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J. Biol. Chem., Vol. 275, Issue 31, 23446-23455, August 4, 2000

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Dopamine D5 Receptor Agonist High Affinity and Constitutive Activity Profile Conferred by Carboxyl-terminal Tail Sequence^*

Lidia L. Demchyshyn^§¶, Fortunata McConkey^§, and Hyman B. Niznik^§**

From the Departments of  Psychiatry and  Pharmacology and ^** Institute of Medical Science, University of Toronto, Ontario, Canada M5S 1AB; and the ^§ Laboratory of Molecular Neurobiology, Center For Addiction and Mental Health, Toronto, Ontario, Canada M5T 1R8

Received for publication, January 10, 2000, and in revised form, May 8, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The mammalian dopamine D1-like receptor gene family is comprised of two members, termed D1/D1A and D5/D1B. In an attempt to define the role of the carboxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacological and constitutive activity profiles, we examined a series of D5 receptor chimeras in which only the CT tail was swapped with corresponding sequences encoding human/vertebrate D1-like receptors. D5/D1_CT or D5/D1D_CT tail substitution mutants displayed a rank order of potency and agonist affinities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, similar to wt D1 receptors, did not exhibit receptor constitutive activity or responsiveness to inverse agonists. D1/D5_CT or D1/D1D_CT tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1_CT and D5/D1D_CT receptors. The affinities for numerous antagonists remained essentially unchanged for all receptor chimeras relative to parental wt receptors. A series of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438-448 and particularly Gln⁴³⁹, as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conserved D5/D1B receptor residue Gln⁴³⁹-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affinities for discriminating agonists ~4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP accumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino acid in this region can further regulate D5 agonist high affinity binding interactions independent of receptor constitutive activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Dopamine is a neurotransmitter that regulates a variety of physiological functions. Acting through D1- and D2-like receptors, dopamine exerts a major role in regulating neuronal motor control, cognition, event prediction, and emotion and has been implicated in the maintenance and expression of neuropsychiatric disease states such as addiction and schizophrenia (1-5).

In mammals, dopamine D1-like receptors are encoded by two distinct genes, termed D1 (6) and D5 (7), and belong to a superfamily of single polypeptide seven-transmembrane (TM)¹ domain receptors that exert their biological effects via intracellular G-protein-coupled signaling cascades. Dopamine D1 and D5 receptors can be distinguished from each other on the basis on a number of inherent attributes (8). These include obvious differences in primary structure, chromosomal localization, and, at an anatomical level, distinct mRNA and cellular and subcellular protein distribution profiles. Despite this, little is known with regard to the molecular determinants that modulate the functional properties of these receptors. This is in large part due to the relatively low expression levels of D5 receptors in neuronal populations and the lack of available pharmacological tools and ligands that can selectively identify this molecule.

When expressed in a variety of mammalian cell lines, a few unique distinguishing pharmacological features between D1 and D5 receptors are evident. The endogenous neurotransmitter dopamine and nonselective dopaminergic agonist 6,7-dihydroxy-2-aminotetralin (6,7-ADTN) display up to 10-fold higher affinity and efficacy for D5/D1B receptors, whereas nonselective antagonists such as (+)-butaclamol, flupenthixol, and spiperone exhibit much higher affinities for D1/D1A than for D5/D1B receptors. In contrast, the benzazepine class of molecules, in general, does not appear to discriminate between the various D1-like receptor subtypes and may be considered as generic nonselective ligands for all the members of the D1 receptor gene family. These characteristics appear invariant, because cloned vertebrate dopamine D1A and D1B receptors express pharmacological profiles for dopaminergic agonists and antagonists virtually identical to their mammalian counterparts (reviewed in Ref. 9).

Although all cloned members of the D1 receptor family stimulate cAMP accumulation, a defining differential characteristic of mammalian D5/D1B receptors is that they exhibit higher levels of constitutive or agonist-independent adenylyl cyclase activity, which is inhibited by the inverse agonists (+)-butaclamol and flupenthixol (10, 11). Relative to D1/D1A receptors (12), this characteristic again appears to be inherent to this receptor subtype, because this property is absolutely conserved in all cloned vertebrate members of the D1B receptor subclass (9, 13). To date, unlike that of other naturally occurring constitutively active receptors, which maintain various pathological states (14, 15), the physiological significance of increased basal D5 receptor activity is unknown, but suggests that the D5 receptor may exhibit distinct intrinsic functional correlates relative to D1-like receptors (11).

A number of studies have shown that particular amino acids and regions of both the third intra- and extracellular loop of D1-like receptors play an important role in allowing for the expression of subtype-specific pharmacological and functional attributes of these receptors (reviewed in Ref. 16). In particular, mutation of one conserved amino acid residue (Ile²⁸⁸) in the third cytoplasmic loop of mammalian D1B receptors appears to allow for the expression of agonist-independent receptor activity as well as ligand binding properties (17). In addition, a recent elegant study based on chimeric D1-D5 receptors suggests that regions encoding TM-6/7 and the carboxyl-terminal (CT) tail of D5 and D1 receptors confer the observed unique pharmacological and functional characteristics found in respective cognate wild-type (wt) receptor subtypes (18). Despite these insights, the swapping of relatively large receptor domains obfuscates the contribution of specific regions and amino acid sequence motifs that may confer subtype-specific D1 versus D5 receptor pharmacological and functional profiles.

