amino acid alignment of avian D1 receptors to other cloned members of
the mammalian and vertebrate dopamine D1-like receptor family. Boxed and shadedareas denote conserved
amino acid residues between the avian D1A, D1B, and D1D receptors and
their mammalian counterparts. Putative TM domains are demarcated by boxed regions. Putative N-linked glycosylation sites
are indicated by arrows. Potential phosphorylation sites for
protein kinase A (cAMP-dependent) and protein kinase C are indicated by closedcircles () and closedsquares (
), respectively. Single-letter amino acid
code used. Sequences encoding the monkey (56) and opossum (57) D1A receptors are omitted from this alignment due to the
relatively high degree of conservation in deduced amino acid sequence
to human/rat D1/D1A receptors.
The third genomic clone, LDChi-1, initially believed to encode for a D1C -like receptor was found to be particularly distinct. Sequence identity between this potentially novel dopamine receptor and other cloned members of the D1 receptor family was found to be highest within transmembrane domains: 75% to mammalian D1/D1A, 77% to D5/D1B, and 81% to vertebrate D1C receptors. Sequence homologies within TM domains increased considerably when D1D was compared with all cloned vertebrate members of either the D1A (86%) or D1B (88%) receptor subfamily listed in Fig. 1. Overall similarities were considerably lower with identities of 49, 46, and 54% to the D1, D5, and D1C receptor, respectively. At the nucleotide level, LDChi-1 displays a similar profile of overall sequence identity with 57% to mammalian D1/D1A receptor genes, 60% to the D5/D1B receptor subfamily, and 60% to the amphibian D1C gene. Based on both the nucleotide and deduced amino acid sequence homologies, it was concluded that this clone did not possess a particularly striking similarity to any of the previously cloned D1-like receptors and was not likely to be a species homolog. This gene product is, therefore, referred to as an avian dopamine D1D receptor, in line with the alphabetical nomenclature scheme suggested for nonhuman dopamine D1-like receptor genes(4) .
Consensus sequences for putative post-translation
modifications have been conserved in the avian D1 like receptor family.
As outlined in Fig. 1, of the three avian receptors, only D1A
and D1B contain consensus sites for N-linked glycosylation.
The D1A receptor possesses two sites, at Asn in the amino
terminus and Asn
located in the third extracellular loop
similar to mammalian or vertebrate counterparts. The D1B receptor,
however, only contains one site, at Asn
, in the third
extracellular loop. As illustrated in Fig. 1, several putative
phosphorylation sites for protein kinase C and cAMP-dependent protein
kinase A were found in the third cytoplasmic loops of all three
receptors, comparable with their mammalian counterparts. Unlike the
mammalian dopamine D1/D1A or D5/D1B like receptors, but similar to the
amphibian D1C and avian D1B and D1A receptor, the D1D receptor contains
additional putative protein kinase C sites within the carboxyl
terminus. Conserved amino acid residues characteristic of dopamine
receptors are also found within the avian clones, such as the 2 serine
residues within TM5, believed to mediate binding of the endogenous
neurotransmitter dopamine ( (43) and references therein).
Interestingly, while all D1-like receptors contain three sequential
serine residues within TM5, D1D is the first dopamine receptor to
possess a conserved substitution for the first of these serines at
position Thr
in TM5. The functional significance of this
serine residue for mammalian dopamine D1-like receptor activity has not
been addressed in site-directed mutagenesis studies(43) . Also
present is a conserved cysteine residue, Cys
D1D;
Cys
D1A; Cys
D1B, located in the carboxyl
terminus, which is believed to function as a site for
palmitoylation(44) .
In order to further justify our
nomenclature scheme for avian genes encoding D1-like receptors, we
compared the avian D1 receptors to their mammalian counterparts in
terms of their exhibited pharmacological profiles and ligand binding
specificity. A 3-kb XbaI fragment of LDChi-1 (D1D) and two
3-kb PstI fragments of LDCh2 (D1A) and 6 (D1B) were subcloned
into the expression vector pcD-ps. Membranes prepared from COS-7 cells
transiently expressing these genes were assessed for their ability to
bind the D1-selective antagonist, [H]SCH-23390.
All three receptors bound [
H]SCH-23390
(0.25-4.0 nM) in a concentration-dependent and saturable
manner with high affinity (data not shown). Scatchard transformation of
the data revealed a single class of binding site for D1A and D1B
receptors with an estimated K
of 180 ± 21
and 171 ± 18 pM and B
values
which on average were of 0.9 ± 0.2 and 0.6 ± 0.14 pmol/mg
of protein, respectively. The expressed D1D receptor also bound
[
H]SCH-23390 with high affinity with an estimated K
of 322 ± 58 pM and somewhat
lower level of receptor expression ranging from
0.2-0.4
pmol/mg of protein.
