Introduction

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Thus far, five different genes encoding distinct dopamine receptor subtypes have been cloned and characterized (Neve and Neve, 1997). Using pharmacological and structural criteria, the protein products of these genes can be divided into two major subfamilies referred to as D₁-like and D₂-like receptors. The D₁-like receptor subfamily consists of two members, the D₁ and D₅ subtypes, also referred to as the D_1A and D_1B receptors. In contrast, the D₂ subfamily consists of three subtypes, the D₂, D₃, and D₄ receptors. In addition to their differences in structure and pharmacology, the D₁-like and D₂-like subfamilies differ in their G protein-coupling and signal transduction pathways (Huff, 1997; Robinson and Caron, 1997). The D₁-like receptors couple to activation of adenylyl cyclase activity and increased levels of the second-messenger cAMP, whereas the D₂-like receptors exhibit coupling to G_i/o-like proteins, resulting in modulation of K⁺ and/or Ca²⁺ channels and depression of adenylyl cyclase activity. As with other G protein-coupled receptors (GPCRs), dopamine receptors are subject to a variety of regulatory mechanisms that can either positively or negatively modulate their expression and functional activity (Sibley and Neve, 1997).

One of the most important forms of regulatory mechanisms that modulate signaling by GPCRs is that of agonist-induced desensitization. Defined as the tendency of receptor mediated responses to wane over time despite continued agonist stimulation, GPCR desensitization has been extensively investigated using adrenergic receptor systems (Hausdorff et al., 1989; Krupnick and Benovic, 1998; Lefkowitz, 1998). One important mechanism that has been established for desensitizing adrenergic receptors is their phosphorylation by GPCR kinases (GRKs), which phosphorylate only the agonist occupied or activated form of the receptor and are critical for homologous or agonist-specific forms of desensitization (Krupnick and Benovic, 1998; Lefkowitz, 1998). In addition, there are second-messenger-activated protein kinases, such as the cAMP-dependent protein kinase (PKA), that can phosphorylate adrenergic receptors in a largely agonist-independent fashion (Hausdorff et al., 1989). Originally thought to be important in only heterologous or nonspecific forms of receptor desensitization, recent data have suggested that second-messenger-activated protein kinases, such as PKA, may play important roles in homologous or agonist-specific forms of receptor desensitization (Chuang et al., 1996; Moffett et al., 1996; Post et al., 1996).

Recent studies have indicated that similar, but not completely identical, pathways may be operative in agonist-induced regulation of dopamine receptors, with great variability being observed among the subtypes. For instance, agonist-induced desensitization is not always observed with the D₂ dopamine receptor, and in some instances, agonist occupancy of this subtype results in increased receptor expression (reviewed in Sibley and Neve, 1997). In contrast, the D₁ receptor has been shown to exhibit agonist-induced refractoriness in both endogenous and recombinant/heterologous cellular expression systems (reviewed in Sibley and Neve, 1997). Recent data have provided support for a GRK-mediated phosphorylation pathway underlying agonist-induced desensitization of the D₁ receptor. Studies involving the expression of D₁ receptors in Sf9 (Ng et al., 1994) or human embryonic kidney 293 cells (Tiberi et al., 1996) have shown that the D₁ receptor undergoes agonist-induced phosphorylation and that in the human embryonic kidney 293 cells, this phosphorylation is enhanced by coexpression of GRK isoforms 2, 3, and 5. In contrast, the role of PKA-mediated phosphorylation events in agonist-induced D₁ receptor desensitization is less clear. Some studies have shown that intracellular activation of PKA can partially mimic agonist-induced desensitization of D₁ receptors, thereby suggesting a role for this kinase in regulating D₁ receptor function (Bates et al., 1991; Black et al., 1994). In addition, Zhou et al. (1991) found that intracellular inhibitors of both PKA and GRKs could attenuate D₁ receptor desensitization, thus implying a role for both GRK and PKA kinase systems. In contrast, Bates et al. (1993) and Lewis et al. (1998) provided data arguing that PKA is not important for agonist-induced D₁ receptor desensitization. To investigate this further, we thought that it would be informative to express the D₁ receptor in cells that possess defective PKA isozymes and exhibit little to no cellular PKA activity to see what effect, if any, this would have on D₁ receptor regulation. We find that in such PKA-deficient cells, agonist-induced desensitization and down-regulation of receptor binding activity are significantly, but not completely, attenuated. These results strongly imply a role for PKA-mediated phosphorylation in agonist-induced regulation of the D₁ dopamine receptor.

