Abstract
Chronic dopamine receptor activation is implicated in several central nervous system disorders. Although acute activation of -coupled D2 dopamine receptors inhibits adenylyl cyclase, persistent activation enhances adenylyl cyclase activity, a phenomenon called heterologous sensitization. Previous work revealed a requirement for in D2-induced heterologous sensitization of AC5. To elucidate the mechanism of dependency, we expressed mutants in -deficient cells. Neither -palmitoylation nor -Gฮฒฮณ interactions were required for sensitization of AC5. Moreover, we found that coexpressing ฮฒARKct-CD8 or Sar1(H79G) blocked heterologous sensitization. These studies are consistent with a role for -AC5 interactions in sensitization however, Gฮฒฮณ appears to have an indirect role in heterologous sensitization of AC5, possibly by promoting proper signalosome assembly.
1. Introduction
Dopamine receptors and dopamine signaling have been implicated in various neurological and psychiatric disorders including Parkinsonโs disease, schizophrenia, and drug abuse [1โ3]. Dopamine receptors are divided into two subfamilies, the Gฮฑ s-coupled D1 and D5 receptors and the Gฮฑ i/o-coupled D2, D3, and D4 dopamine receptors that have stimulatory and inhibitory effects on adenylyl cyclase (AC), respectively (see [3] for a recent review). Acute stimulation of D2 dopamine receptors leads to inhibition of AC activity, however, persistent activation of this Gฮฑ i/o-coupled receptor paradoxically results in its enhancement [4]. This phenomenon, called heterologous sensitization of AC, is also known as cAMP overshoot, supersensitization, or superactivation of AC. D2 dopamine receptor-induced heterologous sensitization of cyclic AMP signaling has been demonstrated in several cellular systems as well as in animal models and has also been suggested to occur in humans [4โ6]. For example, it was observed that repeated administration of the D2 receptor agonist quinpirole enhances AC activity in the caudate putamen, increases CREB phosphorylation, and also alters behavior in rodents [5, 6]. Although this mode of AC regulation has been recognized for over three decades [7], the molecular signaling mechanism causing heterologous sensitization of AC is only partially understood, attributed to some extent to differences in AC isoform-specific regulation [4].
There are nine differentially regulated membrane-bound AC isoforms in mammalian cells [4, 8]. Whereas all AC isoforms are stimulated by stimulatory Gฮฑ s, only a subset is inhibited by inhibitory Gฮฑ i, and some AC isoforms are differentially regulated by Gฮฒฮณ [4, 8]. Here, we studied human adenylyl cyclase type 5 (AC5) that is potently stimulated by Gฮฑ s, inhibited by acute activation of Gฮฑ i, and conditionally activated by Gฮฒฮณ [8]. AC5 is expressed at high levels in the central nervous system and has been identified as a primary effector of D2 dopamine receptors in the striatum [9, 10].
The aim of the current study was to investigate the role(s) of heterotrimeric G proteins in D2 receptor-mediated heterologous sensitization of AC5. By exploring sensitization in cells devoid of endogenous Gฮฑ s [11], we were able to examine the ability of Gฮฑ s mutants to support sensitization without interference from endogenous Gฮฑ s. Additionally, this Gฮฑ s-deficient cellular model expresses very low levels of AC5 making them a reasonable model for studies of recombinant AC5 [12]. Heterologous sensitization of AC5 was readily rescued by wild-type Gฮฑ s and by mutants deficient in palmitoylation [13] or Gฮฒฮณ interaction [14]. We also assessed the role of Gฮฒฮณ and the signalosome in D2 receptor-induced heterologous sensitization of AC5 by sequestering Gฮฒฮณ subunits with ฮฒARKct-CD8 [15, 16] and coexpressing a dominant-negative mutant of the Sar1 GTPase [17]. These experiments revealed that both ฮฒARKct-CD8 and Sar1(H79G) attenuated sensitization, suggesting that the components of the signaling complex utilized in heterologous sensitization, presumably AC5 and Gฮฑ s, assemble postsynthesis in the endoplasmic reticulum (ER). Together with previous findings, the present data support a model in which Gฮฑ s directly interacts with AC5. In contrast, Gฮฒฮณ appears to have an indirect role in heterologous sensitization of AC5.