[H]SCH-23390 binding to
COS-7 cell membranes expressing D1A, D1B, and D1D receptors was
inhibited in a concentration-dependent, stereoselective, and uniphasic
manner (as indexed by Hill coefficients close to unity) by a variety of
dopaminergic agonists and antagonists with a rank order of potency and
pharmacological profile clearly consistent with a D1-like receptor.
Estimated K
values for these agents are listed in Table 1.
With regard to agonist compounds, a unique
pharmacological characteristic that differentiates D1/D1A from D5/D1B
receptors is the inherent ability of the D5/D1B receptor to exhibit
higher affinity for the neurotransmitter dopamine and the
aminotetralin, ADTN(45, 46) . Comparable with both
mammalian and Xenopus D1/D1A receptor homologs, both dopamine
and ADTN display a 10-fold higher affinity for the avian D1B (261/550
nM) than D1A (2000/4500 nM) receptor, consistent with
our proposed classification of these receptors based on amino acid
sequence homologies depicted in Fig. 1. Due to the absence of
guanine-nucleotide-sensitive agonist high affinity forms following the
expression of D1-like receptor genes in COS-7
cells(38, 39, 45) , direct comparisons
between the avian and mammalian dopamine D1-like receptors can be made.
Other compounds, listed in Table 1, that further differentiate
the two receptors include, (+)-butaclamol and fluphentixol, which
inhibit [H]SCH-23390 binding to avian D1A
receptors at concentrations 2-5-fold lower than D1B, paralleling
their mammalian/vertebrate counterparts. The estimated inhibitory
constants (K
) for the inhibition of
[
H]SCH-23390 binding by a series of dopaminergic
compounds at the avian D1A correlate (r 0.989) strongly with
those K
values obtained for these agents at the
mammalian/vertebrate D1/D1A receptors with a virtual 1:1 correspondence
in estimated receptor affinity. Although a general tendency for some
agonists to be more potent at the avian D1A was noted, receptor
antagonists displayed very little preferential affinity for either the
human or avian D1A receptor. Similarly, estimated K
values for the avian D1B receptor also correlate extremely well (r 0.976.) with the mammalian or vertebrate D5/D1B receptor.
Again, while some agonists (SKF-76783, N-propylnorapomorphine,
and bromocriptine) displayed slightly higher affinities for the avian
D1B receptor, virtually all other compounds tested exhibited affinities
that were equipotent with the mammalian D1B receptor.
Although the
exhibited pharmacological profile of D1D is consistent with a D1-like
receptor, it displayed many unique characteristics not previously
observed with other cloned members of the D1 receptor family. As
depicted in Fig. 2, the D1D receptor displays a 10-fold higher
affinity for dopamine than the D1A receptor. Similar results were
obtained for ADTN and is suggestive of the contention that the D1D
receptor shares characteristics of the D5/D1B subfamily of receptors.
Interestingly, compounds that normally do not discriminate D1A from D1B
receptors were particularly more potent for the D1D receptor. The most
significant of these, as shown in Fig. 2, is the benzazepine,
SKF-38393. Thus, while D1D does not bind the benzazepine, fenoldopam,
(SKF-82526) with much higher affinity than vertebrate D1/D1A, D5/D1B,
or D1C receptors, it exhibits a 13-25-fold increase in affinity
for the ``partial'' agonist SKF-38393 compared with either
mammalian or amphibian receptors. Similarly, while all other agonists
tested displayed somewhat higher affinity (2-6-fold) for the D1D
receptor compared with avian D1A or D1B receptors, nonselective D1/D2
receptor agonists, such as, N-propylnorapomorphine,
apomorphine, and
(-)-4,6,6a,7,8,12b-hexahydro-7-methyl-indolo[4,3-ab]phenanthridine
were significantly more potent at the D1D receptors compared with any
other D1-like receptor and may provide the molecular basis for some of
the observed D1-like actions obtained with these D2 receptor-preferring
agonists(47, 48) . Moreover, lisuride and pergolide,
compounds used as adjunct therapies in the treatment of
Parkinson's disease(49, 50, 51) , are
also significantly more potent at the D1D receptor, with
4-10-fold higher affinities than for vertebrate D1A, D1B, or D1C
receptors. As noted below, both lisuride and pergolide behave as
agonists at D1D receptors stimulating cAMP accumulation. Interestingly
enough, in native brain membranes a small proportion (15%) of
D1-like sites labeled by [
H]SCH-23390 have been
reported to bind lisuride and pergolide and some of the other dopamine
D2 agonists reported here with high affinity(52) .
