Abstract
To investigate the role of the cAMP-dependent protein kinase (PKA) in the desensitization and down-regulation of the D1 dopamine receptor, we stably expressed the rat cDNA for this receptor in mutant Chinese hamster ovary (CHO) cell lines deficient in PKA activity. The 10260 mutant CHO cell line has been characterized as expressing less than 10% of type I and type II PKA activities relative to the parental 10001 CHO cell line. The 10248 mutant CHO line lacks type II PKA activity and expresses a defective type I PKA. The transfected parental and mutant cell lines were found to express ∼1 pmol/mg D1receptor binding activity (Bmax) as determined using [3H]SCH-23390 binding assays. All three cell lines demonstrated similar levels of dopamine-stimulated adenylyl cyclase activity. Pretreatment of all three CHO cells with dopamine resulted in desensitization of the adenylyl cyclase response, although the maximum desensitization was attenuated by 20 and 40% in the 10260 and 10248 cell lines, respectively. Dopamine also promoted, in a time- and dose-dependent fashion, a >90% down-regulation of D1receptors in the parental cell line but only a 50 and 30% decrease in the 10260 and 10248 cells, respectively. Similarly, treatment of the cells with the membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP induced functional desensitization and down-regulation of the D1 receptor, although it was not as great as that observed with agonist pretreatment. As with the agonist pretreatments, the 8-(4-chlorophenylthio)-induced responses were attenuated in the mutant cells with the 10248 line exhibiting the least desensitization/down-regulation. Our results suggest that PKA significantly contributes to the desensitization and down-regulation of D1 receptors in CHO cells and that type II PKA may be the more relevant isoform with respect to regulating D1receptor function.
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 D1A and D1B 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 Gi/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 inSibley 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 inSf9 (Ng et al., 1994) or human embryonic kidney 293 cells (Tiberi et al., 1996) have shown that the D1receptor 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 D1receptors, 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 D1receptor 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
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-SRα (Takebe et al., 1988), and the complete D1 receptor sequence was confirmed by DNA sequencing. The pCD-SRα 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 KDand Bmax, as well as the EC50 values for dopamine, were obtained using the software program Prism (GraphPad Software, San Diego, CA).
Results
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 (Bmax 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 (KD) 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.
We also investigated the ability of the three transfected cell lines to respond to dopamine in terms of cAMP accumulation (Fig. 1B). Incubation of the cells with increasing concentrations of dopamine resulted in a dose-dependent accumulation of cAMP with a similar maximum response of ∼50 to 60 pmol/80,000 cells in all three cell lines. The calculated EC50 values for dopamine stimulation were also similar for the wild-type and mutant cell lines, although the 10248 line exhibited a slightly lower value: wild-type (10001), 0.16 ± 0.07 μM (n = 4); 10260, 0.12 ± 0.03 μM (n = 4); and 10248, 0.08 ± 0.03 μM (n = 4). These results indicate that the D1 dopamine receptor is functionally coupled to a similar extent in all three cell types and that PKA activity is not required for D1 receptor stimulation of cAMP accumulation.
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.
We were also interested in examining agonist-induced receptor down-regulation (defined as a loss of radioligand binding activity without implying any mechanism) in the mutant CHO cell lines. Figure3A shows that pretreatment with dopamine results in a 95% loss in the maximum radioligand binding of the D1 receptor in the wild-type CHO cells without any change in the affinity for [3H]SCH-23390. The lack of effect on the radioligandKD value implies that dopamine is being completely washed out. Figure 3, B and C, shows that the receptor binding activity is also reduced in the mutant cell lines on dopamine pretreatment, although the reduction is not as great as that observed with the wild-type cells. For the 10260 cell line, dopamine preincubation results in a 50% loss of D1receptor binding activity (Fig. 3B), whereas for the 10248 cell line, this treatment results in only a 30% reduction in receptor binding (Fig. 3C). As for the wild-type cells, neither of the mutant cells exhibits an alteration in the affinity of [3H]SCH-23390 subsequent to dopamine exposure. Thus, as for the desensitization response (Fig. 2), the mutant CHO cells also exhibit agonist-induced receptor down-regulation; however, this response is attenuated compared with that exhibited with wild-type CHO cells.