	Experimental Procedures

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Materials. Parental (10001) and PKA mutant (10260 and 10248) Chinese hamster ovary (CHO) cells were a gift from Dr. M. Gottesman (National Institutes of Health). [³H]SCH-23390 [(R)-(+)-6-chloro-7,8-dihydroxy-3-allyl-1-phenly-2,3,4,5-tetrahydro-1H-3-benzazepine; 70-71.3 Ci/mmol] and [³H]cAMP (31.4 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston, MA). Dopamine, forskolin, Ro 20-17244 ([(butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone), and (+)-butaclamol were purchased from Research Biochemicals Inc. (Natick, MA). cAMP assay kits were obtained from Diagnostic Products Corp. (Los Angeles, CA). Cell culture media and reagents were purchased from Life Technologies (Grand Island, NY). FCS was purchased from Summit Biotechnology (Purchase, CO). Calcium phosphate transfection kits were obtained from InVitrogen (San Diego, CA). Phosphoenolpyruvate, GTP, ATP, myokinase, and pyruvate kinase were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest quality available and were obtained from commercial suppliers.

Cell Cultures and Transfections. CHO cells were cultured in F12 nutrient media (Life Technologies) supplemented with 1 mM pyruvate and containing 10% FCS and 50 µg/ml penicillin, streptomycin, and gentamycin. The full-length rat D₁ receptor cDNA (Monsma et al., 1990a) was subcloned into the NotI site of the mammalian expression vector pCD-SR (Takebe et al., 1988), and the complete D₁ receptor sequence was confirmed by DNA sequencing. The pCD-SR plasmid (30 µg of DNA) was then cotransfected with the pMAM-neo plasmid (3 µg of DNA) into CHO cells according to the calcium phosphate precipitation method (calcium phosphate transfection kit; InVitrogen). In brief, cells were seeded onto 150-mm² plates, and transfection was carried out after 30 to 40% confluency was achieved. DNA and 60 µl of 2 M CaCl₂ were mixed in H₂O in a total volume of 500 µl, which was then slowly mixed with 500 µl of HEPES-buffered saline. The reaction mixture was incubated at room temperature for 30 min and then evenly added to the cell culture dish containing 15 ml of fresh media. After overnight incubation at 37°C, the transfection medium was replaced by 25 ml of standard medium. The cultures were split after an additional 2 to 3 days, and G418 (500 µg/ml) was added to the medium. G418-resistant clones were selected after 2 weeks, expanded, and further screened and characterized by a radioligand binding assay.

Radioligand Binding Assay. Cells were harvested by incubation with 5 mM EDTA in Earle's balanced salt solution (EBSS) and collected through centrifugation at 300g for 10 min. The cells were resuspended in lysis buffer (5 mM Tris, pH 7.4 at 4°C, 5 mM MgCl₂) and were disrupted using a Dounce homogenizer followed by centrifugation at 34,000g for 10 min. The resulting membrane pellet was resuspended in binding buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl₂, 4 mM MgCl₂, and 120 mM NaCl). The membrane suspension (final protein concentration, 50 µg/tube) was then added to assay tubes containing 0.015 to 2 nM [³H]SCH-23390 in a final volume of 0.5 ml. (+)-Butaclamol was added at the final concentration of 1 µM to determine nonspecific binding. The assay tubes were incubated at room temperature for 1 h, and the reaction was terminated by rapid filtration through GF/C filters pretreated with 0.3% polyethyleneimine. Radioactivity bound to the filters was quantified by liquid scintillation spectroscopy at a counting efficiency of 47%.