The carboxyl-terminal region of many G-protein-coupled receptors (GPCRs) has been studied and implicated in regulating many receptor-mediated events, including desensitization, phosphorylation, internalization, and constitutive activity (19-23). Moreover, this region appears to impart functional attributes to a variety of GPCRs by allowing coupling to distinct protein effector systems not classically associated with G-protein activation (24). Structurally, although D1 and D5 display amino acid sequence homology of approximately 80% within transmembrane domains, particular divergence of amino acid sequence identity occurs within the CT tails of these receptors (~30%). Recent work has identified that D5, but not D1, receptor stimulation can regulate ligand-gated channel activity independent of classically defined receptor signaling cascades and this receptor cross-talk is solely dependent upon sequence-specific motifs of the D5-CT tail (25). Although sequences within the CT tail of dopamine D5 receptors allow the functional differentiation of dopamine D5- from D1-like receptors, attempts to define the role of the D5-CT tail in the expression and maintenance of D5 subtype-specific pharmacological and constitutive activity profiles have yet to be made. Here we report, based on a series of D1 and D5-CT tail receptor chimeras, truncation, and deletion mutants, as well as site-directed mutagenesis studies, that the pharmacological and functional signature of the D5/D1B receptor is governed by sequence-specific motifs within the CT tail and that one particular amino acid residue in this region can independently dissociate modifications of D5 receptor constitutive activity from the enhancement of the expressed agonist high affinity pharmacological profile.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mutant Receptor Construction

CT-tail Chimeric Receptors-- Wild-type human D1 and D5 and vertebrate D1D receptors were used as described previously (6, 7, 26) except that the D1 and D5 receptor constructs were shortened via polymerase chain reaction (PCR; see below) to yield a parental 1.5-kilobase EcoRI/SpeI fragment and subcloned into pCD-ps as described (26). Mutant D1 and D5 chimeric receptors in which the CT tails were swapped were constructed using an overlap PCR extension methodology as described previously (27). Oligonucleotide primers were chosen in the 5'-region upstream to the start site of the parent receptor and the 3'-terminal region downstream of the stop codon of the tail receptor. Primers were also constructed overlapping each other on both DNA strands in a highly conserved region (YAFNADF) downstream of TM7. Approximately 500 ng of cesium chloride-purified DNA containing either human D1, D5, or vertebrate D1D receptors were used as templates, denatured for 5 min at 95 °C, and submitted to 30 cycles of PCR (1 min at 95 °C, 1.5 min at 58 °C, 1 min at 72 °C) with 2.5 units of Vent Taq DNA polymerase (Perkin-Elmer/Cetus) and 500 ng of synthesized specific oligonucleotide primers. The first round of PCR generated two fragments coding the amino-terminal to TM7 of the first receptor and the carboxyl region of the second receptor (D1, D5, or D1D). The amino portion of the D1 receptor was amplified using primers 5'-CTGGAATTCCTTAGGAACTTGAGGGGT-3' and 5'-CCGGAAGTCAGCATTGAAGGCATA-3'. The amino portion of the D5 receptor was generated using specific primers 5'-TGAGAATTCAGTTGGGATCGCGCACAA-3' and 5'-CCGGAAGTCAGCATTGAAGGCATA-3'. The carboxyl tail of the chimera was generated using specific primer 5'-TATGCTTTGAATGCTGACTTC-3' and either 5'-TGGACTAGTCAGGATTCATCTGCGAGT-3' for the D1 receptor, 5'-GAGACTAGTGGGGTTTCTTAATGCAGT-3' for the D5 receptor, or 5'-GAGGAATTCATTTTTTGGGAGTACAGGCAT-3' for the D1D receptor. Amplified fragments were electrophoresed on 1% agarose gels and the appropriately sized bands were excised and purified by gel extraction. Aliquots (~500 ng) of the two fragments were combined and subjected to a second round of PCR for 30 cycles as described above, utilizing only the 5'-start site and 3'-stop codon primers, which incorporated EcoRI and SpeI restriction sites, respectively, to facilitate direction subcloning. PCR amplification generated full-length chimeric receptors of ~1.5-kilobase fragments, which were excised, purified, and directionally subcloned into the expression vector pCD-ps.

D5 Receptor Truncation-- Stepwise truncation mutants of the human D5 receptor CT tail were generated by PCR amplification from the full-length receptor. Primers were constructed in various regions 3' of the carboxyl tail, and successive shorter fragments were selectively amplified from a full-length D5 receptor template under high stringency conditions. Eight CT-truncated receptors were constructed using specific oligonucleotide primers 5'-TGAGAATTCAGTTGGGATCGCGCACAA-3' and either 5'-CCGGAAGTCAGCATTGAAGGCATA-3' (D5-Phe³⁶⁵), 5'-TGGACTAGTGTTCACCGTCTCCACCGG-3' (D5-Asn³⁸⁸), 5'-TGGACTAGTAACGGCGTTGGGCATCAT-3' (D5-Val⁴¹⁸), 5'-TGGACTAGTCTGTCGTTGTCCACCTC-3' (D5-Glu⁴²⁹), 5'-TGGACTAGTGAACATGCGATCGAAAGG-3' (D5-Phe⁴³⁸), 5'-TGGACTAGTCTGGAACATGGATCGAA-3' (D5-Gln⁴³⁹), 5'-TGGACTAGTGATCTGGAACATGCGATC-3' (D5-Ile⁴⁴⁰), or 5'-TGGACTAGTGTCACCATCTGGGGACGT-3' (D5-Asp⁴⁴⁸). Approximately 500 ng of DNA plasmid containing the full-length D5 receptor was used as template, denatured for 5 min at 95 °C, and submitted to 30 cycles of PCR (1 min at 95 °C, 1.5 min at 58 °C, 1 min at 72 °C) as described above with the listed combination of specific oligonucleotide primers. Amplified products were gel-purified and subcloned as described above.

Site-directed Mutagenesis-- Glutamine-deleted and -substituted mutants of the human D5 receptor CT tail were generated by PCR amplification from the full-length receptor. Primers were constructed in regions 3' of the carboxyl tail specifically surrounding the glutamine amino acid and selectively amplified from a full-length D5 receptor template under high stringency conditions. Four deleted and substituted mutant receptors were constructed using specific oligonucleotide primers 5'-TGAGAATTCAGTTGGGATCGCGCACAA-3' and either 5'-CTGCTGATAGATGAACATGCGATC-3' (D5-Gln⁴³⁹deletion), 5'-GGACGTATAGATGAACATGCGATC-3' (D5-Gln⁴³⁹-Gln⁴⁴² deletion), 5'-CTGATAGATCGGAACATGCGATC-3' (D5-Gln⁴³⁹ substitution to Ala), 5'-CTGATAGATGATGAACATGCGATC-3' (D5-Gln⁴³⁹ substitution to Ile) to generate amino-terminal fragments, and either 5'-GATCGCATGTTATCTATCAGAG-3' (D5-Gln⁴³⁹ deletion), 5'-GATGCATGTTCATCTATACGTCC-3' (D5-Gln⁴³⁹-Gln⁴⁴² deletion), 5'-GATCGCATGTTCGCGATCTATCAG-3' (D5-Gln⁴³⁹ substitution to Ala) or 5'-GATCGATGTTCATCATCTATCAG-3' (D5-Gln⁴³⁹ substitution to Ile) with oligo 5'-GAGACTAGTGGGGTTTCTTAATGCAGT-3' to generate the carboxyl-terminal fragment. Approximately 500 ng of DNA plasmid containing the full-length D5 receptor was used as template, utilizing the same PCR amplification conditions described above. Gel-purified aliquots (~500 ng) of the two fragments were combined and subjected to a second round of PCR for 30 cycles as described above, utilizing only the 5'-start site and 3'-stop codon primers, which incorporated EcoRI and SpeI restriction sites, respectively, to facilitate direction subcloning.