Because the effects of dopamine treatment on D1receptor regulation were attenuated in the PKA mutant cell lines, we decided to investigate whether this attenuation reflected a decrease in the potency or efficacy (or both) of the agonist to promote the desensitization and down-regulation responses. Figure4A shows a dose-response experiment for dopamine-induced desensitization of the cAMP response in all three CHO cell lines. As can be seen, this is a dose-dependent response with dopamine exhibiting a similar potency among all three cell lines. Interestingly, the potency of dopamine for producing desensitization is similar to that for stimulating cAMP accumulation (cf. Figs. 1B and 2). Also, as observed previously (Fig. 2), the extent of desensitization is significantly reduced in the mutant cell lines, with the 10248 line showing the greatest attenuation relative to the wild-type cells (Fig.4A). Most importantly, it can be observed that the attenuation of desensitization in the mutant cell lines is due to a reduction in the maximum effectiveness of dopamine for producing this response.
Figure 4B shows dose-response experiments for dopamine-induced down-regulation of receptor binding. As can be seen, this regulatory response is also clearly dose-dependent. Interestingly, the EC50 values for producing receptor down-regulation are somewhat lower than those for producing desensitization for the wild-type and 10260 cell lines, whereas these values are more similar for the 10248 cell line. Nevertheless, there is a clear gradation of responses among the wild-type, 10260, and 10248 cell lines, with the wild-type cells showing the greatest down-regulation and the 10248 cells showing the least. Moreover, as observed for the desensitization response, the diminished receptor down-regulation in the mutant cell lines is due to a reduction in the efficacy of dopamine for producing this response. From these results, we can further conclude that the attenuated regulatory responses previously observed in the mutant cell lines (Figs. 2 and 3) are not due to a less-than-maximally effective dose of dopamine.
Because the attenuated regulatory responses in the mutant cells could be also due to a reduction in the rate of agonist-induced desensitization, we decided to perform time course experiments for the dopamine-induced effects. Figure 5A shows the rate of dopamine-induced desensitization and down-regulation in the wild-type 10001 cell line. Both responses exhibit similar rates, each with a t1/2 of ∼5 h, and both are maximal by ∼20 h of pretreatment. Figure 5B shows the results with the 10260 cell line. With these cells, the rates of desensitization and down-regulation are similar to those seen with the wild-type cells except that the extent of these responses is attenuated. As with the wild-type cells, both responses are maximal by 20 h. Finally, Fig.5C shows data from the 10248 cell line. As with the other two cell lines, the rates of desensitization and down-regulation exhibitt1/2 values of 5 to 10 h and are maximal by 20 h of pretreatment. In contrast to the other two cell lines, however, the 10248 cells exhibit significantly reduced desensitization and down-regulation responses, as previously noted. Also, there appears to be a greater separation of the rates of desensitization and down-regulation in the 10248 cells compared with the other cell lines. This may be due to the higher receptor expression level in the 10248 cells (Fig. 1A) resulting in “spare” receptors such that the desensitization rate is somewhat slower than the down-regulation rate. Most importantly, however, the data in Fig. 5indicate that the attenuated agonist-induced desensitization observed in the mutant cell lines is due to a reduction in the extent and not the rate of onset of these regulatory responses.
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. Figure6A 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. Figure6B 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).
In Fig. 7, the effects of CPT-cAMP treatment on the radioligand binding activity of the D1 dopamine receptor are shown. Figure 7A shows that CPT-cAMP treatment of the 10001 wild-type cells results in a 50% loss of ligand binding activity without a significant change in receptor affinity. Conversely, CPT-cAMP treatment results in only a 30% loss of receptor binding activity in the 10260 cell line (Fig.7B), whereas the 10248 cells show only a negligible down-regulation response to CPT-cAMP treatment (Fig. 7C). Thus, as with the cAMP accumulation assays (Fig. 6), the 10001, 10260, and 10248 cell lines show graded down-regulation responses to CPT-cAMP treatment that overall are less than those observed with agonist treatment (cf. Figs.3 and 7).
Discussion
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 ofBates et al. (1991), it was argued that PKA was required for agonist-induced down-regulation of the D1receptor 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 D1receptor 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 β-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.
Footnotes
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Send reprint requests to: Dr. David R. Sibley, Experimental Therapeutics Branch, NINDS/National Institutes of Health, Bldg. 10, Rm. 5C108, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. E-mail: sibley{at}helix.nih.gov
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↵1 A.L.M.V. is the recipient of a fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico—CNPq.
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↵2 Present address: Departamento de Neurobiologia, Universidade Federal Fluminense, Niteroi, Cx. Postal 100180-RJ, 24001-970 Brasil.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- PKA
- protein kinase A
- GRK
- G protein-coupled receptor kinase
- EBSS
- Earle's balanced salt solution
- CPT
- 8-(4-chlorophenylthio)
- CHO
- Chinese hamster ovary
- Received October 5, 1999.
- Accepted January 18, 2000.
- U.S. Government