Figure 2:
Pharmacological specificity of
[H]SCH-23390 binding to membranes prepared from
COS-7 cells expressing avian D1A, D1B, and D1D receptors. COS-7 cells
were transfected with genes encoding D1A, D1B, and D1D receptors,
membranes prepared and assayed for D1 receptor activity as described
under ``Experimental Procedures.'' Representative curves are
illustrated for the concentration-dependent inhibition of
[
H]SCH-23390 binding (200-400 pM)
to expressed avian D1 receptors by the dopaminergic agonists dopamine
and SKF-38393. For this particular experiment, D1A, D1B, and D1D
receptor concentrations varied less than 2-fold. Estimated inhibitory
constants (K
) for these compounds,
included in Table 1, were determined by KALEIDAGRAPH and are
representative of at least three independent experiments each conducted
in duplicate and which varied by less than
15%.
With
regard to antagonists, the D1D receptor displays affinities for
compounds truly reflective of both D1A and D1B receptor profiles. Thus,
D1D receptors discriminate and exhibit poor affinity for butaclamol,
similar to D1B/D5 receptors, yet is bound by fluphentixol with high
affinity, characteristic of D1/D1A receptors. Of particular interest is
the observed low affinity (2 µM) of haloperidol for
the D1D receptor. By comparison, all other D1-like receptors display
relatively high affinity (
70-150 nM) for the
classical D2-like receptor antagonist. As illustrated in Fig. 3,
direct comparison of the estimated K
values of
numerous agonists and antagonists at the D1D receptor with human D1,
D5, and vertebrate D1C receptors, although correlative, clearly depict
the lack of a simple one to one correspondence in estimated affinities
for a number of compounds. As such, the D1D receptor is the first
member of the D1 gene family that clearly deviates from the classical
D1-like pharmacology with respect to differentiating agonist
benzazepine ligands and binding D2-like agonists with high affinity.
Whether the D1D receptor can further discriminate and display exquisite
sensitivity for other dopaminergic compounds or ``second''
generation D1-like agonists and antagonists is currently under
investigation.
Figure 3:
Pharmacological homology between the avian
D1D receptor and other cloned D1 receptor subtypes. Correlational plots
of estimated inhibitory constants (K) of
various dopaminergic agonists (
) and antagonists (
) to
inhibit [
H]SCH-23390 binding to for the avian D1D
receptor and the human dopamine D1 (A), D5 (B), and Xenopus D1C (C) receptors transiently expressed in
COS-7 cells. K
values for D1, D5, and D1C
receptors were taken from Refs. 38, 39, and 45. Compounds displaying
major discrepancies in estimated affinities between these receptors
were reassayed under identical conditions and reported here. The line
of identity or equimolarity is indicated.
As classically defined, the D1 receptor stimulates
adenylate cyclase affinity. We assessed the ability of various
compounds to alter cAMP accumulation in COS-7 cells transiently
expressing the avian D1A, D1B, and D1D receptors. As summarized in Fig. 4, all three avian dopamine receptors stimulated the
production of cAMP. The addition of 10 µM dopamine or 1
µM SKF82526 to cells expressing D1D resulted in a
10-fold stimulation over basal levels, which was sensitive to
antagonism by the addition of 1 µM SCH-23390. cAMP
production in cells expressing avian D1A and D1B receptors increased,
on average, 7- and 9-fold with the addition of dopamine (10
µM) and 5- and 6-fold with the addition of SKF82526 (1
µM), respectively. Both lisuride and pergolide (10
µM) stimulated cAMP accumulation at all three avian D1
receptors with estimated EC
values at the D1D receptor of
9 and 90 nM, respectively. As such, all cloned members of the
dopamine D1 receptor gene family including the amphibian D1C receptor
are coupled to the stimulation of adenylate cyclase activity. While
evidence suggests the existence of native dopamine D-1 like receptor
that are functionally coupled to effector elements other than adenylate
cyclase, particularly the activation of phosphatidylinositol hydrolysis
(see above), we were unable to show in COS-7 cells any consistent
stimulation of phosphatidylinositol turnover by either avian D1A, D1B,
or D1D receptors in response to varying concentrations of dopamine or
SKF-82526 up to 100 µM (data not shown). It is unknown, at
present, whether this lack of response is due to the inappropriate
complement, in COS-7 cells, of subtype-specific G protein
or
subunits needed for specific avian D1-like receptor
stimulation of phospholipase C or the appropriate molecular form of the
enzyme. Coexpression studies with these proteins in COS-7 cells may
resolve this issue(53) .