To ensure the appropriate splice fusion, site-directed mutation/truncation, and absence of PCR-generated spurious sequence mutations/substitutions, all generated receptor constructs were sequenced in both directions using the dideoxynucleotide chain termination method with 7-deaza-GTP, Sequenase (version 2.0, U.S. Biochemical Corp.) and specific oligonucleotide primers (Biotechnology Service Center, Hospital for Sick Children, Toronto).

Transfection and Ligand Binding Analysis

COS-7 cells were transiently transfected with cesium chloride-purified wt or mutant/truncated DNA constructs by electroporation (60-80 µg of DNA per 2.5 × 10⁷ cells; 48 ohms, 135 mA, 500 microfarads), placed into 150-mm plates, and cultured for 4-5 days. COS-7 cells were maintained in Dulbecco's -minimal essential media supplemented with 10% fetal calf serum at 37 °C and 5% CO₂. Cells were then collected, and membranes were prepared for binding assays as described previously (26). For saturation experiments, 0.5-ml aliquots of tissue homogenate (~50-100 µg of membrane protein) were incubated in duplicate with increasing concentrations of [³H]SCH-23390 (85.5 Ci/mmol, 25-4000 pM final concentration) for 120 min at room temperature in a total volume of 1.5 ml. For competition binding studies, 0.5 ml of membranes were incubated in duplicate with [³H]SCH-23390 (250-400 pM) and increasing concentrations of competing ligands (10¹³ to 10⁴ M) for 120 min. Experiments were terminated by rapid filtration, and filters were monitored for tritium. Nonspecific binding was defined in the presence of 1 µM (+)-butaclamol. Binding data were analyzed by the nonlinear least-square curve-fitting program KALEIDAGRAPH (Abelbeck Software) as described previously (26). For all experiments, direct assay comparisons between relevant wt and mutant/chimeric receptor preparations were conducted on the same day, using the same serial drug dilutions and on the same batch of transfected cells. To further "normalize" assay comparisons, transfection conditions were optimized to yield receptor ligand binding densities that were essentially equivalent between wt and mutant/chimeric receptor preparations.

cAMP Accumulation Assay

COS-7 cells were transiently transfected as described above, placed in 24-well plates, and grown for 72 h. Cells were washed with 0.5 ml of prewarmed Dulbecco's -minimal essential media containing 1-methyl-3-isobutylxanthine and 1 µM propranolol, then incubated in the above media in the presence or absence of antagonist or inverse agonist for 15 min at 37 °C and 5% CO₂. Increasing concentrations of dopamine or other agonists were added, and the cells were incubated for an additional 15 min at 37 °C and 5% CO₂. The reaction was terminated by the addition of 0.5 ml of 0.2 N HCl and incubation for 20 min at 4 °C. Cellular debris was pelleted, and aliquots of the supernatant were used to determine the cAMP content via immunodetection (Amersham Pharmacia Biotech) as described previously (26). Estimated EC₅₀ and IC₅₀ values were obtained as described previously. To ensure equivalence of whole cell cAMP assay comparisons, receptor densities were monitored for [³H]SCH-23390 binding (3.0 nM final concentration) and wt/mutant receptor assays were conducted on the same day, using the same serial drug dilutions and on the same batch of transfected cells. Data were analyzed using a two-tailed t test set at 0.1 level of significance.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

To determine what potential role sequences within the highly sequence divergent carboxyl tails may play in the pharmacological and functional differentiation of the human D1 and D5 receptors, D1/D5 receptor chimeras were generated in which the CT tail of the parent receptor was removed and substituted by complementary CT-tail receptor sequences encoded by other members of the mammalian/vertebrate D1 receptor gene family. Dideoxynucleotide sequence analysis confirmed the absence of any spurious PCR-generated sequence mutations either within the CT-tail splice site or throughout the entire full-length mutant receptor sequence.

We first assessed the effects of swapping the CT tails of human D1 and D5 receptors on their ability to bind the selective D1-like receptor antagonist [³H]SCH-23390 with high affinity. As listed in Table I, D5/D1_CT or D1/D5_CT receptor mutants bound to [³H]SCH-23390 in a saturable, concentration-dependent, and uniphasic manner with estimated K_d values that were not significantly different from either parental wt human D5 or D1 receptors. Transfection conditions were established that allowed for mutant/wt receptors to be expressed (ranging from 850 to 1300 fmol/mg of protein) at essentially equivalent levels, at least as indexed by [³H]SCH-23390 binding, with estimated B_max values for wt and mutant receptors similarly listed in Table I. For reasons outlined below, we constructed D5- and D1-CT receptor mutants to encode the highly unique sequence of CT tail of the vertebrate dopamine D1D receptor (26) as an additional control. These D5 and D1 chimeric mutants similarly bound [³H]SCH-23390 with high affinity and in a saturable manner, with estimated K_d and B_max values (Table I) not significantly different from either parental D1/D5 or CT mutant receptors.