Determination of cAMP Production. The cAMP formation was determined using either intact cell or membrane homogenate assays as indicated in Results. For intact cell assays, CHO cells were harvested, washed three times in EBSS, and resuspended in AC buffer (250 mM sucrose, 75 mM Tris-HCl, pH 7.4 at 37°C, 12.5 mM MgCl₂, 1.5 mM EDTA, 1 mM dithiothreitol, 200 µM sodium metabisulfite, and 100 µM Ro 20-1724, a phosphodiesterase inhibitor). Cell suspensions (50 µl) containing 80,000 cells were added to a 10 µl solution of dopamine (0-100 µM final concentration). cAMP generation was allowed to proceed for 5 min at 37°C, and the reaction was terminated at 100°C for 3 min. The cAMP generated was quantified with a competitive binding assay previously described (Monsma et al., 1990b) except that PKA isolated from bovine heart (Sigma Chemical Co.) was used in lieu of adrenal cAMP binding protein. The cAMP concentrations produced in this assay were determined by comparison to a standard curve that was linear in the range of 0.5 to 25 pmol cAMP/assay tube. Basal cAMP levels were 0.94 ± 0.43 pmol/80,000 cells (n = 3, line 10001), 0.95 ± 0.03 pmol/80,000 cells (n = 3, line 10260), and 0.67 ± 0.52 pmol/80,000 cells (n = 3, line 10248). For some experiments, cAMP production was assessed using a broken cell/membrane assay as follows. CHO cells were harvested and membranes were prepared as described for the radioligand binding assays above. Final resuspension of the membranes was performed in AC buffer supplemented with 2.75 mM phosphoenolpyruvate, 53 µM GTP, 0.12 mM ATP, 1.0 U of myokinase, and 0.2 U of pyruvate kinase. cAMP generation and quantification were subsequently accomplished as described for intact cells. In all experiments, protein content was determined with the bicinchoninic acid protein assay (Pierce, Rockville, MD), using BSA (Sigma Chemical Co.) as the standard.

Data Analysis. All radioligand binding assays were routinely performed in triplicate and repeated three or four times. cAMP experiments were performed in duplicate (intact cells) or triplicate (membrane homogenates) and repeated three or four times. Estimation of the radioligand binding parameters K_D and B_max, as well as the EC₅₀ values for dopamine, were obtained using the software program Prism (GraphPad Software, San Diego, CA).

	Results

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Expression of D₁ Receptor in Wild-Type and Mutant CHO Cell Lines. To investigate the role of PKA-mediated phosphorylation events in D₁ receptor regulation, we transfected the cloned rat D₁ receptor cDNA into two mutant CHO cell lines that have been well characterized as exhibiting defective PKA activities. The first of these is mutant cell line 10248, which has been shown to lack type II PKA activity and whose type I PKA regulatory subunit exhibits a greatly diminished affinity for cAMP (Singh et al., 1985). The second is cell line 10260, which exhibits a >95% reduction in both type I and II PKA activities (Singh et al., 1981). In addition, as a control, we transfected the parental (wild-type) CHO cell line (10001), which possesses normal levels of type I and II PKA activities (Singh et al., 1981, 1985). Stably transfected cell lines were selected for all CHO variants and were initially characterized using radioligand binding and cAMP accumulation assays (Fig. 1). Saturation radioligand binding analysis (Fig. 1A) using the D₁-selective antagonist [³H]SCH-23390 revealed the following receptor densities (B_max values) in membranes prepared from three stably transfected CHO cell lines: wild-type (10001), 0.75 ± 0.079 pmol/mg (n = 17); 10260, 0.95 ± 0.16 pmol/mg (n = 5); and 10248, 1.6 ± 0.44 pmol/mg (n = 7). The affinity (K_D) for [³H]SCH-23390 binding to the D₁ receptor was almost identical in the three cell lines: wild-type (10001), 0.21 ± 0.01 nM (n = 17); 10260, 0.18 ± 0.02 nM (n = 5); and 10248, 0.19 ± 0.013 nM (n = 7). These results indicate that all three cell lines are capable of expressing the D₁ receptor to a similar extent and that PKA activity is not required for D₁ receptor expression in CHO cells.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Expression of the D1 dopamine receptor in wild-type and mutant CHO cell lines. A, saturation binding isotherms for [3H]SCH-23390 were generated using membranes prepared from wild-type (), 10260 (), and 10248 () cell lines. The radioligand binding assays were performed as described in Experimental Procedures with the saturation isotherm data being plotted in Scatchard coordinates of bound/free versus free radioligand. In this representative experiment, the KD values for [3H]SCH-23390 were identical among the three cell lines at 0.16 nM, whereas the Bmax values were 0.9, 1.1, and 1.4 pmol/mg for the wild-type, 10260, and 10248 cell lines, respectively. Average binding parameters from multiple experiments are provided in the text. B, dose-response curves for dopamine-stimulated cAMP accumulation in wild-type (), 10260 (), and 10248 () CHO cell lines. Cells were washed with EBSS and incubated with increasing concentration of dopamine in the presence of 0.1 mM Ro-201724 and 0.2 mM sodium metabisulfite. The cAMP was determined as described in Experimental Procedures. In this representative experiment, the dopamine EC50 values were 0.3, 0.15, and 0.11 µM in the wild-type, 10260, and 10248 cell lines, respectively. Average data from multiple experiments are given in the text.

