Introduction

Summary Introduction Procedures Results Discussion References

Brain dopamine receptors that couple to stimulation of adenylyl cyclase have been classified as members of the D₁ dopamine receptor family, which includes the cloned D_1A and D_1B dopamine receptor subtypes (1, 2). Diverse neurochemical, electrophysiological, and behavioral observations have, however, suggested that other transduction systems for dopamine D₁ receptors exist in both the central and peripheral nervous systems (3-8). In a series of investigations, we demonstrated a D₁ dopaminergically mediated stimulation of IP formation in rat brain regions that does not parallel the distribution of the dopamine D₁/cyclase receptor activity (9, 10). Furthermore, the mRNA coding for the phosphatidylinositol-linked receptor site was found to differ markedly in size from that for the classic D_1A dopamine receptor (11). Also, the stimulation of phosphoinositide metabolism by the D₁-like dopamine receptor seems to be distinct from the classic D₁ receptor that is coupled to stimulation of adenylyl cyclase in terms of both receptor and the transducing G protein (12). Although coupling of striatal D₁-like dopamine receptors to IP formation was demonstrated to be mediated by G_q, the coupling of the D_1A receptor to cAMP formation was shown to occur via G_s (12). In the current study, we sought further evidence to test whether the two actions of dopamine are transduced by distinct molecular entities. The experiments were performed in tissues derived from homozygous D_1A-deficient mutant mice, which were produced by homologous recombination (13).

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Animals. Homologous recombination was used to generate mutant mice lacking functional D_1A dopamine receptors, as previously described (13). Homozygous mice matched for sex (seven females and one male) and age (9.0 ± 0.9 months) with wild-type animals (age, 9.3 ± 0.8 months) were singly housed with free access to food and water under standard conditions of humidity (60%), room temperature (22°), and 12-hr light/dark cycle for 5 days after arrival at the animal facility and before the experiments.

IP formation in cerebral cortex slices. The experimental procedures have been previously described in detail (9). Briefly, the cerebral cortices were chopped into 350 × 350-µm slices. The resulting slices were weighed and transferred into a 25-ml screw-capped polypropylene tube containing HEPES bicarbonate buffer at 35°, which was composed of 122 mM NaCl, 1.2 mM MgCl₂, 4.9 mM KCl, 1.2 mM KH₂PO₄, 3.6 mM NaHCO₃, 30 mM HEPES, and 10 mM glucose and bubbled with 95% oxygen/5% carbon dioxide, pH 7.4. The slices were washed twice, resuspended in 5 ml of buffer, and incubated at 37° for 30 min. Then, the slices were resuspended in fresh buffer containing 1.3 mM CaCl₂ and labeled with 10 µl of 66.67 µM 2-[³H] inositol/ml (15 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO) at 37° for 60 min before being washed twice with 2 volumes of fresh buffer. The slices were finally suspended in fresh calcium containing buffer (3 ml/80 mg of fresh tissue).

Adenylyl cyclase assay. Striatum and frontal cortex were homogenized using a Teflon/glass homogenizer in 10 volumes (w/v) of prechilled buffer containing 10 mM imidazole, 2 mM EGTA, and 10% sucrose, pH 7.3. The homogenate was centrifuged at 1,000 × g for 10 min, and the supernatant was centrifuged at 27,000 × g for 20 min. The pellet was washed twice with 10 mM cold imidazole and suspended in 10 mM imidazole buffer, pH 7.3. Membrane protein was determined according to the method of Bradford (14). The adenylyl cyclase assay was performed by a modification of the method described by Salomon (15). The reaction mixture included 0.5 mM MgCl₂, 0.5 mM 3-isobutyl-1-methylxanthine, 0.2 mM EGTA, 0.5 mM dithiothreitol, 10 µM pargyline, 1 µM GTP, 0.1 mM ATP, 2 mM phosphocreatine, 5 units of creatine phosphokinase, and 1 µCi of [-³²P]ATP (~2.2 × 10⁶ cpm) in 10 mM imidazole buffer, pH 7.3, with or without dopamine or SKF38393. After preincubation at 30° for 5 min, the reaction was started by the addition of 50 µg of membrane protein. The reaction was terminated 10 min later by the addition of 300 µl of a solution containing 2% SDS, 25 mM ATP, and 1.3 mM cAMP. Formed [³²P]cAMP was separated by Dowex and alumina columns. [³H]cAMP was included in each reaction for estimation of column recovery (typically ~70-80%).