As summarized in Table II, wt D5 binds the endogenous neurotransmitter dopamine and some other dopamine agonists such as 6,7-ADTN with 10-fold higher affinity than D1 while displaying a 2- to 7-fold preference for dopaminergic agonists such as apomorphine-, N-propylnorapomorphine (NPA)-, and benzazepine-like compounds, respectively. With regard to antagonists, D1 receptors in contrast display somewhat higher affinity for non-benzazepine antagonist compounds, particularly butaclamol and flupenthixol. To assess the potential contribution of sequence motifs within the CT tail to the overall pharmacological differentiation of human D5 versus D1 receptors, we analyzed the ability of numerous dopaminergic agonists and antagonists to inhibit the binding of [³H]SCH-23390 to generated receptor CT tail substitution mutants relative to parental/wt D1 and D5. Replacement of human D5-CT sequence with amino acids encoded by the D1 receptor CT tail resulted in a mutant receptor in which [³H]SCH-23390 binding was inhibited in a concentration-dependent and uniphasic manner by a variety of dopaminergic agonists with a rank order of potency and pharmacological profile comparable to that exhibited by the human D1 receptor. Thus, as illustrated in Fig. 1A, D5/D1_CT receptor mutants exhibited estimated K_i values for discriminating agonist dopamine 10-fold lower than wt D5 receptors and virtually identical to K_i values displayed for these compounds by the human D1 receptor. Similar reductions in affinity for other discriminating dopaminergic agonists, such as 6,7-ADTN and apomorphine, were noted with estimated K_i values listed in Table II. The effects of CT-tail substitution are specific for D1-like agonist compounds, because the pharmacological profile and estimated affinities for numerous antagonist ligands, including D1/D5 discriminating agents such as butaclamol and flupenthixol, were retained by mutant receptors relative to wt receptors (see Fig. 1B) consistent with estimated K_d values for [³H]SCH-23390 binding to these mutants (Table I). Estimated K_i values for a number of antagonists for this particular chimera are listed in Table II.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Effects of swapping the carboxyl-terminal tail domain on the agonist and antagonist ligand binding profiles of dopamine D5 receptors. COS-7 cells were transfected with DNA encoding D1 or D5 receptors or their chimeras, and membranes were prepared and assayed for receptor activity as described under "Experimental Procedures." Representative curves are illustrated for the concentration-dependent inhibition of [3H]SCH-23390 binding (200-400 pM) to the discriminating endogenous agonist dopamine (A) and antagonist butaclamol (B) to wt D5 and D1 receptors and D5/D1CT chimera. For this particular experiment, receptor concentrations as indexed by [3H]SCH-23390 (3.5 nM final concentration) binding was 0.55, 0.78, and 0.69 pmol/mg of protein for D5, D1, and D5/D1CT receptors, respectively. Estimated inhibitory constants (Ki) for these compounds are listed in Table II. Data are representative of at least three independent experiments, each conducted in duplicate. C and D, correlation plots estimating Ki values of various dopaminergic () agonists and () antagonists to inhibit [3H]SCH-23390 binding to the human wt (C) D1 versus D1/D5CT and (D) D5 versus D5/D1CT chimeric receptors. Ki values were taken from Table II. The line of identity or equimolarity is also drawn. Substitution of the D5-CT tail significantly reduced agonist, but not antagonist Ki values, respectively (p < 0.05).

To determine if the selective loss of agonist high affinity by the D5/D1_CT mutant receptor was due to the removal of sequence motifs encoded by the D5 receptor tail or a result of the addition of specific D1-tail sequence domains that may confer inherent D1 agonist low affinity characteristics, we analyzed the pharmacological profiles of a D5 receptor mutant in which the CT tail was substituted with highly divergent but corresponding sequences of the vertebrate D1D receptor (26). The dopamine D1D receptor displays an overall pharmacological profile and affinity for dopaminergic agonists similar to that of the human D5 receptor. Despite differences in overall length and net charge, the generated D5/D1D_CT chimera displayed agonist affinity profiles identical to that exhibited by either the wt human D1 receptor or D5/D1_CT mutant with a loss of affinities for the agonists dopamine, 6,7-ADTN, NPA, and apomorphine greater than 10-fold, whereas SKF-38393 and SKF-82526 only displayed a 2-fold loss in affinity. As with the human D5/D1_CT mutant described above, the D5/D1D_CT receptor exhibited antagonist binding profiles identical with that observed for wt D5 receptors with estimated K_i values listed in Table II. These data suggest that it is the removal of CT sequence motifs of the D5 receptor, rather than the addition of D1- or D1D-CT-tail sequence domains, that allow for the expression of agonist D1 receptor characteristics and is consistent with the notion that the pharmacological profile of the D1/D1A receptor is an inherent default characteristic of members of the D1 receptor family (9, 13, 27).

The contention is further supported by the fact that sequence motifs encoded within the CT tail of the D5 receptor cannot fully confer D5 receptor pharmacological characteristics to other members of the D1 receptor family. As outlined in Table II, the D1/D5_CT chimera retained a pharmacological profile consistent with that observed for the wt D1 receptor for both agonists and antagonists, although agonist potencies were observed to slightly, albeit nonsignificantly, shift to the left. Similar results were obtained with the D1/D1D_CT receptor mutant in which the substitution of D1-CT tail with that encoded by the D1D receptor did not affect either agonist or antagonist potencies relative to wt D1 or mutant D1/D5_CT receptor (Table II). The selective modification of D5 agonist high affinity binding interactions by swapping the CT-tail sequence but not that of antagonists or both agonists and antagonists at D1 receptors is depicted graphically in Fig. 1, C and D. Correlation plots of estimated K_i values for agonists obtained at wt D5 versus D5/D1_CT chimeric receptors significantly deviate from unity and are distinct from that observed for D1 versus D1/D5_CT tail mutants, which exhibit a virtual one-to-one correspondence in both estimated agonist and antagonist affinity values (Fig. 1C). Thus, estimated K_i values for agonists but not antagonists at D5 versus D5/D1_CT chimeric receptors (Fig. 1D) significantly diverge from this one-to-one correspondence in affinity values (Z value of 2.9, p < 0.05). The inability to reciprocally confer D5 receptor characteristics to D1 receptors by D5-CT sequences suggests that additional D5 receptor domains must be present for the full expression of its pharmacological signature to occur, a finding similar to that previously reported for D1/D5 receptor substitution mutants within the third cytoplasmic loop (17). These may include domains encoded by the last two transmembrane domains and third extracellular loop (18). In any event, the data suggest that CT-tail substitution alone has little effect in altering D1 receptor pharmacological profiles and agonist affinity characteristics unlike that observed for the human D5 or the vertebrate D1B receptors (27).