Agonist-Induced Regulation of D₁ Receptor in Wild-Type and Mutant CHO Cell Lines. As an initial approach to examining the agonist-induced desensitization (defined as a diminution of the cAMP response) of the D₁ receptor in the wild-type and mutant cell lines, we pretreated the cultures with dopamine for 24 h (a maximally effective time for desensitization; see later) and determined the dopamine-dependent cAMP accumulation subsequent to this treatment. Preincubation of the cells with dopamine resulted in a near-total loss of the dopamine-stimulated cAMP response in the wild-type (10001) cell line (Fig. 2A). In the mutant cell lines, dopamine pretreatment also resulted in desensitization of the D₁ receptor response; however, the loss of activity was not as profound as that observed with the wild-type cells (Fig. 2, B and C). In the 10260 cell line, the maximal stimulation of cAMP accumulation induced by dopamine was decreased by ~80% relative to the control response, whereas in the 10248 cells, this response was diminished by only 60%. In both mutant cell lines, the EC₅₀ value for dopamine stimulation of cAMP accumulation was shifted by ~10-fold (to lower potency) subsequent to the dopamine pretreatment. Thus, although both mutant cell lines support agonist-induced desensitization of the D₁ receptor, this regulatory response is attenuated compared with that observed with the wild-type CHO cells.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Agonist-induced desensitization of the D1 dopamine receptor in wild-type and mutant CHO cell lines. Cells were cultured in the presence of 0.2 mM sodium metabisulfite and in the absence (control, ) or presence (treated, ) of 100 µM dopamine for 24 h. The cells were then extensively washed with EBSS and further incubated with the indicated concentrations of dopamine in the presence of 0.2 mM sodium metabisulfite for 5 min at 37°C. cAMP assays were performed as described in Experimental Procedures. The EC50 values for dopamine stimulation of cAMP accumulation determined in these experiments were as follows: a, 10001 cells, control EC50 = 0.097 µM; treated EC50 = not determinable. b, 10260 cells, control EC50 = 0.083 µM; treated EC50 = 0.78 µM. c, 10248 cells, control EC50 = 0.038 µM; treated EC50 = 0.37 µM. Data are presented as mean ± S.E. values of triplicate determinations from one representative experiment that was repeated three times with similar results.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Dopamine-induced down-regulation of D1 receptors in CHO cell lines. Cells were treated with 100 µM dopamine for 24 h as described in Fig. 2 before harvesting and preparation of membranes. The radioligand binding assays were performed as described in Experimental Procedures with the saturation isotherm data being plotted in Scatchard coordinates of bound/free versus free radioligand. Indicated are data from control cultures () and the dopamine-treated cultures (). A, [3H]SCH-23390 binding parameters in the wild-type (10001) cells are as follows: control KD = 0.22 nM, Bmax = 0.9 pmol/mg; dopamine-treated KD = 0.16 nM, Bmax = 0.041 pmol/mg. B, [3H]SCH-23390 binding parameters in the 10260 cells are as follows: control KD = 0.21 nM, Bmax = 0.51 pmol/mg; dopamine-treated KD = 0.2 nM, Bmax = 0.26 pmol/mg. C, [3H]SCH-23390 binding parameters in the 10248 cells are as follows: control KD = 0.18 nM, Bmax = 1.8 pmol/mg; dopamine-treated KD = 0.19 nM, Bmax = 1.2 pmol/mg. All data are from a representative experiment that was performed three times with similar results.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dopamine dose-response curves for D1 receptor desensitization and down-regulation in CHO cell lines. Cells were cultured in the presence of 0.2 mM sodium metabisulfite and increasing concentrations of dopamine for 24 h before assessment of dopamine-stimulated cAMP stimulation and receptor binding as described in Experimental Procedures. A, desensitization of dopamine-dependent cAMP accumulation in the wild-type (), 10260 (), and 10248 () cell lines. Subsequent to pretreatment, dopamine-stimulated cAMP production was assessed using a single maximally effective dose of dopamine (100 µM), and the data are expressed as the percent loss (or desensitization) of activity relative to a control (untreated) group. The data shown are from a single representative experiment that was performed three times with similar results. The EC50 values for dopamine-induced desensitization are as follows: wild-type, 0.1 µM; 10260, 0.18 µM; 10248, 0.28 µM. B, down-regulation of D1 receptor binding activity in the wild-type (), 10260 (), and 10248 () cell lines. Subsequent to pretreatment, [3H]SCH-23390 binding was assessed using a single concentration (0.5 nM), and the data are expressed as the percent decline of activity relative to a control (untreated) group. The data represent the mean ± S.E. values of three independent experiments. The EC50 values for dopamine-induced down-regulation are wild-type, 0.012 µM; 10260, 0.026 µM; and 10248, 0.22 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Time courses for dopamine-induced D1 receptor desensitization () and down-regulation () in CHO cell lines. Cells were cultured in the presence of 0.2 mM sodium metabisulfite and 100 µM dopamine (except for a control group with no dopamine) for the indicated times before assessment of dopamine-stimulated cAMP stimulation and receptor binding as described in Experimental Procedures. Subsequent to pretreatment, dopamine-stimulated cAMP production was assessed using a single maximally effective dose of dopamine (100 µM), and [3H]SCH-23390 binding was assessed using a single concentration (0.5 nM) of radioligand. The data are expressed as the percent decline of activity relative to a control (untreated) group. The data shown are from single representative experiments that were performed three times with similar results. A, wild-type cells. B, 10260 cells. C, 10248 cells.