Coprecipitation of [³H]SCH23390-bound receptor with discrete G proteins. Determination of the linkage between receptor and G proteins was carried out as previously described (12). Crude striatal membranes were prepared by homogenizing brain striata in 10 volumes of 25 mM HEPES, pH 7.5, buffer containing 2 mM MgCl₂, 1 mM EDTA, 0.2% 2-mercaptoethanol, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 0.01 unit/ml soybean trypsin inhibitor, and 0.04 mM phenylmethylsulfonyl fluoride with the use of a glass/glass homogenizer. The homogenate was centrifuged at 750 × g for 5 min, and the supernatant was centrifuged for 10 min at 48,200 × g. Membranes were washed and resuspended in 100 mM Tris·HCl immunoprecipitation buffer, pH 7.5, containing 200 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, 0.2% 2-mercaptoethanol, 50 µg/ml leupeptin, 25 µg/ml pepstatin A, 0.01 unit/ml soybean trypsin inhibitor, and 0.04 mM phenylmethylsulfonyl fluoride. The concentration of membrane proteins was determined (16), and 200 µg of membrane proteins was solubilized in 1 ml of immunoprecipitation buffer with 0.2% cholate and 0.5% digitonin. Solubilized tissues were precleared by incubation with normal rabbit serum (1:100 dilution) at 4° for 60 min followed by an additional 30 min with 100 µl of a 10% suspension of protein A-bearing Staphylococcus aureus cells (Pansorbin cells, Calbiochem, San Diego, CA). The suspension was centrifuged at 4°, and the supernatant was combined with antisera (1:1000 dilution) raised against specific peptides of G proteins (New England Nuclear Research Products, Boston, MA) for 3 hr at 4° followed by an additional 30-min incubation with 100 µl of Pansorbin. The specificity of antisera used was previously defined (17). The mixture was centrifuged and washed, and the pellet was suspended and incubated for 30 min at 30° in 500 µl of 50 mM Tris·HCl binding buffer, pH 7.5, which included 5 mM MgCl₂, 1 µM mesulergine, and 1 nM [³H]SCH23390. Nonspecific binding was defined by the addition of 1 µM cis-(Z)-flupenthixol. The reaction was terminated by the addition of 9 ml of ice-cold buffer and immediately vacuum filtered over Whatman GF/F filters. The amount of radioactivity on the filter was assessed by liquid scintillation counting, and specific [³H]SCH23390 binding was determined.

Immunoblot analysis. Twenty-five micrograms of membrane proteins was solubilized in sample preparation buffer, and proteins were separated by SDS-polyacrylamide gel electrophoresis (12%) according to the method of Laemmli (18). Proteins were transferred electrophoretically to a nitrocellulose membrane. The completeness of transfer was checked by Coomassie blue staining of the gel. The membranes were incubated at 4° overnight with 10% nonfat dry milk in 0.1% TBS to block nonspecific sites, washed with 0.1% TBS, and incubated for 2 hr with antisera directed against G_s, G_i1/2, G_o, or G_q (New England Nuclear Research Products) at 1:2,000 dilution or with affinity-purified G protein antibody at 0.25 µg/ml (Santa Cruz Biochemicals, Santa Cruz, CA) in 0.1% TBS. The unbound antibody was washed out with 0.1% TBS. After a 60-min incubation with horseradish perioxidase-conjugated anti-rabbit IgG (Amersham, Arlington Heights, IL) (1:10,000 in 1% TBS), the blots were washed with 3% TBS for 20 min followed by four 5-min washes. The immunoreactive proteins were detected with the enhanced chemiluminescence Western blot detection system (Amersham/Searle, Des Plaines, IL) and visualized by a 2-min exposure to film.

Materials. For these experiments, dopamine HCl, pargyline HCl, soybean trypsin inhibitor, and the buffer reagents were purchased from Sigma Chemical (St. Louis, MO). The chemicals used for IP isolation and determination were purchased from Fisher Scientific (Pittsburgh, PA). Mesulergine HCl [N-[(8)-1,6-dimethylergolin-8-yl]N,N-dimethylsulfamide HCl], S-()-sulpiride [()5-aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]2-methoxybenzamide], cis-(Z)-flupenthixol dihydrochloride [(Z)-4-[3-[2-(trifluormethyl)-9H-thioxanthen-9-ylidene]propyl]-1-piperazine-ethanol dihydrochloride], and SKF38393 HCl [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride] were purchased from Research Biochemicals (Natick, MA). Normal rabbit serum and Pansorbin were purchased from Calbiochem. Prazosin HCl and SCH23390 hemimaleate (8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin hemimaleate) were generously supplied by Pfizer (New York, NY) and Schering (Bloomfield, NJ), respectively. SCH23390 [N-methyl-³H](71.3 Ci/mmol) and antisera for G_s (RM/1), G_i(1, 2) (AS/7), G_o (GC/2), and G_q (QL) were purchased from DuPont-New England Nuclear (Boston, MA).