To assess whether the observed selective reductions in D5 agonist ligand binding affinity by CT-tail substitutions were functionally relevant, we examined the ability of D5-D1_CT mutant receptors to stimulate cAMP accumulation constitutively and in response to agonist stimulation. Transfection protocols were optimized to maintain comparatively similar levels of expression between wt and chimeric receptors. Although previous studies have demonstrated the importance of amino acids in the third cytoplasmic loop and regions encoding TM6 to the receptor carboxyl terminus for the maintenance and expression of constitutive nature of the D5 receptor (17, 18, 28), we show here that the replacement of the D5 receptor CT tail alone converts D5 receptor agonist-independent stimulation of cAMP accumulation and responsivity to the inverse agonists butaclamol and flupenthixol to one displayed by wt D1 receptors. As illustrated in Fig. 2A, constitutive activity generated by the wt D5 receptor was approximately 2- to 3-fold higher relative to basal cAMP levels generated by wt D1 receptors under similar levels of expression and consistent with previous observations (10, 17, 18). Basal cAMP levels generated by either D1 or D5 receptors were blocked by the inverse agonist butaclamol (10 µM) and flupenthixol (not shown) and not by SCH-23390 (10 µM). D5/D1_CT receptor mutants resulted in the significant reduction of constitutive basal cAMP activity and responsivity to inverse agonists resembling levels and activity inherent of wt D1 receptors. Similar results were obtained with D5/D1D_CT receptor mutants (data not shown). In contrast, and consistent with ligand binding data described above, replacement of the D1-CT tail with that of the D5-CT tail did not significantly effect mutant receptor constitutive basal cAMP accumulation or responsiveness to inverse agonists and displayed characteristics virtually identical to wt D1 receptors. These data again suggest that dopamine D5-CT sequence motifs alone cannot confer functional D5 receptor characteristics to D1-like receptors.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Effects of swapping the carboxyl-terminal tail domain on the functional profiles of dopamine D5 receptors. A, constitutive activity and responsivity to inverse agonists and antagonists by wt and chimeric receptors. COS-7 cells expressing D1 and D5 receptors and D1/D5CT and D5/D1CT chimeras were assayed for basal whole cAMP accumulation in the absence or presence of the indicated concentrations of SCH-23390 or butaclamol. The D5 receptor agonist-independent activity and sensitivity to the inverse agonist butaclamol is lost by substituting sequences encoded by D1-CT, whereas D5-CT sequences do not influence inherent D1 agonist-independent cAMP accumulation or responsivity to butaclamol. B, maximal whole cell cAMP production following D1, D5, and CT-chimeric receptor stimulation by 10 µM dopamine. Values given for maximal cAMP accumulation with 10 µM dopamine are those obtained following the subtraction of either basal cAMP or that inhibited by SCH-23390 (1 µM). Results shown are means ± S.E. of four to six independent experiments, each conducted in duplicate. C, dose-response curves for dopamine-stimulated whole cell cAMP accumulation by wt and chimeric receptors. Data points are expressed as percentage of maximal response obtained by either wt and chimeric receptors after subtracting basal or SCH-23390 (1 µM)-inhibited activity as described above. Data shown are representative of three to four independent experiments with estimated EC50 values for dopamine and other agonists listed in Table III. Receptor expression levels for the data depicted above were for D5, 0.793 ± 0.16; D5/D1CT, 0.902 ± 0.177; and D1, 1.0 ± 0.150 in pmol/mg of protein as indexed by [3H]SCH-23390 binding (3.5 nM, final concentration).

We next assessed the ability of dopamine to maximally stimulate cAMP in both wt and mutant chimeric receptors, because previous work has indicated differences in the potency of agonist-mediated coupling profiles of D1 and D5 receptors (10, 17, 18). Under the present experimental conditions, dopamine stimulated maximal whole cell cAMP accumulation by both wt D1, wt D5, and CT-tail chimeric receptors to virtually identical levels the effects of which were blocked by the D1/D5 receptor antagonist SCH-23390 (Fig. 2B). These data suggest that sequences encoded by the CT tails of D5 or D1 receptors do not influence the intrinsic activity of dopamine at these receptors similar to that seen with vertebrate D1A/D1B_CT-tail substitution mutants (27) but distinct from that observed with D1 and D5 receptors in which other sequence domains have been swapped or replaced (17, 18, 28, 29). We next assessed dose-response curves for dopamine-stimulated whole cell cAMP accumulation by both wt D1, wt D5 and CT-tail chimeric receptors. These are illustrated in Fig. 2C, and as previously reported, the potency of dopamine is ~3-fold higher for dopamine D5-mediated cAMP accumulation than for that with D1 with estimated EC₅₀ values as listed in Table III. The selective reduction of D5 receptor constitutive activity by replacement of its CT tail by D1-CT sequence was accompanied by a concomitant but small rightward shift (~2-fold) in the potency for dopamine-stimulated cAMP accumulation with an estimated EC₅₀ value somewhat intermediate to that observed for wt D1 and D5 receptors and without any change in the maximal value of cAMP production. These data suggest that, in addition to the observed loss of agonist high affinity binding characteristics and constitutive activity profiles, D5 receptor mutants display functional properties more reminiscent of wt D1 receptors following substitution of CT-tail sequences with corresponding sequences of either D1 or D1D receptors. Similarly, as listed in Table III, D1/D5_CT receptor chimeras displayed an apparent ~2-fold enhancement in the potency of dopamine to stimulate cAMP accumulation relative to wt D1 receptors, albeit, with estimated EC₅₀ values intermediate to that exhibited by D1 and D5 receptors and not significantly distinct from D5/D1_CT receptor mutants. The fact that sequence motifs on either D1- or D5-CT tails do not fully confer or reconstitute dopamine agonist-prompted coupling of D1/D5_CT or D5/D1_CT receptor mutants to mimic wt D5 or D1 receptors, respectively, would again suggest that additional molecular determinants are necessary for the full expression of both D1 and D5 dopamine-mediated receptor coupling (18) but not for exhibited D5 ligand binding and constitutive activity profiles. This notion is further attested to by the observation that the observed shifts in the potency for dopamine-stimulated cAMP accumulation with CT-tail chimeric receptors appear specific to the endogenous neurotransmitter dopamine itself, because results obtained for the discriminating agonist 6,7-ADTN and both SKF-82526 and partial agonist SKF-38393 revealed no consistent modification in the estimated EC₅₀ values of these compounds at wt versus chimeric receptor (Table III). Thus we conclude that despite the trend for D5/D1_CT and D1/D5_CT chimeric receptors to display EC₅₀ values for dopamine-stimulated cAMP accumulation (Table III), more reminiscent of those exhibited by wt D1 and D5 receptors, respectively, these are particular for the endogenous neurotransmitter and that CT-tail sequences cannot alone impart dopamine-mediated receptor coupling characteristics significantly different from their cognate receptors. Moreover, these data suggest that the reported hallmarks of constitutively active mutant or native receptor agonist-independent activity, increased binding affinity and functional potency for agonists as well as lower affinity for antagonists/inverse agonists (14, 15), all characteristics of the naturally occurring dopamine D5 receptor, can be dissociated from each other by sequences encoded within the D5-CT tail. As such, it appears that only constitutive activity and agonist but not antagonist ligand binding profiles are maintained by sequences within the D5-CT tail. Full expression of dopamine D5 functional agonist potencies and modification of antagonist/inverse agonist binding affinities, and possibly potencies, would require the participation of additional molecular determinants and amino acid domains as attested to in the literature for other GPCRs (19).