2. Materials and Methods
2.1. Constructs
The human D2L receptor and AC5 or ฮAC5 [18] were cloned into the dual expression vector pBUDCE4 (Invitrogen, Carlsbad, CA) creating pBUD/hAC5, D2R and pBUD/ฮAC5, D2R. pcDNA3/ฮฒARKct-CD8 [15, 16] and pcDNA/vsvg-Sar1 (wild type and H79G) [19] were used. pcDNA1/Gฮฑ s-CFP [20] was a gift from Dr. Catherine Berlot. The pcDNA3.1/Gฮฑ s-IEK+ mutant [21] was a gift from Dr. Philip Wedegaertner. The C3S mutation was created by site-directed mutagenesis, and the fragment containing the IEK+ mutations was amplified by PCR. The resulting constructs, pcDNA1/Gฮฑ s-CFP(C3S) and pcDNA1/Gฮฑ s-CFP(IEK+) were sequenced.
2.2. Cell Culture and Transient Transfection
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Gฮฑ s-deficient murine embryonic fibroblast cells, cells [11, 12], were a gift from Dr. Murat Bastepe. Cells were cultured in 50โ:โ50 mix of F12โ:โDMEM media supplemented with 5% FBS (HyClone, Logan, UT), 1% Ant-Anti (Invitrogen, Carlsbad, CA) in a humidified incubator at 33ยฐC with 5% CO2. Approximately 80,000โcells/well were seeded in 24-well plates the day before transient transfection. DNA (400โng pBUD/hAC5 or ฮAC5, D2R alone or in combination with 10โng pcDNA/Gฮฑ s-CFP, 300โng pcDNA3/ฮฒARKct-CD8, or 300โng pcDNA/vsvg-Sar1) was mixed with Opti-MEM and 1โฮผL/well Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The medium was replaced with 200โฮผL/well prewarmed Opti-MEM, and the DNA/Lipofectamine mixture was added to the cells. After 4โhr, culture medium (500โฮผL/well) was added, and the cells were analyzed after 48โhr. For microscopy, the amount of pcDNA/Gฮฑ s-CFP was increased to 100โng/well.
2.3. Acute cAMP Accumulation
The assays were carried out in assay buffer (EBSS supplemented with 0.2% ascorbic acid, 15โmM HEPES, and 2% BCS (HyClone, Logan, UT), and 500โฮผM IBMX) with 100โnM forskolin (Tocris Bioscience, Ellisville, MO) as noted for 37ยฐC for 15โmin. The media was decanted, ice-cold trichloroacetic acid was added, and the lysates were stored at 4ยฐC. Cyclic AMP was quantified using a competitive binding assay as described previously [22]. Data were collected from a minimum of three independent experiments carried out in duplicate and were normalized to either basal or vehicle conditions. The GraphPad Prism 5 software (GraphPad Software Inc., LaJolla, CA) was used for data and statistical analyses. A value of โค0.05 was considered statistically significant.
2.4. Heterologous Sensitization
The cells were pretreated with 1โฮผM quinpirole or vehicle in assay buffer (without IBMX) for 2โhr followed by three washes. cAMP was measured as described above for acute cAMP accumulation, with the addition of 1โฮผM spiperone to block the action of any residual quinpirole.
2.5. Microscopy
Cells were seeded in cover glass slides (Nunc, Rochester, NY). A 12 bit photometric CoolSNAP (Roper Scientific) CCD camera mounted on a TE-2000 inverted epifluorescence microscope (Nikon Instruments Inc., Melville, NY) with filters (ex. 500/20, em. 535/30) from Chroma (Rockingham, VT) was used. Images were acquired with the MetaMorph software (Molecular Devices, Sunnyvale, CA) and analyzed using Image J (http://rsbweb.nih.gov/ij/).
3. Results and Discussion
3.1. Gฮฑ s Mutants Rescue Heterologous Sensitization of AC5
Our laboratory has previously shown that mutants of canine AC5 that do not interact with Gฮฑ s are deficient in sensitization [23, 24] and that D2-mediated heterologous sensitization of AC5 has an absolute requirement for Gฮฑ s [12]. Our present objective was to elucidate the mechanism of Gฮฑ s-dependent heterologous sensitization of human AC5 by utilizing two different Gฮฑ s-CFP [20] mutants (Figure 1(a)). The C3S substitution eliminates the N-terminal palmitoylation site, which causes Gฮฑ s to mislocalize to the cytosolic fraction [13]. The IEK+ mutant contains a series of substitutions, yielding a Gฮฒฮณ-binding deficient Gฮฑ s that also displays a reduction in palmitoylation [21].