cAMP-Induced Regulation of D₁ Receptor in Wild-Type and Mutant CHO Cell Lines. Because dopamine treatment is likely to be activating more than one regulatory pathway in the cells, we wanted to assess the effects of selectively stimulating only the PKA-mediated pathway. To do this, we used a membrane-permeable analog of cAMP, 8-(4-chlorophenylthio) (CPT)-cAMP, for cell treatments. Due to interference of residual CPT-cAMP in our whole-cell cAMP accumulation assays, subsequent assessments of dopamine-stimulated cAMP levels were conducted using membrane homogenate assays. Figure 6A shows cAMP accumulation assays in membranes prepared from control and CPT-cAMP-treated 10001 wild-type cells. As shown, CPT-cAMP treatment results in a 10-fold shift in the EC₅₀ value for dopamine-stimulated cAMP accumulation as well as a 60% decrease in the maximum response. Figure 6B shows results using the 10260 cell line. In these cells, CPT-cAMP treatment results in a 3-fold shift in the dopamine dose-response curve and a ~40% decrease in the maximum response. Finally, in Fig. 6C, it can be seen that CPT-cAMP treatment of the 10248 cells promotes only a 2-fold shift in the EC₅₀ value for dopamine and a ~20% decrease in the maximum response. In separate experiments, it was determined that these CPT-cAMP treatments were maximally effective with respect to both dose and time of treatment (data not shown). Notably the 10001, 10260, and 10248 cell lines show a graded desensitization response to CPT-cAMP, as was observed with dopamine treatment (Fig. 2). However, the desensitization responses evoked with the cAMP analog are considerably less that those induced by agonist treatment for all three cell lines (cf. Figs. 2 and 6).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   CPT-cAMP-induced desensitization of the D1 dopamine receptor in wild-type and mutant CHO cell lines. Cells were cultured in the presence of Ro-201724 and in the absence (control, ) or presence () of 1 mM CPT-cAMP for 24 h. The cells were then extensively washed, and the membranes were prepared as for the binding assays. cAMP stimulation in the membrane preparations was subsequently performed as described in Experimental Procedures. The data are from single representative experiments that were performed three times with similar results. The EC50 values for dopamine-stimulation of cAMP accumulation determined in these experiments were as follows: A, 10001 wild-type cells, control EC50 = 5 µM; treated EC50 = 53 µM. B, 10260 cells, control EC50 = 0.7 µM; treated EC50 = 1.7 µM. C, 10248 cells, control EC50 = 2.4 µM; treated EC50 = 4.4 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   CPT-cAMP-induced down-regulation of D1 receptors in CHO cell lines. Cells were treated with 1 mM CPT-cAMP for 24 h as described in the legend to Fig. 6 subsequent to harvesting and preparation of membranes. The radioligand binding assays were performed as described in Experimental Procedures with the saturation isotherm data being plotted in Scatchard coordinates of bound/free versus free radioligand. Indicated are data from control cultures () and the CPT-cAMP-treated cultures(). A, [3H]SCH-23390 binding parameters in the wild-type (10001) cells are as follows: control KD = 0.15 nM, Bmax = 1.4 pmol/mg; CPT-cAMP-treated KD = 0.18 nM, Bmax = 0.77 pmol/mg. B, [3H]SCH-23390 binding parameters in the 10260 cells are as follows: control KD = 0.14 nM, Bmax = 1.3 pmol/mg; CPT-cAMP-treated KD = 0.17 nM, Bmax = 1.0 pmol/mg. C, [3H]SCH-23390 binding parameters in the 10248 cells are as follows: control KD = 0.14 nM, Bmax = 3.7 pmol/mg; CPT-cAMP-treated KD = 0.17 nM, Bmax = 3.5 pmol/mg. All data are from a representative experiment that was performed three times with similar results.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The involvement of PKA in agonist-induced desensitization of the D₁ dopamine receptor has been relatively uncertain, with various studies reporting results of both a positive and negative nature. For instance, some investigators have reported that direct activation of PKA, through treatment of cells with membrane-permeable cAMP analogs or forskolin, will result in functional desensitization (Bates et al., 1991; Black et al., 1994), whereas others have reported opposite results (Bates et al., 1993; Lewis et al., 1998). Similarly, it has been observed that treatment of cells with PKA inhibitors can either attenuate agonist-induced D₁ receptor desensitization (Zhou et al., 1991) or have no effect (Lewis et al., 1998). Interestingly, in the study of Bates et al. (1991), it was argued that PKA was required for agonist-induced down-regulation of the D₁ receptor but not for its functional desensitization. Given these discrepant findings, we thought it would be informative to examine D₁ receptor desensitization phenomena in CHO cell lines that are significantly lacking in PKA activity. Our current data using these PKA-deficient CHO cell lines clearly indicate the involvement of PKA in both agonist-induced desensitization and down-regulation of the D₁ receptor.