	Results

Summary Introduction Procedures Results Discussion References

Dopamine- or SKF38393-activated cAMP production in striatal and cortical membranes is absent in D_1A-deficient mice. Incubation of striatal and frontal cerebrocortical membranes obtained from wild-type mice with dopamine or with the D₁-selective agonist SKF38393 resulted in concentration-dependent elevations in cAMP production. The maximal responses for both dopamine and SKF38393 were achieved at 100 µM in both brain areas. The results summarized in Fig. 1 indicate that adenylyl cyclase activity in response to dopamine or SKF38393 was completely absent in both brain regions of D_1A-deficient mice. In contrast, direct enzyme stimulation with forskolin was unchanged in brain membranes obtained from D_1A mutant mice (Table 1), suggesting that the mutation does not affect the activity of adenylyl cyclase per se.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Dopamine- and SKF38393-activated cAMP accumulations in striatal (ST) and cortical (CX) membranes of D1A knockout mice. Membrane protein (50 µg) was exposed to either 100 µM dopamine or SKF38393 in 10 mM imidazole buffer, pH 7.3, containing 1 µCi of [-32P]ATP (2.2 × 106 cpm), 0.5 mM MgCl2, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM GTP, and 0.1 mM ATP at 30° for 10 min. Values represent the rate of accumulated cAMP in stimulated tissue above basal activity. Data are mean ± standard error from seven animals.

Dopamine- or SKF38393-activated IP formation is not altered in cortical slices of D_1A gene-deficient mice. Incubation with the D₁ dopamine receptor agonist SKF38393 of frontal cerebrocortical slices obtained from control mice increased the formation of IPs. This dopaminergic effect was inhibited by the D₁-selective antagonist SCH23390 but not by the ₁-adrenergic antagonist prazosin or by the 5-hydroxytryptamine_2C/A serotonin receptor antagonist mesulergine (Fig. 2). In contrast to the absence of D₁ receptor-mediated cAMP responses in D_1A-deficient mice, the concentration-response curves for dopamine-induced (Fig. 3A) or SKF38393-induced (Fig. 3B) elevations in IP were identical in D_1A-deficient and wild-type mice, suggesting that D₁ dopaminergic stimulations of cAMP and IP formations are mediated by structurally distinct dopamine receptors.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   The effect of receptor antagonists on SKF38393-stimulated IP accumulation. Cortical slices were prelabeled with 3H-inositol and incubated with 10 mM LiCl in the presence of buffer alone or with 50 µM SCH23390, mesulergine, or prazosin for 10 min before the addition of 250 µM SKF38393. The reaction proceeded for 60 min, and accumulated IPs were determined. SCH23390 significantly inhibited the SKF38393-induced IP accumulation (p < 0.01; four animals).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Dopamine- and SKF38393-activated IP formation in cortical slices of D1A knockout mice. Drug-induced IP accumulation was calculated as net change from basal accumulation. Basal accumulation across all groups was 7.21 ± 0.39 dpm/µg of protein (28 animals). Points, mean ± standard error of seven experiments performed in triplicate. The accumulation of IP in responses to increasing concentrations of (A) dopamine or (B) SKF38393 were not different in D1A knockout than in wild-type mice.