(a)
(b)
(c)
(d)
The Gฮฑ s-CFP constructs were coexpressed with AC5 and D2. Since both C3S and IEK+ are deficient in responses to receptor stimulation [13, 21], we used direct stimulation of AC5 with forskolin throughout this study. Basal cAMP accumulation without any Gฮฑ s was โpmol/well, whereas co-expression of Gฮฑ s-CFP increased cAMP accumulation to โpmol/well (wild-type, wt), โpmol/well (C3S), and โpmol/well (IEK+). Forskolin further stimulated cAMP with values 2.5โ3-fold over basal levels (Figure 1(b)), indicating that wild-type and both Gฮฑ s mutants functionally couple to AC5. Next, expression and subcellular localization of the Gฮฑ s-CFP constructs (in the presence of AC5 and D2) were evaluated by fluorescence microscopy (Figure 1(c)). Wild-type Gฮฑ s-CFP showed both plasma membrane and intracellular localization, whereas the C3S and IEK+ mutants were predominantly localized intracellularly (Figure 1(c)), consistent with previous reports [14, 21].
To assess whether the Gฮฑ s-CFP mutants could rescue heterologous sensitization, cells were pretreated with vehicle or quinpirole followed by cAMP accumulation. Consistent with our previous report [12], no sensitization of AC5 was observed in the absence of Gฮฑ s (Figure 1(d), ctrl). In contrast, coexpression of wild-type Gฮฑ s-CFP resulted in robust sensitization of AC5 under both basal and forskolin-stimulated conditions (Figure 1(d)). Surprisingly, expression of the Gฮฑ s mutants also significantly rescued heterologous sensitization under basal conditions (white bars) and to a lesser degree forskolin-stimulated conditions (black bars). As both mutants are deficient in palmitoylation and membrane localization, neither palmitoylation per se, nor membrane localization of Gฮฑ s appears to be essential for heterologous sensitization of AC5.
3.2. Role of Gฮฒฮณ Subunits in Heterologous Sensitization of AC5
Although we have established that Gฮฑ s is required for heterologous sensitization, our findings above for the IEK+ mutant suggest that direct interactions between Gฮฑ s and Gฮฒฮณ are not critical. This prompted us to further investigate the role of Gฮฒฮณ in D2 receptor-mediated heterologous sensitization of AC5. The C-terminus of ฮฒ-adrenergic kinase or GRK2 (ฮฒARKct) has been used to sequester Gฮฒฮณ subunits and inhibit Gฮฒฮณ-mediated signaling events, including heterologous sensitization [15, 25, 26]. In the absence of ฮฒARKct-CD8 (membrane bound ฮฒARKct), AC5 displayed robust heterologous sensitization (open bars, Figure 2(a)). Sequestering Gฮฒฮณ blocked sensitization of AC5, under both basal and forskolin-stimulated conditions, revealing the necessity of Gฮฒฮณ for heterologous sensitization of AC5 (black bars, Figure 2(a)). In contrast, ฮฒARKct-CD8 had no substantial effects on acute D2 receptor activation; quinpirole produced significant inhibition of cAMP accumulation in the presence of ฮฒARKct-CD8 (77 ยฑ 10% inhibition; , data not shown). In an effort to explore the site of action for Gฮฒฮณ-dependent sensitization, we used an N-terminal deletion mutant of AC5, ฮAC5. This mutant is functional and responds to Gฮฑ s stimulation but is deficient in binding Gฮฒฮณ [18]. The ฮAC5 mutant displayed significant sensitization that was also blocked by ฮฒARKct-CD8 (Figure 2(b)), suggesting that N-terminal Gฮฒฮณ binding is not intimately involved in heterologous sensitization of AC5. Instead, there are clearly additional, unidentified Gฮฒฮณ interaction sites in AC5 that are necessary for heterologous sensitization. Such an assumption is supported by FRET and in vitro activation studies of the AC5 deletion mutant [18] as well as studies of AC2, which possesses multiple motifs for Gฮฒฮณ interaction and regulation that are located in the C1b and C2b domains of AC2 [27]. Other possibilities are that ฮAC5 interacts with endogenous AC isoforms in an AC dimer (see [28]) that binds Gฮฒฮณ or that specific Gฮฒ and Gฮณ subunits or Gฮฒฮณ pairs are involved. However, it is also possible that the Gฮฒฮณ mechanisms involving sensitization of AC may be indirect [4].