Although it seems certain that PKA plays a significant role in the agonist-induced regulation of the D₁ receptor, the mechanisms by which PKA exerts its effects are unclear. With respect to agonist-induced down-regulation of the receptor, it is reasonable to speculate that at a minimum, this process involves GRK-mediated phosphorylation of the receptor and arrestin-dependent internalization, eventually leading to degradation of the receptor (Krupnick and Benovic, 1998; Lefkowitz, 1998). This degradative process might also be induced and/or enhanced by phosphorylation of the receptor by PKA. Recently, however, we found that mutagenesis of all potential PKA phosphorylation sites on the D₁ receptor has no effect on agonist-induced down-regulation, although functional desensitization is impaired (Jiang and Sibley, 1999). This observation seems to argue against PKA promoting a degradative pathway, at least through phosphorylation of the receptor protein. Another possibility is that PKA activation may result in inhibition of receptor synthesis, leading to decreased receptor expression. Because the transcription of the receptor is under the control of a strong viral promoter in the transfected cells, any potential regulation of synthesis must occur at the posttranscriptional level. In this regard, it is interesting to note that agonist treatment of cells expressing the D₁ receptor has been shown to decrease receptor mRNA levels (Minowa et al., 1996; Sidhu et al., 1999). Moreover, it has long been known that agonist-induced down-regulation of -adrenergic receptors partially involves a PKA-mediated destabilization of their mRNAs (Bouvier et al., 1989; Hadcock et al., 1989; Pendel et al., 1996; Danner et al., 1998). It seems likely that similar processes are operative for D₁ dopamine receptors. Thus, in the PKA-deficient cells, agonist-induced receptor down-regulation would be attenuated, whereas the cAMP analog-induced down-regulation would be nearly abolished, which is, in fact, what we observed (Figs. 3 and 7).