View this table: [in this window] [in a new window]   Table III Estimated EC50 values for dopaminergic agonist-mediated whole cell cAMP accumulation following wild-type and chimeric receptor stimulation Estimated constants (EC50) of whole cell cAMP accumulation following receptor or chimeric stimulation with various agonists [108-104] in COS-7 cells transfected with the human D1 and D5 receptor genes and D1/D5CT and D5/D1CT chimeras. Values represent the means of three to four independent experiments, each conducted in duplicate.

To identify particular amino acid domains within the CT tail of the D5 carboxyl tail that are critical to both its expressed pharmacological profile and agonist-independent functional signature, a series of CT-truncated D5 receptor mutants was constructed. As schematically illustrated and summarized in Fig. 3A, the shortest construct was terminated at amino acid 365 (D5-Phe³⁶⁵) following the conserved region distal to TM7. Each subsequent stepwise mutant (D5-Asn³⁸⁸, D5-Val⁴¹⁸, D5-Asp⁴⁴⁸) was constructed to contain an additional 30 amino acids from the previous truncation. When transiently expressed in COS-7 cells, however, these truncated D5-Phe³⁶⁵, D5-Asn³⁸⁸, and D5-Val⁴¹⁸ receptors displayed very poor or a total lack of ability to bind [³H]SCH-23390 (Fig. 3, A and B) consistent with previous results on the effects of CT substitution/deletion mutants of the D1 receptor (see Refs. 16 and 29) and possibly as a result of poor receptor processing or trafficking. Despite the fact that either D5 or D1 receptor tail truncation resulted in the loss of [³H]SCH-23390 binding, the functional responsiveness of D5-Phe³⁶⁵, D5-Asn³⁸⁸, and D5-Val⁴¹⁸ receptor mutants to dopamine stimulation was retained. Thus, cAMP production increased with the addition of agonists such as dopamine (Fig. 3C), SKF-82526, and SKF-38393 (data not shown) in COS-7 cells expressing D5-Phe³⁶⁵, D5-Asn³⁸⁸, and D5-Val⁴¹⁸ receptor mutants to levels similar to wt receptors and similar to D5/D1_CT receptor mutants. Similarly, SCH-23390 inhibited dopamine-stimulated cAMP accumulation in D5-CT-truncated mutants at either a comparable affinity or slight rightward shift to wt D5 (Fig. 3D). Estimated IC₅₀ values of 15.0 ± 3.6 nM for D5-Asn³¹⁸, 8.0 ± 2.4 nM for D5-Val⁴¹⁸ were observed and were comparable to wt D5 receptor (6.7 ± 1.5 nM). As such, the lack of carboxyl tail sequences did not interfere with the functional coupling of D5-truncated mutants for agonists or antagonists. Taken together, these data suggest that the observed lack of [³H]SCH-23390 binding in COS-7 cells expressing D5-CT-truncated mutants is likely a result of poor receptor processing to the membrane surface. These truncated receptors also did not appear to display constitutive receptor activity as indexed by the greater than 75% loss of basal cAMP accumulation relative to wt D5 receptors and responsivity to the inverse agonists, butaclamol (Fig. 3C) or flupenthixol (data not shown). Full restoration of inherent wt D5 receptor ligand binding and constitutive activity profiles were evident with the D5 truncation mutant D5-Asp⁴⁴⁸. Thus, as illustrated in Fig. 3B, the affinity of dopamine and butaclamol for this mutant was essentially identical to wt D5 receptors. K_i values for additional compounds are listed in Table IV. Moreover, the D5 truncation mutant D5-Asp⁴⁴⁸ displayed basal cAMP levels, responsiveness to the inverse agonist butaclamol, and maximal dopamine-stimulated cAMP accumulation identical to wt D5 receptors (Fig. 3C). It is of interest to note, however, that the D5-Asp⁴⁴⁸ receptor mutant displayed some inverse agonist activity to SCH-23390 unlike wt D5 receptors and suggests that the CT tail and surrounding regions of D1 and D5 receptors, under certain conditions, influence the expression of inverse agonism (also see Refs. 10, 18, 27, 30). Moreover, the D5-Asp⁴⁴⁸ truncation mutant expressed an estimated EC₅₀ for dopamine-stimulated cAMP accumulation identical to that of wt D5 receptor (390 ± 87 versus 440 ± 98 nM) and distinct from that of wt D1 receptor and D5-Phe³⁶⁵ truncation mutants (1890 ± 512 versus 2100 ± 499 nM).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Dopamine D5 receptor pharmacological and functional characteristics following truncation of the CT-tail domain. A, schematic representation of the generated D5-CT truncation mutants (D5-Phe365, D5-Asn388, D5-Val418, D5-Glu429, D5-Phe438, D5-Asp448) and a synopsis of the data obtained with these mutant receptors relative to wt D5 following transient transfection in COS-7 cells as indexed by [3H]SCH-23390 binding activity (no specific binding detected); maximal dopamine (10 µM)-stimulated cAMP (+ indicates at least 70% of control wt values calculated following subtraction of basal activity); and agonist-independent cAMP accumulation (basal and that inhibited by butaclamol (also see C below). B, restoration of D5 receptor ligand binding characteristics by the D5 truncation mutant D5-Asp448. COS-7 cells expressing wt D5 and various CT tail truncation mutants were incubated with the indicated concentrations of dopamine or butaclamol and assayed for [3H]SCH-23390 (~350 pM) binding activity. Estimated Ki values for these and other compounds at the D5-Asp448 receptor are listed in Table IV. All other CT truncation mutants displayed extremely low levels of specific [3H]SCH-23390 binding. C, maximal dopamine-stimulated and constitutive whole cell cAMP production by various D5-CT truncation mutants. Basal and values for maximal cAMP accumulation with 10 µM dopamine are given as well as whole cell basal cAMP accumulation in the presence of 10 µM SCH-23390 or butaclamol. D5 receptor agonist-independent activity and response to inverse agonists is lost with the truncation of the D5 carboxyl tail (p < 0.01) while still responsive to maximal stimulation by dopamine to 77% of control (basal subtracted) with estimated EC50 values for dopamine listed in the text. Only the D5-Asp448 truncation mutant displays functional characteristics virtually identical to wt D5 receptors. Results shown are means ± S.E. of four to six independent experiments, each conducted in duplicate. D, dose-dependent inhibition of dopamine-stimulated whole cell cAMP accumulation by SCH-23390 in wt and D5-CT-truncated mutants, D5-Asn388 and D5-Val418. Maximal whole cell cAMP production was measured by pretreatment of cells expressing D5, and D5-CT mutants with increasing concentrations of SCH-23390 followed by the addition of 10 µM dopamine. Values are expressed as percentage of maximal response obtained by either wt or truncated mutants after subtracting basal activity as described above. Data shown are representative of three independent experiments, each conducted in duplicate with estimated IC50 values for SCH-23390 described under "Results and Discussion."