(a)
(b)
(c)
3.3. Disruption of Signalosome Assembly Affects Heterologous Sensitization of AC5
Because sequestering Gฮฒฮณ subunits alters signalosome assembly [15], we hypothesized that a specific signaling complex could be required for heterologous sensitization of AC5. Several small GTPases, including Sar1, are involved in signal complex assembly and anterograde protein trafficking [29]. A series of studies using dominant negative mutants of these GTPases shows that Gฮฑ s and Gฮฒฮณ interact with AC2 during trafficking to the plasma membrane [30, 31] and that the Gฮฑ s-AC2 interaction is disrupted by Sar1(H79G) [30].
To study the possibility that interactions between AC5 and its specific signaling partners play a role, we utilized Sar1 and Sar1(H79G) and noted that coexpression with the dominant negative mutant prevented heterologous sensitization of AC5 (Figure 2(c)). In contrast, acute D2 receptor-mediated inhibition of AC5 was not significantly blocked in the presence of Sar1(H79G) (data not shown). Our data are consistent with the findings that Sar1(H79G) disrupts AC-Gฮฑ s interactions (as measured by BRET or coimmunoprecipitation) to a larger degree than AC-Gฮฑ i interactions [30]. In contrast, Sar1(H79G) did not affect the interactions between AC and Gฮฒฮณ [30], suggesting that the AC interacts with Gฮฒฮณ at an early step in the endoplasmic reticulum (ER), but that the interaction with Gฮฑ s occurs after ER export. The observation that signaling mechanisms of acute activity and heterologous sensitization are differentially affected further supports the hypothesis that heterologous sensitization and acute stimulation are dependent on separate mechanisms and possibly separate signalosome components.
4. Conclusion
The present data support a complex model of D2 dopamine receptor-induced heterologous sensitization of AC5 where Gฮฑ s appears to directly interact with AC5. A role for Gฮฒฮณ was confirmed; however, our observations suggest an indirect role for Gฮฒฮณ that may be involved during the formation of the sensitization signaling complex. A critical role for AC5 in mediating dopamine responses has been previously demonstrated in AC5 deficient mice, which show impaired responses to D2 receptor activation [9]. Therefore, these results have implications in brain regions where D2 dopamine receptors and AC5 are coexpressed, such as the striatum [32], which is implicated in drug addiction, motivation, mood, and voluntary movement. Persistent D2 dopamine receptor activation has also been linked to psychiatric disorders (e.g., schizophrenia and drug abuse) and to the adaptive responses associated with drug therapy in Parkinsonโs disease. Enhancing our understanding of the underlying components and mechanisms of heterologous sensitization and regulation of specific AC activity (in the striatum) may aid in the development of improved and future therapies for these disorders. For example, recent studies have identified small molecule inhibitors of Gฮฒฮณ-mediated signaling [33] and AC isoform-specific inhibitors [34] that may offer novel therapeutic strategies for modulating complex CNS behaviors involving dopamine receptor signaling.
Abbreviations
| ACโ: | Adenylyl cyclase |
| cAMPโ: | Cyclic adenosine monophosphate |
| CFPโ: | Cyan fluorescent protein |
| D2Rโ: | Dopamine D2L receptor |
| GPCRโ: | G protein-coupled receptor |
| ฮฒARKctโ: | ฮฒ-adrenergic kinase c-terminus |
| ERโ: | Endoplasmic reticulum |
Acknowledgments
This work was supported by Purdue University and by NIH grants MH060397 (V. J. Watts) and GM60419 (C. W. Dessauer). T. E. Hebรฉrt is a Chercheur National of the Fonds de la Recherche en Santรฉ du Quรฉbec (FRSQ). The authors would like to thank Dr. Pierre-Alexandre Vidi for help with cloning of constructs. They would also like to thank Jason Conley and Elisabeth Garland-Kuntz for proofreading and making suggestions to improve the present paper.