With respect to desensitization of the cAMP response, this presumably involves a functional uncoupling of the receptor such that it is less able to activate downstream G proteins as well as a loss of cell surface receptors through internalization or down-regulation. Tiberi et al. (1996) showed that the D₁ receptor is phosphorylated via GRK-mediated mechanisms and that this correlates with the agonist-induced desensitization. As mentioned earlier, we have recently obtained evidence, via site-directed mutagenesis methods, suggesting that PKA-mediated phosphorylation of the D₁ receptor is also involved in agonist-induced desensitization (Jiang and Sibley, 1999). Thus, treatment of the cells with agonists would be expected to functionally desensitize the D₁ receptor through GRK- and PKA-mediated phosphorylation as well as to induce down-regulation through GRK/arrestin-dependent internalization and a PKA-mediated decrease in receptor synthesis. Indeed, down-regulation of receptor number could play a major role in the desensitization response, especially for that induced by CPT-cAMP treatment, which does not involve agonist activation of the receptor. Obviously, each of these regulatory steps must be independently verified, but we can use such a model to predict experimental outcomes and to compare our present data with such predictions. For instance, it would be expected that agonist-induced desensitization (GRK plus PKA pathways) would be greater than that produced by cAMP analog-induced (PKA pathway only) desensitization, which was, in fact, observed (cf. Figs. 2 and 6). Similarly, in the PKA-deficient cells, agonist-induced desensitization would be attenuated, whereas the cAMP analog-induced response would be nearly abolished, which, again, is what we observed (Figs. 2 and 6). Thus, our current data are entirely consistent with these proposed mechanisms of PKA in regulating D₁ receptor function.

A final interesting observation was that the cells that were more deficient in type II PKA activity exhibited the greatest attenuation of agonist- and cAMP analog-induced receptor regulation. Thus, the CHO line 10248, which is completely lacking type II PKA activity, showed less desensitization and receptor down-regulation than the CHO line 10260, which contains some residual type II activity. This might suggest that type II PKA is more relevant than type I with respect to regulating D₁ receptor function. In this regard, it is interesting to note that PKA type II is the predominant isoform in the corpus striatum, the brain region showing the highest D₁ receptor expression, where it is present in postsynaptic densities (Brandon et al., 1998). Given this observation, it will be interesting and important to perform further experiments addressing the cellular colocalization of PKA type II and the D₁ receptor in the striatum and other brain regions.