Figure 4: cAMP accumulation following avian D1A, D1B and D1D receptor stimulation. COS-7 cells expressing pcD-D1A, pcD-D1B, and pcD-D1D were assayed for cAMP accumulation as described under ``Experimental Procedures.'' Following treatment with either 10 µM dopamine or 1 µM SKF-82526, avian D1A, D1B, and D1D receptors stimulate cAMP production from 7-10-fold above basal levels. Maximal stimulation with 10 µM dopamine for all three avian D1-like receptors is reversed by the addition of the D1-selective antagonist SCH-23390 (1 µM). Results shown are representative of at least two independent experiments each conducted in duplicate.
Northern blot analysis of brain and
peripheral regions from chicken total RNA revealed the expression of
all three receptors. As depicted in Fig. 5, an mRNA species of
3 kb was observed for the D1D receptor specifically localized only
in the brain. D1A receptor mRNA (
3.4 kb) was found within the
brain and to a much lesser extent in the kidney. The D1B receptor mRNA
(
2.7 kb) was more widely distributed and found in brain, spleen,
and kidney and to a much lesser extent in the heart. Whether the
slightly different mRNA transcript sizes for D1B receptor mRNA in the
various tissues may possibly be attributable to multiple transcription
initiation or poly-adenylation sites is currently unknown. In any
event, the mRNA distribution patterns of the cloned D1 receptors are in
line with the known distribution patterns of both dopamine-containing
cells and D1 binding sites in the avian brain. Further work, via in
situ hybridization analysis, will be necessary to clearly define
the tissue-specific cellular distribution profile of multiple D1
receptor mRNAs in this species.
Figure 5:
Tissue-specific distribution of avian D1A,
D1B, and D1D receptor mRNAs. Autoradiogram depicting the mRNA
distribution of cloned avian D1D, D1A, and D1B receptor. Total chicken
RNA, from various tissues, was denatured with formaldehyde,
electrophoresed, transferred to nylon membrane, and probed with
-
P-labeled receptor fragments as described under
``Experimental Procedures.'' Blots were subjected to
autoradiography for approximately 72 h. Estimated molecular size of
receptor mRNAs are listed in the text.
In addition to D1A and D1B receptor
genes, the existence of distinct genes encoding for Xenopus D1C and avian D1D receptors raises the intriguing question as to
whether these genes are truly reflective of distinct D1-like receptor
subfamilies, as may be the case with D1A and D1B receptors, or merely
associated with the particular evolutionary pressures and constraints
of these particular phyla or species. Clearly the identification of all
four genes within one species or the isolation of mammalian
counterparts to these receptors will help establish the distinctive
molecular nature of these genes. To that end, preliminary molecular
evidence suggests the existence of a D1C-like receptor gene in the
avian species G. domesticus. Thus, Southern blots of BamHI-HindIII-digested avian DNA, hybridized under
stringency conditions described under ``Experimental
Procedures'' with human D1 and D5 receptor probes, revealed the
presence of a 4 kb fragment hybridizing to both D5 and D1. Under
high stringency conditions, however, this band was strongly hybridized
to a Xenopus D1C receptor probe, but not at all to avian D1D,
D1A, or D1B receptor probes. (
)Although further work will be
necessary to fully ascertain the molecular nature of this DNA fragment,
these data suggest that at least in this avian species, an additional
D1-like receptor gene more homologous to D1C may exist and is
supportive of the contention that four distinct genes encoding D1-like
receptors may be found in a single genome. Although the evolutionary
relationship between mammals and birds is weak, (54, 55) the availability of both D1C and D1D receptor
genes may provide the necessary molecular tools for the successful
isolation of mammalian homologs of these or related D1 receptor
variants that have, to date, eluded detection following screening with
gene fragments encoding mammalian D1A or D1B receptors alone.
In any event, the availability of these vertebrate genes may provide a more practical approach by which to identify sequence specific motifs that may underlie the maintenance and expression of unique D1-like receptor characteristics. Given the fairly high degree of exhibited sequence homology between members of the D1-like receptor family, observed receptor-specific and selective pharmacological profiles would suggest that changes in key amino acid residues or domains may translate into major shifts in potency and substrate specificity. As such, the construction of human D1 or D5/avian D1D receptor chimeras may aid in the identification of those regions involved in the unique pharmacological specificity, affinity, and ability of the avian D1D receptor to differentiate agonists and antagonists of various structural classes and may ultimately provide the molecular basis for the rationale design of therapeutic compounds acting at D1-like receptors for the treatment of various psychomotor diseased states.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L36877[GenBank]-L36879[GenBank].