Coprecipitation of D₁ dopamine receptors with G_q and G_s in striatal and cortical membranes. The results summarized in Fig. 4 demonstrate that G_s and G_q antisera coimmunoprecipitated specific D₁ dopamine receptor binding sites labeled by the selective D₁ receptor ligand [³H]SCH23390 in striatal or frontal cortex membranes of wild-type mice; antisera recognizing G_i and G_o proteins or normal rabbit serum did not immunoprecipitate [³H]SCH23390 binding sites. Fig. 4 also illustrates that coupling of D₁ dopamine receptors to G_s is reduced by 75-82%, whereas the association of [³H]SCH23390 binding sites with G_q were unaltered in tissues from D_1A mutant mice. The reduction in coupling of specific [³H]SCH23390 binding sites to G_s in brains of D_1A receptor-deficient mice does not result from reduced G_s because similar levels of G_s were found in membranes of wild-type and D_1A-deficient mice (Fig. 5). The results demonstrate that G_s-coupled D₁ dopamine sites are selectively reduced in D_1A receptor-deficient mice.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Copurification of [3H]SCH23390 binding sites with Gs or Gq in D1A-deficient mutant mice tissue. Frontal cortical or striatal membranes obtained from D1A knockout or wild-type mice were solubilized and subjected to immunoprecipitation with the indicated anti-G antisera. The immunocomplexes were incubated with 1 nM [3H]SCH23390 and 1 µM mesulergine with or without 1 µM cis-(Z)-flupenthixol for 30 min at 30°. Bound [3H]SCH23390 was assessed by counting the radioactivity collected on GF/F filters. Bar, mean ± standard error obtained from seven determinations, each performed in duplicate. Significant reduction in specific [3H]SCH23390 binding was observed to be associated only with Gs in the membranes of D1A knockout mice (p < 0.05), whereas [3H]SCH23390 binding sites coupled with Gq remained unchanged.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Immunoblots of Gs, Gi1/2, Go, Gq, and G in frontal cortical and striatal membranes of wild-type (W) and D1A knockout (K) mice. Twenty-five micrograms of membrane proteins was separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. G proteins and G were immunoblotted with antibodies specific for the G protein subunits. The immunocomplexes were detected using anti-rabbit IgG and enhanced chemiluminescence. Densitometry showed no significant changes in the level of G protein subunits in brain membranes of D1A knockout mice.

	Discussion

Summary Introduction Procedures Results Discussion References

The current findings clearly demonstrate that the dopamine receptor that stimulates the formation of IPs is completely independent of the D_1A dopamine receptor system, which is known to couple to adenylyl cyclase. In addition, the data confirm our previous conclusion that the D_1A dopamine receptors couple to adenylyl cyclase via G_s protein, whereas G_q protein links D₁-like dopamine receptors to the activation of phosphoinositide hydrolysis.

The results of pharmacological and neurochemical investigations have previously suggested that the D₁ dopamine receptors that are coupled to phospholipase C and adenylyl cyclase are distinct receptors that are linked to their respective effector systems via different coupling proteins. Evidence demonstrating size differences for mRNAs coding for the two receptors first suggested that the D₁ dopamine receptor sites that couple to phospholipase C and adenylyl cyclase may be distinguishable molecular moieties (11). Differential order of potencies and efficacies for a series of benzazepine derivatives in activating striatal phosphoinositide hydrolysis and adenylyl cyclase (10) and the unique regional distributions of the two D₁ dopaminergic transduction systems in the rat brain (9) further support this possibility. D₁ dopamine receptors, which activate cyclase and phospholipase C, were also shown to couple to their respective effectors via G_s and G_q (12). Both of these G proteins were in turn found to interact with [³H]SCH23390 binding sites. However, the sites that were coupled to G_s were identified as being the D_1A receptors, whereas those that were linked to G_q were not recognized by the same selective monoclonal antibody that recognizes D_1A receptors (12). The G_q/phosphatidylinositol-linked dopaminergic receptor site therefore seems to be a subtype of the D₁ dopamine receptor family.

The current data demonstrating that the D_1A-deficient mutant mice are dramatically impaired in dopamine-stimulated adenylyl cyclase without a parallel loss in dopamine-stimulated phosphoinositide metabolism directly support the conclusion that the two D₁ dopaminergic signal transduction systems are independently activated by two dopamine receptors. The discrepancy between the total absence of dopamine-mediated cyclase activation and a residual coupling of [³H]SCH23390 binding sites to G_s is probably a function of the greater sensitivity of the binding experiment in comparison to the measurement of dopamine-stimulated adenylyl cyclase. Alternatively, the residual G_s/[³H]SCH23390 coupling found in mutant mice may reflect the coupling of G_s to other members of the D₁ dopamine receptor family that are not linked to adenylyl cyclase but activate other effector systems (7, 8).

The findings presented here lend support to the suggested molecular heterogeneity of the signaling pathways for the D₁ dopamine receptors. The results indicate that in addition to the classic dopamine D_1A receptor/G_s/adenylyl cyclase cascade, an unidentified dopamine D₁ receptor also couples to G_q protein and that this interaction may in turn modulate dopamine-stimulated phosphoinositide hydrolysis.