A further series of CT-tail truncation mutants of 10 amino acids each was generated to pinpoint those amino acid motifs within positions 418 and 448 that reconstituted D5 receptor characteristics. These truncated mutants, termed D5-Glu⁴²⁹ and D5-Phe⁴³⁸, similar to D5-Phe³⁶⁵, D5-Asn³⁸⁸, and D5-Val⁴¹⁸ did not display [³H]SCH-23390 ligand binding characteristics or constitutive activity but did exhibit dopamine-stimulated cAMP accumulation (data not shown). These data suggest that amino acids 438-448 are absolutely required to allow for the full expression of D5 receptor ligand binding profiles, which are pharmacological and functional attributes that distinguish it from wt members of the D1 receptor family, and that the last 30 amino acids of the D5 receptor do not partake in these events. These amino acids are thought to confer additional functional attributes to the D5 receptor, namely, the modulation of -aminobutyric acid (type A) receptor-mediated ligand-gated channel activity (25).

Alignment of the amino acid sequence of all available cloned and pharmacologically characterized members of the vertebrate D5/D1B receptor between D5-Phe⁴³⁸ and D5-Asp⁴⁴⁸ reveals significant sequence variability in this area. As depicted in Fig. 4A, only one amino acid (Gln) is absolutely conserved between all cloned members of this receptor subclass. To assess whether Gln⁴³⁹ is involved in either the expression and/or maintenance of inherent D5 receptor pharmacological and functional characteristics, we first constructed D5-CT-truncated tail mutants terminating at Gln⁴³⁹ or the following amino acid Ile⁴⁴⁰. D5-CT-tail truncation mutants terminating at Gln⁴³⁹ or I⁴⁴⁰ bound [³H]SCH-23390 with high affinity (~500 pM, see Table V) and displayed pharmacological profiles and estimated K_i values for dopamine and butaclamol (Fig. 4, B and C) as well as numerous other dopaminergic agonists and antagonists (listed in Table IV) indistinguishable from that observed for wt D5 receptors. Similarly, these truncation mutants increased cAMP production following stimulation with agonists such as dopamine to levels equivalent to wt D5 receptors. As depicted in Fig. 4D, enhanced constitutive basal cAMP accumulation rates and sensitivity to the inverse agonist butaclamol were observed, similar to that of wt D5 receptors despite the fact that Gln⁴³⁹/Ile⁴⁴⁰ truncation mutants displayed relative poorly expression rates or B_max values (Table V). These data suggest that the addition of just one amino acid to truncated D5-Phe⁴³⁸ receptor mutants (which display functional characteristics of D1-like receptors and no [³H]SCH-23390 ligand binding activity; see above) fully restores all the pharmacological and functional characteristics inherent in wt D5 receptors.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   A, alignment of deduced amino acid sequences of a portion of the CT tail of cloned and characterized members of the mammalian and vertebrate dopamine D5/D1B receptor gene family. Amino acid numbering refers to the human D5 receptor and reveals that only one amino acid within the region encoded by Phe338 and Asp448 is absolutely conserved (Gln439) in all members of the D5/D1B receptor family. B and C, pharmacological profiles of D5-CT receptor mutants. COS-7 cells were transiently transfected with wt D5, D5-CT truncation mutant or full-length receptors in which only Gln439 was deleted or substituted with the indicated concentrations of dopamine (B) or butaclamol (C). Estimated Ki values for these and other compounds at all the mutants tested are in Table IV. Estimated Bmax and Kd values for [3H]SCH-23390 binding to these and other generated D5-CT mutant receptors are listed in Table V. Data depicted are representative of three independent experiments, each conducted in duplicate. D, maximal dopamine-stimulated and constitutive whole cell cAMP production by various D5-CT mutants. Basal values for maximal cAMP accumulation with 10 µM dopamine are given as well as whole cell based cAMP accumulation in the presence of either 10 µM SCH-23390 or butaclamol as described above. No significant enhancement of either basal or agonist-stimulated cAMP accumulation was observed by any of the mutants listed despite mutation-induced increases in agonist ligand binding affinities.

To assess the relative contribution of Gln⁴³⁹ to the expressed pharmacological and functional profile of human D5 receptors, we constructed full-length D5 receptor mutants in which Gln⁴³⁹ was deleted or substituted with alanine or isoleucine. D5 receptor site-directed mutants in which either Gln⁴³⁹ was deleted or substituted by Ala or Ile bound [³H]SCH-23390 with high affinity and expressed estimated receptor densities not significantly different from wt D5 receptors (Table V). In contrast to expectation, these receptor mutants displayed agonist affinity values for discriminating agonists from 3- to 6-fold higher than that exhibited by wt receptors. Thus, as depicted in Fig. 4B, dopamine inhibited the binding of [³H]SCH-23390 to these mutants in a concentration-dependent and uniphasic manner with estimated K_i values of ~60 nM. Similar results were obtained for other D1 versus D5 discriminating agonists, including 6,7-ADTN and NPA (~3 fold) but not for benzazepine-like agonist compounds (see Table IV). Antagonist affinities, including that of butaclamol, remained unchanged (Fig. 4C). All estimated K_i values for these and other dopaminergic compounds at various single amino acid D5 receptor deletion/substitution mutants are listed in Table IV. These data suggest that the highly conserved D5/D1B receptor residue Gln⁴³⁹ exerts a tonic negative modulatory influence on the pharmacological signature of native D5 receptors, which without this braking influence can display "super" D5 characteristics with expressed affinities for discriminating agonists ~5 fold higher than wt D5 receptors. Again, as with the D1/D5_CT mutants described above, D1/D5_CT chimeras expressing the mutant Gln⁴³⁹ deletion or substitution sequence cannot confer super D5 receptor pharmacological characteristics to members of the D1 receptor family. As outlined in Table IV, the D1/D5_CTGln⁴³⁹-deleted or D1/D5_CTGln-Ala⁴³⁹ full-length mutant chimera retained a pharmacological profile consistent with that observed for the wt D1 receptor for both agonists and antagonists. The inability to reciprocally confer D5 or super D5 receptor characteristics to D1 receptors by D5-CT sequences suggests, as above, that additional D5 receptor domains indeed partake in the full expression of its pharmacological signature. The full complement of these expressions has yet to be elucidated.

In any event, to assess whether enhanced agonist affinity values for these mutant receptors are associated with corresponding augmentation of functional receptor characteristics, we analyzed the basal and dopamine-stimulated cAMP accumulation as well as the expression of inverse agonist activity exhibited by these receptors. As depicted in Fig. 4D, relative to wt D5 receptors (see Figs. 2A and 3C), there is no concomitant increase in the intrinsic activity of agonist-independent basal cAMP accumulation or responsivity to inverse agonists. Likewise, D1/D5_CTGln⁴³⁹-deleted or D1/D5_CTGln-Ala⁴³⁹ full-length mutant chimeric receptors displayed functional characteristics not significantly different from that of wt D1 receptors (data not shown). These data clearly suggest that, in addition to well characterized receptor and G-protein binding domains, the pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that single amino acid residues within this region can independently modulate D5 receptor constitutive activity from its expressed agonist high affinity pharmacological profile.

To date, although many studies have addressed the roles played by various amino acids and sequence-specific domains within the D1 receptor that relate to both its expressed pharmacology and overall function (16), few have examined the issue of which molecular determinants govern and allow for the discrimination of human D5 from D1 receptors. In summary, we conclude: 1) that a region exists within the CT tail of the dopamine D5 receptor that allows for the full expression of two of the major distinguishing pharmacological and functional features inherent of this subclass, namely, higher agonist affinities and constitutive activity relative to the D1 receptor family; 2) that the last 40 amino acids of the D5 receptor are not necessary for its observed pharmacological and functional signature or expression; 3) a highly conserved amino acid within the D5/D1B receptor subclass can further enhance and modulate agonist ligand binding affinities but independently maintain normative constitutive activity profiles; 4) that sequences within the CT tail confer the receptors ability to bind [³H]SCH-23390 with high affinity, yet conserve its ancient functional status as a dopamine-stimulating adenylate cyclase receptor (31); and 5) that these sequence motifs alone cannot fully impart to other members of the D1 receptor subfamily characteristics attributable to the D1B/D5 receptor.

Clues to the possible functional differentiation of D1- and D5-like receptors have only recently begun to be unraveled. These have been based primarily on data obtained from targeted gene mutations of D1 or D5 receptors (see Refs. 32-34), the evolutionary history of these genes (31), and the observed acquisition of neural expression territories and cellular and subcellular distribution profiles (35-37) of these receptors in specific neural populations. The data presented here suggest that, despite the observed functional redundancy of multiple members of the D1-like receptor gene family, which include also the D1C and D1D receptor family in vertebrates, the highly variable amino acid sequence motifs encoded within the CT tails of these receptor subtypes can impart highly selective and exquisite subtype-specific receptor functions and suggest that members of the D1-like gene family are not fully capable of reconstituting each others function. Given the existence of D5 receptor point mutations (38), some of which may influence agonist high affinity binding reactions (39) and be associated with specific behavioral manifestations of neuropsychiatric disease states (40), further work on the role these CT sequences may play in the expression of subtype-specific dopamine D1 versus D5 receptor-mediated events in both health and disease appears warranted.

	FOOTNOTES

^* This work was supported in part by grants from National Institute on Drug Abuse, the Medical Research Council of Canada (MT-15581), and the Ontario Mental Health Foundation (to H. B. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by an Ontario Mental Health Foundation Fellowship. To whom correspondence should be addressed: Dept. of Discovery Biology and Pharmacology, NPS Allelix Corp., 6850 Goreway Dr., Mississauga, Ontario, Canada L4V 1V7. Tel.: 905-677-0831; Fax: 905-677-9595; E-mail: l.demchyshyn@allelix.com.

Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M000157200

	ABBREVIATIONS

The abbreviations used are: TM, transmembrane; 6, 7-ADTN, (±)-6,7-dihydroxy-2-aminotetralin; CT, carboxyl-terminal; CY208-243, ()-4,6,6,7,8,12-hexahydro-7-methylindolo[4,3-ab]phenanthridine; GPCR, G-protein coupled receptor; NPA, N-propylnorapomorphine; SCH-23390, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SCH-39166, ()-trans-6,7,7,8,9,13-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo[d-2,1]napthobenzazepine; SKF-38393, 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; SKF-82526, 6-chloro-7,8-dihydroxy-1-(p-hydroxyphenyl)-2,3,4,5-tetrahydro-(1H)-3-benzazepine; wt, wild-type; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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